Angiotensin-converting enzyme 2 activation improves endothelial function.

PubMed

Fraga-Silva, Rodrigo A; Costa-Fraga, Fabiana P; Murça, Tatiane M; Moraes, Patrícia L; Martins Lima, Augusto; Lautner, Roberto Q; Castro, Carlos H; Soares, Célia Maria A; Borges, Clayton L; Nadu, Ana Paula; Oliveira, Marilene L; Shenoy, Vinayak; Katovich, Michael J; Santos, Robson A S; Raizada, Mohan K; Ferreira, Anderson J

2013-06-01

Diminished release and function of endothelium-derived nitric oxide coupled with increases in reactive oxygen species production is critical in endothelial dysfunction. Recent evidences have shown that activation of the protective axis of the renin-angiotensin system composed by angiotensin-converting enzyme 2, angiotensin-(1-7), and Mas receptor promotes many beneficial vascular effects. This has led us to postulate that activation of intrinsic angiotensin-converting enzyme 2 would improve endothelial function by decreasing the reactive oxygen species production. In the present study, we tested 1-[[2-(dimetilamino)etil]amino]-4-(hidroximetil)-7-[[(4-metilfenil)sulfonil]oxi]-9H-xantona-9 (XNT), a small molecule angiotensin-converting enzyme 2 activator, on endothelial function to validate this hypothesis. In vivo treatment with XNT (1 mg/kg per day for 4 weeks) improved the endothelial function of spontaneously hypertensive rats and of streptozotocin-induced diabetic rats when evaluated through the vasorelaxant responses to acetylcholine/sodium nitroprusside. Acute in vitro incubation with XNT caused endothelial-dependent vasorelaxation in aortic rings of rats. This vasorelaxation effect was attenuated by the Mas antagonist D-pro7-Ang-(1-7), and it was reduced in Mas knockout mice. These effects were associated with reduction in reactive oxygen species production. In addition, Ang II-induced reactive oxygen species production in human aortic endothelial cells was attenuated by preincubation with XNT. These results showed that chronic XNT administration improves the endothelial function of hypertensive and diabetic rat vessels by attenuation of the oxidative stress. Moreover, XNT elicits an endothelial-dependent vasorelaxation response, which was mediated by Mas. Thus, this study indicated that angiotensin-converting enzyme 2 activation promotes beneficial effects on the endothelial function and it is a potential target for treating cardiovascular disease.

Molecular diversity of tuliposide B-converting enzyme in tulip (Tulipa gesneriana): identification of the third isozyme with a distinct expression profile.

PubMed

Nomura, Taiji; Kuchida, Ryo; Kitaoka, Naoki; Kato, Yasuo

2018-02-23

6-Tuliposide B (PosB), a major secondary metabolite that accumulates in tulip (Tulipa gesneriana), is converted to the antibacterial lactone, tulipalin B (PaB), by PosB-converting enzyme (TCEB). TgTCEB1 and TgTCEB-R, which encode TCEB, are specifically expressed in tulip pollen and roots, respectively, but are hardly expressed in other tissues (e.g. leaves) despite the presence of substantial PosB-converting activity, suggesting the existence of another TCEB isozyme. Here, we describe the identification of TgTCEB-L ("L" for leaf), a paralog of TgTCEB1 and TgTCEB-R, from leaves via native enzyme purification. The enzymatic characters of TgTCEB-L, including catalytic activity and subcellular localization, were substantially the same as those of TgTCEB1 and TgTCEB-R. However, TgTCEB-L did not exhibit tissue-specific expression. Identification of TgTCEB-L explains the PosB-converting activity detected in tissues where TgTCEB1 and TgTCEB-R transcripts could not be detected, indicating that tulip subtilizes the three TgTCEB isozymes depending on the tissue.

Degradation of neurotensin by rat brain synaptic membranes: involvement of a thermolysin-like metalloendopeptidase (enkephalinase), angiotensin-converting enzyme, and other unidentified peptidases.

PubMed

Checler, F; Vincent, J P; Kitabgi, P

1983-08-01

Neurotensin was inactivated by membrane-bound and soluble degrading activities present in purified preparations of rat brain synaptic membranes. Degradation products were identified by HPLC and amino acid analysis. The major points of cleavage of neurotensin were the Arg8-Arg9, Pro10-Tyr11, and Tyr11-Ile12 peptide bonds with the membrane-bound activity and the Arg8-Arg9 and Pro10-Tyr11 bonds with the soluble activity. Several lines of evidence indicated that the cleavage of the Arg8-Arg9 bond by the membrane-bound activity resulted mainly from the conversion of neurotensin1-10 to neurotensin1-8 by a dipeptidyl carboxypeptidase. In particular, captopril inhibited this cleavage with an IC50 (5.7 nM) close to its K1 (7 nM) for angiotensin-converting enzyme. Thiorphan inhibited the cleavage at the Tyr11-Ile12 bond by the membrane-bound activity with an IC50 (17 nM) similar to its K1 (4.7 nM) for enkephalinase. Both cleavages were inhibited by 1,10-phenanthroline. These and other data suggested that angiotensin-converting enzyme and a thermolysin-like metalloendopeptidase (enkephalinase) were the membrane-bound peptidases responsible for cleavages at the Arg8-Arg9 and Tyr11-Ile12 bonds, respectively. In contrast, captopril had no effect on the cleavage at the Arg8-Arg9 bond by the soluble activity, indicating that the enzyme responsible for this cleavage was different from angiotensin-converting enzyme. The cleavage at the Pro10-Tyr11 bond by both the membrane-bound and the soluble activities appeared to be catalyzed by an endopeptidase different from known brain proline endopeptidases. The possibility is discussed that the enzymes described here participate in physiological mechanisms of neurotensin inactivation at the synaptic level.

Glutamic acid is an active site residue of angiotensin I-converting enzyme. Use of the Lossen rearrangement for identification of dicarboxylic acid residues.

PubMed

Harris, R B; Wilson, I B

1983-01-25

A set of chemical reactions was used to show that one glutamic acid residue at the active site of bovine lung angiotensin I-converting enzyme is esterified with the alkylating agent p-[N,N-bis(chloroethyl)amino] phenylbutyryl-L-Pro (chlorambucyl-L-Pro), an affinity label for this enzyme (Harris, R. B., and Wilson, I. B. (1982) J. Biol. Chem. 257, 811-815). The same procedure was used to confirm that a glutamic acid residue of carboxypeptidase A alpha is esterified by reaction with bromoacetyl-N-methyl-L-phenylalanine (Haas, G. M., and Neurath, H. (1971) Biochemistry 10, 3535-3546). In the procedure described in this paper, the esterified residue at the active site is converted to the hydroxamic acid by reaction with hydroxylamine and the hydroxamic acid is subject to the Lossen rearrangement. If a glutamic acid residue was esterified, 1 eq of 2,4-diaminobutyric acid will be formed. Aspartyl esters will give 2,3-diaminopropionic acid. The diamino acids can be quantitatively measured using the short column of an amino acid analyzer if the amount of lysine and histidine is largely decreased by modification with suitable side chain protecting groups. With carboxypeptidase A, the reactions were done on the whole undigested enzyme. With the converting enzyme, we first cleaved the esterified enzyme with cyanogen bromide. Twenty-nine cleavage peptides were separated on high performance liquid chromatography and one of these contained all of the bound radioactive inhibitor. This active site peptide was then subjected to the derivatization and Lossen procedures, and 1 eq of 2,4-diaminobutyric acid was obtained.

Angiotensin-converting enzyme: I. New strategies for assay

PubMed Central

Ryan, James W.; Chung, Alfred; Ryan, Una S.

1980-01-01

The disposition of converting enzyme (kininase II) on the luminal surface of pulmonary endothelial cells is well established. Further, it is known that there is a net conversion of angiotensin I into angiotensin II as blood passes through the lungs. However, little is known about modulations of converting enzyme activity that may arise through, e.g., changes in the quality of inhalants, blood flow, or blood oxygenation. There are few data on the effects of lung disease. A major barrier to studies to examine for pathophysiologic modulations of converting enzyme is that of assay. The enzyme can be measured in terms of the rate of formation of angiotensin II from a known quantity of angiotensin I. However, both peptides are biologically active, and lungs contain other enzymes capable of degrading them. We have developed a series of radiolabeled, acylated tripeptides to improve our ability to examine for changes in the net converting enzyme of intact lungs. The enzyme, a dipeptidyl carboxypeptidase, is capable of removing C-terminal dipeptides from a variety of oligopeptides. We have prepared benzoyl-Gly-Gly-Gly (I), benzoyl-Pro-Phe-Arg (II), benzoyl-Gly-His-Leu (III), benzoyl-Phe-Ala-Pro (IV), and benzoyl-Phe-His-Leu (V), each containing a 3H-atom in the para position of the benzoyl moiety. Substrates I and III have been used previously in photometric assays of low sensitivity. II is the acylated C-terminal tripeptide of bradykinin, IV is an acylated tripeptide analog of BPP5a (

A human lung mast cell chymotrypsin-like enzyme. Identification and partial characterization.

PubMed

Wintroub, B U; Kaempfer, C E; Schechter, N M; Proud, D

1986-01-01

We have used a high performance liquid chromatography assay, which detects chymotryptic cleavage of the phe8-his9 bond of angiotensin I to yield angiotensin II, in order to examine human lung mast cells for the presence of chymotryptic activity. Mast cells, purified from human lung by enzymatic dispersion, countercurrent elutriation, and Percoll gradient centrifugation, were lysed or challenged with goat anti-human IgE. In multiple experiments angiotensin II-converting activity was detected in lysates of 10-99% pure mast cell preparations. Regression analysis of net percent release values of histamine and the angiotensin I-converting activity from dose-response experiments demonstrated a correlation between the two parameters, indicating that the chymotrypsin-like enzyme is a constituent of the mast cell secretory granule. The chymotryptic activity was completely inhibited by 10(-3) M phenylmethylsulfonylfluoride but not by 10(-3) M Captopril, and the pH optimum of activity was 7.5-9.5. Gel filtration of released material separated the activity from tryptase and demonstrated an approximate molecular weight of 30-35,000. The mast cell enzyme, like a human skin chymotrypsin-like proteinase, can be distinguished from leukocyte cathepsin G by lack of susceptibility to inhibition by bovine pancreatic trypsin inhibitor. Thus, an enzyme with limited chymotryptic specificity is present in human lung mast cells. The Michaelis constant of the enzyme for angiotensin I of 6.0 X 10(-5) M is similar to that of endothelial cell angiotensin-converting enzyme and is consistent with a reaction of physiologic importance.

In Vitro Studies on the Antioxidant Property and Inhibition of α-Amylase, α-Glucosidase, and Angiotensin I-Converting Enzyme by Polyphenol-Rich Extracts from Cocoa (Theobroma cacao) Bean

PubMed Central

Ademosun, Ayokunle O.; Ademiluyi, Adedayo O.; Omojokun, Olasunkanmi S.; Nwanna, Esther E.; Longe, Kuburat O.

2014-01-01

Background. This study sought to investigate the antidiabetic and antihypertensive mechanisms of cocoa (Theobroma cacao) bean through inhibition of α-amylase, α-glucosidase, angiotensin-1 converting enzyme, and oxidative stress. Methodology. The total phenol and flavonoid contents of the water extractable phytochemicals from the powdered cocoa bean were determined and the effects of the extract on α-amylase, α-glucosidase, and angiotensin-1 converting enzyme activities were investigated in vitro. Furthermore, the radicals [1,1-diphenyl-2 picrylhydrazyl (DPPH), 2,2..-azino-bis(3-ethylbenzthiazoline-6-sulphonic acid) (ABTS), hydroxyl (OH), and nitric oxide (NO)] scavenging ability and ferric reducing antioxidant property of the extract were assessed. Results. The results revealed that the extract inhibited α-amylase (1.81 ± 0.22 mg/mL), α-glucosidase (1.84 ± 0.17 mg/mL), and angiotensin-1 converting enzyme (0.674 ± 0.06 mg/mL [lungs], 1.006 ± 0.08 mg/mL [heart]) activities in a dose-dependent manner and also showed dose-dependent radicals [DPPH (16.94 ± 1.34 mg/mL), NO (6.98 ± 0.886 mg/mL), OH (3.72 ± 0.26 mg/mL), and ABTS (15.7 ± 1.06 mmol/TEAC·g] scavenging ability. Conclusion. The inhibition of α-amylase, α-glucosidase, and angiotensin-1 converting enzyme activities by the cocoa bean extract could be part of the possible mechanism by which the extract could manage and/or prevent type-2 diabetes and hypertension. PMID:25295218

In Vitro Studies on the Antioxidant Property and Inhibition of α-Amylase, α-Glucosidase, and Angiotensin I-Converting Enzyme by Polyphenol-Rich Extracts from Cocoa (Theobroma cacao) Bean.

PubMed

Oboh, Ganiyu; Ademosun, Ayokunle O; Ademiluyi, Adedayo O; Omojokun, Olasunkanmi S; Nwanna, Esther E; Longe, Kuburat O

2014-01-01

Background. This study sought to investigate the antidiabetic and antihypertensive mechanisms of cocoa (Theobroma cacao) bean through inhibition of α-amylase, α-glucosidase, angiotensin-1 converting enzyme, and oxidative stress. Methodology. The total phenol and flavonoid contents of the water extractable phytochemicals from the powdered cocoa bean were determined and the effects of the extract on α-amylase, α-glucosidase, and angiotensin-1 converting enzyme activities were investigated in vitro. Furthermore, the radicals [1,1-diphenyl-2 picrylhydrazyl (DPPH), 2,2..-azino-bis(3-ethylbenzthiazoline-6-sulphonic acid) (ABTS), hydroxyl (OH), and nitric oxide (NO)] scavenging ability and ferric reducing antioxidant property of the extract were assessed. Results. The results revealed that the extract inhibited α-amylase (1.81 ± 0.22 mg/mL), α-glucosidase (1.84 ± 0.17 mg/mL), and angiotensin-1 converting enzyme (0.674 ± 0.06 mg/mL [lungs], 1.006 ± 0.08 mg/mL [heart]) activities in a dose-dependent manner and also showed dose-dependent radicals [DPPH (16.94 ± 1.34 mg/mL), NO (6.98 ± 0.886 mg/mL), OH (3.72 ± 0.26 mg/mL), and ABTS (15.7 ± 1.06 mmol/TEAC·g] scavenging ability. Conclusion. The inhibition of α-amylase, α-glucosidase, and angiotensin-1 converting enzyme activities by the cocoa bean extract could be part of the possible mechanism by which the extract could manage and/or prevent type-2 diabetes and hypertension.

Angiotensin-I Converting Enzyme (ACE) Inhibitory and Anti-Oxidant Activities of Sea Cucumber (Actinopyga lecanora) Hydrolysates

PubMed Central

Ghanbari, Raheleh; Zarei, Mohammad; Ebrahimpour, Afshin; Abdul-Hamid, Azizah; Ismail, Amin; Saari, Nazamid

2015-01-01

In recent years, food protein-derived hydrolysates have received considerable attention because of their numerous health benefits. Amongst the hydrolysates, those with anti-hypertensive and anti-oxidative activities are receiving special attention as both activities can play significant roles in preventing cardiovascular diseases. The present study investigated the angiotensin-I converting enzyme (ACE) inhibitory and anti-oxidative activities of Actinopyga lecanora (A. lecanora) hydrolysates, which had been prepared by alcalase, papain, bromelain, flavourzyme, pepsin, and trypsin under their optimum conditions. The alcalase hydrolysate showed the highest ACE inhibitory activity (69.8%) after 8 h of hydrolysis while the highest anti-oxidative activities measured by 2,2-diphenyl 1-1-picrylhydrazyl radical scavenging (DPPH) (56.00%) and ferrous ion-chelating (FIC) (59.00%) methods were exhibited after 24 h and 8 h of hydrolysis, respectively. The ACE-inhibitory and anti-oxidative activities displayed dose-dependent trends, and increased with increasing protein hydrolysate concentrations. Moreover, strong positive correlations between angiotensin-I converting enzyme (ACE) inhibitory and anti-oxidative activities were also observed. This study indicates that A. lecanora hydrolysate can be exploited as a source of functional food owing to its anti-oxidant as well as anti-hypertension functions. PMID:26690117

A novel lactone-forming carboxylesterase: molecular identification of a tuliposide A-converting enzyme in tulip.

PubMed

Nomura, Taiji; Ogita, Shinjiro; Kato, Yasuo

2012-06-01

Tuliposides, the glucose esters of 4-hydroxy-2-methylenebutanoate and 3,4-dihydroxy-2-methylenebutanoate, are major secondary metabolites in tulip (Tulipa gesneriana). Their lactonized aglycons, tulipalins, function as defensive chemicals due to their biological activities. We recently found that tuliposide-converting enzyme (TCE) purified from tulip bulbs catalyzed the conversion of tuliposides to tulipalins, but the possibility of the presence of several TCE isozymes was raised: TCE in tissues other than bulbs is different from bulb TCE. Here, to prove this hypothesis, TCE was purified from petals, which have the second highest TCE activity after bulbs. The purified enzyme, like the bulb enzyme, preferentially accepted tuliposides as substrates, with 6-tuliposide A the best substrate, which allowed naming the enzyme tuliposide A-converting enzyme (TCEA), but specific activity and molecular mass differed between the petal and bulb enzymes. After peptide sequencing, a novel cDNA (TgTCEA) encoding petal TCEA was isolated, and the functional characterization of the recombinant enzyme verified that TgTCEA catalyzes the conversion of 6-tuliposide A to tulipalin A. TgTCEA was transcribed in all tulip tissues but not in bulbs, indicating the presence of a bulb-specific TgTCEA, as suggested by the distinct enzymatic characters between the petal and bulb enzymes. Plastidial localization of TgTCEA enzyme was revealed, which allowed proposing a cytological mechanism of TgTCE-mediated tulipalin formation in the tulip defensive strategy. Site-directed mutagenesis of TgTCEA suggested that the oxyanion hole and catalytic triad characteristic of typical carboxylesterases are essential for the catalytic process of TgTCEA enzyme. To our knowledge, TgTCEA is the first identified member of the lactone-forming carboxylesterases, specifically catalyzing intramolecular transesterification.

A Novel Lactone-Forming Carboxylesterase: Molecular Identification of a Tuliposide A-Converting Enzyme in Tulip1[W

PubMed Central

Nomura, Taiji; Ogita, Shinjiro; Kato, Yasuo

2012-01-01

Tuliposides, the glucose esters of 4-hydroxy-2-methylenebutanoate and 3,4-dihydroxy-2-methylenebutanoate, are major secondary metabolites in tulip (Tulipa gesneriana). Their lactonized aglycons, tulipalins, function as defensive chemicals due to their biological activities. We recently found that tuliposide-converting enzyme (TCE) purified from tulip bulbs catalyzed the conversion of tuliposides to tulipalins, but the possibility of the presence of several TCE isozymes was raised: TCE in tissues other than bulbs is different from bulb TCE. Here, to prove this hypothesis, TCE was purified from petals, which have the second highest TCE activity after bulbs. The purified enzyme, like the bulb enzyme, preferentially accepted tuliposides as substrates, with 6-tuliposide A the best substrate, which allowed naming the enzyme tuliposide A-converting enzyme (TCEA), but specific activity and molecular mass differed between the petal and bulb enzymes. After peptide sequencing, a novel cDNA (TgTCEA) encoding petal TCEA was isolated, and the functional characterization of the recombinant enzyme verified that TgTCEA catalyzes the conversion of 6-tuliposide A to tulipalin A. TgTCEA was transcribed in all tulip tissues but not in bulbs, indicating the presence of a bulb-specific TgTCEA, as suggested by the distinct enzymatic characters between the petal and bulb enzymes. Plastidial localization of TgTCEA enzyme was revealed, which allowed proposing a cytological mechanism of TgTCE-mediated tulipalin formation in the tulip defensive strategy. Site-directed mutagenesis of TgTCEA suggested that the oxyanion hole and catalytic triad characteristic of typical carboxylesterases are essential for the catalytic process of TgTCEA enzyme. To our knowledge, TgTCEA is the first identified member of the lactone-forming carboxylesterases, specifically catalyzing intramolecular transesterification. PMID:22474185

Discovery of piperonal-converting oxidase involved in the metabolism of a botanical aromatic aldehyde

PubMed Central

Doi, Shiori; Hashimoto, Yoshiteru; Tomita, Chiaki; Kumano, Takuto; Kobayashi, Michihiko

2016-01-01

Piperonal-catabolizing microorganisms were isolated from soil, the one (strain CT39-3) exhibiting the highest activity being identified as Burkholderia sp. The piperonal-converting enzyme involved in the initial step of piperonal metabolism was purified from strain CT39-3. Gene cloning of the enzyme and a homology search revealed that the enzyme belongs to the xanthine oxidase family, which comprises molybdoenzymes containing a molybdopterin cytosine dinucleotide cofactor. We found that the piperonal-converting enzyme acts on piperonal in the presence of O2, leading to formation of piperonylic acid and H2O2. The growth of strain CT39-3 was inhibited by higher concentrations of piperonal in the culture medium. Together with this finding, the broad substrate specificity of this enzyme for various aldehydes suggests that it would play an important role in the defense mechanism against antimicrobial compounds derived from plant species. PMID:27905507

Synthesis and evaluation of chalcone analogues based pyrimidines as angiotensin converting enzyme inhibitors.

PubMed

Bukhari, S N A; Butt, A M; Amjad, M W B; Ahmad, W; Shah, V H; Trivedi, A R

2013-11-01

Hypertension is a widespread and frequently progressive ailment that imparts a foremost threat for cardiovascular and renal disorders. Mammoth efforts are needed for the synthesis of innovative antihypertensive agents to combat this lethal disease. Chalcones have shown antihypertensive activity through inhibition of Angiotensin Converting Enzyme (ACE). Hence, a series of chalcone analogues is synthesized and used as precursor for the synthesis of novel series of pyrimidines. Precursor chalcones were prepared by reacting aldehydes and ketones in presence of sodium hydroxide followed by synthesis of corresponding pyrimidines by reaction with urea in presence of potassium hydroxide. Both groups were then evaluated for their effects on ACE. The results depicted that pyrimidines were more active than chalcones with methoxy (C5 and P5) substitution showing best results to inhibit ACE. Given that chalcone analogues and pyrimidines show a potential as the angiotensin converting enzyme inhibitors.

LECITHINASE AND LYSOLECITHINASE ACTIVITY OF RAT INTESTINAL MUCOSA AFTER WHOLE-BODY X-IRRADIATION

DOE Office of Scientific and Technical Information (OSTI.GOV)

Ottolenghi, A.; Bernheim, F.

1961-11-01

Twenty-four hours after whole-body x irradiation the lecithinase activity of rat intestinal mucosa has markedly decreased and the lysolecithinase activity has decreasecp to a lesser extent. Addition of normal mucosa or chyi'otrypsin to the irradiated mucosa restores the activity of both enzymes. This indicates that irradiation eithei produces an inhibitor or inactivates a mechanism necessarly to convert pro-enzymes into active enzymes. Since chymo trypsin can increase to some extent the activity of the enzymes in normal mucosa, the second possibility seems more probable. (auth)

Angiotensin-I converting enzyme (ACE): structure, biological roles, and molecular basis for chloride ion dependence.

PubMed

Masuyer, Geoffrey; Yates, Christopher J; Sturrock, Edward D; Acharya, K Ravi

2014-10-01

Somatic angiotensin-I converting enzyme (sACE) has an essential role in the regulation of blood pressure and electrolyte fluid homeostasis. It is a zinc protease that cleaves angiotensin-I (AngI), bradykinin, and a broad range of other signalling peptides. The enzyme activity is provided by two homologous domains (N- and C-), which display clear differences in substrate specificities and chloride activation. The presence of chloride ions in sACE and its unusual role in activity was identified early on in the characterisation of the enzyme. The molecular mechanisms of chloride activation have been investigated thoroughly through mutagenesis studies and shown to be substrate-dependent. Recent results from X-ray crystallography structural analysis have provided the basis for the intricate interactions between ACE, its substrate and chloride ions. Here we describe the role of chloride ions in human ACE and its physiological consequences. Insights into the chloride activation of the N- and C-domains could impact the design of improved domain-specific ACE inhibitors.

Functional Activities and Immunohistochemical Distribution of Superoxide Dismutase in Normal, Dysplastic and Squamous Cell Carcinoma Oral Tissues

DTIC Science & Technology

2001-07-26

antioxidant enzymes: SOD, catalase ( CAT ), and glutathione peroxidase (GPO).2 ° SOD converts superoxide radical into hydrogen peroxide, while CAT and GPO convert...in endometriosis and adenomyosis. Fertility and Sterility 1999; 1:129-134. 45. Muse KE, Oberley TD, Sempf JM, Oberley LW. Immunolocalization of antioxidant enzymes in adult hamster kidney. Histochem J 1994;9:734-753. 43

Measurement of crude-cell-extract glycerol dehydratase activity in recombinant Escherichia coli using coupled-enzyme reactions.

PubMed

Sankaranarayanan, Mugesh; Seol, Eunhee; Kim, Yeonhee; Chauhan, Ashish Singh; Park, Sunghoon

2017-03-01

Glycerol dehydratase (GDHt), which converts glycerol to 3-hydroxypropionaldehyde, is essential to the production of 1,3-propanediol (1,3-PDO) or 3-hydroxypropionic acid (3-HP). A reliable GDHt activity assay in crude-cell extract was developed. In the assay, GDHt converted 1,2-propanediol (1,2-PDO) to propionaldehyde, which was further converted to 1-propionic acid by aldehyde dehydrogenase (KGSADH) or to 1-propanol by yeast-alcohol dehydrogenase (yADH), while the NADH concentration change was monitored spectrophotometrically. Cells should be disintegrated by Bead Beater/French Press, not by chemical methods (BugBuster ® /B-PER™), because the reagents significantly inactivated GDHt and coupling enzymes. Furthermore, in the assay mixture, a much higher activity of KGSADH (>200-fold) or yADH (>400-fold) than that of GDHt should have been maintained. Under optimal conditions, both KGSADH and yADH showed practically the same activity. The coupled-enzyme assay method established here should prove to be applicable to recombinant strains developed for the production of 3-HP and/or 1,3-PDO from glycerol.

Enzymatic activity and partial purification of solanapyrone synthase: first enzyme catalyzing Diels-Alder reaction.

PubMed

Katayama, K; Kobayashi, T; Oikawa, H; Honma, M; Ichihara, A

1998-05-19

In cell-free extracts of Alternaria solani, an enzymatic activity converting prosolanapyrone II to solanapyrones A and D via oxidation and subsequent Diels-Alder reaction has been found. Chromatography with DEAE-Sepharose provided two active fractions, pools 1 and 2. The former fraction converted prosolanapyrone II to solanapyrones A and D in a ratio of 2.2:1 with optical purities of 99% and 45% ee, respectively. The latter fraction did so in a ratio of 7.6:1 with 99% and nearly 0% ee, respectively. The enzyme partially purified from pool 2 native molecular weight of 40-62 kD and a pl of 4.25. The high reactivity of prosolanapyrone III in aqueous solution and the chromatographic behavior of the enzyme in pool 2 suggest that a single enzyme catalyzes both the oxidation and Diels-Alder reaction.

High endothelin-converting enzyme-1 expression independently predicts poor survival of patients with esophageal squamous cell carcinoma.

PubMed

Wu, Ching-Fang; Lee, Ching-Tai; Kuo, Yao-Hung; Chen, Tzu-Haw; Chang, Chi-Yang; Chang, I-Wei; Wang, Wen-Lun

2017-09-01

Patients with esophageal squamous cell carcinoma have poor survival and high recurrence rate, thus an effective prognostic biomarker is needed. Endothelin-converting enzyme-1 is responsible for biosynthesis of endothelin-1, which promotes growth and invasion of human cancers. The role of endothelin-converting enzyme-1 in esophageal squamous cell carcinoma is still unknown. Therefore, this study investigated the significance of endothelin-converting enzyme-1 expression in esophageal squamous cell carcinoma clinically. We enrolled patients with esophageal squamous cell carcinoma who provided pretreated tumor tissues. Tumor endothelin-converting enzyme-1 expression was evaluated by immunohistochemistry and was defined as either low or high expression. Then we evaluated whether tumor endothelin-converting enzyme-1 expression had any association with clinicopathological findings or predicted survival of patients with esophageal squamous cell carcinoma. Overall, 54 of 99 patients with esophageal squamous cell carcinoma had high tumor endothelin-converting enzyme-1 expression, which was significantly associated with lymph node metastasis ( p = 0.04). In addition, tumor endothelin-converting enzyme-1 expression independently predicted survival of patients with esophageal squamous cell carcinoma, and the 5-year survival was poorer in patients with high tumor endothelin-converting enzyme-1 expression ( p = 0.016). Among patients with locally advanced and potentially resectable esophageal squamous cell carcinoma (stage II and III), 5-year survival was poorer with high tumor endothelin-converting enzyme-1 expression ( p = 0.003). High tumor endothelin-converting enzyme-1 expression also significantly predicted poorer survival of patients in this population. In patients with esophageal squamous cell carcinoma, high tumor endothelin-converting enzyme-1 expression might indicate high tumor invasive property. Therefore, tumor endothelin-converting enzyme-1 expression could be a good biomarker to identify patients with worse survival and higher risks of recurrence, who might benefit from the treatment by endothelin-converting enzyme-1 inhibitor.

Pharmacologic modulation of ACE2 expression.

PubMed

Soler, María José; Barrios, Clara; Oliva, Raymond; Batlle, Daniel

2008-10-01

Angiotensin-converting enzyme 2 (ACE2) is an enzymatically active homologue of angiotensin-converting enzyme that degrades angiotensin I, angiotensin II, and other peptides. Recent studies have shown that under pathologic conditions, ACE2 expression in the kidney is altered. In this review, we briefly summarize recent studies dealing with pharmacologic interventions that modulate ACE2 expression. ACE2 amplification may have a potential therapeutic role for kidney disease and hypertension.

Quantitative Structure-Activity Relationship Modeling Coupled with Molecular Docking Analysis in Screening of Angiotensin I-Converting Enzyme Inhibitory Peptides from Qula Casein Hydrolysates Obtained by Two-Enzyme Combination Hydrolysis.

PubMed

Lin, Kai; Zhang, Lanwei; Han, Xue; Meng, Zhaoxu; Zhang, Jianming; Wu, Yifan; Cheng, Dayou

2018-03-28

In this study, Qula casein derived from yak milk casein was hydrolyzed using a two-enzyme combination approach, and high angiotensin I-converting enzyme (ACE) inhibitory activity peptides were screened by quantitative structure-activity relationship (QSAR) modeling integrated with molecular docking analysis. Hydrolysates (<3 kDa) derived from combinations of thermolysin + alcalase and thermolysin + proteinase K demonstrated high ACE inhibitory activities. Peptide sequences in hydrolysates derived from these two combinations were identified by liquid chromatography-tandem mass spectrometry (LC-MS/MS). On the basis of the QSAR modeling prediction, a total of 16 peptides were selected for molecular docking analysis. The docking study revealed that four of the peptides (KFPQY, MPFPKYP, MFPPQ, and QWQVL) bound the active site of ACE. These four novel peptides were chemically synthesized, and their IC 50 was determined. Among these peptides, KFPQY showed the highest ACE inhibitory activity (IC 50 = 12.37 ± 0.43 μM). Our study indicated that Qula casein presents an excellent source to produce ACE inhibitory peptides.

More Nuts and Bolts of Michaelis-Menten Enzyme Kinetics

ERIC Educational Resources Information Center

Lechner, Joseph H.

2011-01-01

Several additions to a classroom activity are proposed in which an "enzyme" (the student) converts "substrates" (nut-bolt assemblies) into "products" (separated nuts and bolts) by unscrewing them. (Contains 1 table.)

Molecular identification of tuliposide B-converting enzyme: a lactone-forming carboxylesterase from the pollen of tulip.

PubMed

Nomura, Taiji; Murase, Tatsunori; Ogita, Shinjiro; Kato, Yasuo

2015-07-01

6-Tuliposides A (PosA) and B (PosB), which are the major secondary metabolites in tulip (Tulipa gesneriana), are enzymatically converted to the antimicrobial lactonized aglycons, tulipalins A (PaA) and B (PaB), respectively. We recently identified a PosA-converting enzyme (TCEA) as the first reported member of the lactone-forming carboxylesterases. Herein, we describe the identification of another lactone-forming carboxylesterase, PosB-converting enzyme (TCEB), which preferentially reacts with PosB to give PaB. This enzyme was isolated from tulip pollen, which showed high PosB-converting activity. Purified TCEB exhibited greater activity towards PosB than PosA, which was contrary to that of the TCEA. Novel cDNA (TgTCEB1) encoding the TCEB was isolated from tulip pollen. TgTCEB1 belonged to the carboxylesterase family and was approximately 50% identical to the TgTCEA polypeptides. Functional characterization of the recombinant enzyme verified that TgTCEB1 catalyzed the conversion of PosB to PaB with an activity comparable with the native TCEB. RT-qPCR analysis of each part of plant revealed that TgTCEB1 transcripts were limited almost exclusively to the pollen. Furthermore, the immunostaining of the anther cross-section using anti-TgTCEB1 polyclonal antibody verified that TgTCEB1 was specifically expressed in the pollen grains, but not in the anther cells. N-terminal transit peptide of TgTCEB1 was shown to function as plastid-targeted signal. Taken together, these results indicate that mature TgTCEB1 is specifically localized in plastids of pollen grains. Interestingly, PosB, the substrate of TgTCEB1, accumulated on the pollen surface, but not in the intracellular spaces of pollen grains. © 2015 The Authors The Plant Journal © 2015 John Wiley & Sons Ltd.

Angiotensin-converting enzyme inhibition and novel cardiovascular risk biomarkers: results from the Trial of Angiotensin Converting Enzyme Inhibition and Novel Cardiovascular Risk Factors (TRAIN) study.

PubMed

Cesari, Matteo; Kritchevsky, Stephen B; Atkinson, Hal H; Penninx, Brenda W; Di Bari, Mauro; Tracy, Russell P; Pahor, Marco

2009-02-01

Beneficial effects of angiotensin-converting enzyme (ACE) inhibitors seem to be mediated by mechanisms that are partly independent of blood pressure lowering. The present study evaluates effects of an ACE inhibitor (ie, fosinopril) intervention on novel cardiovascular risk factors. Data are from the Trial of Angiotensin Converting Enzyme Inhibition and Novel Cardiovascular Risk Factors (TRAIN) study, a double-blind, crossover, randomized, placebo-controlled trial enrolling subjects > or =55 years old with high cardiovascular disease risk profile. Biomarkers of hemostasis (ie, plasminogen activator inhibitor 1, D-dimer), inflammation (ie, C-reactive protein, interleukin-6), and endothelial function (ie, endothelin 1, vascular cell adhesion molecule 1) were measured at the baseline, at the midterm, and at end of follow-up (after 1 year) clinic visits. Paired t test analyses (after Sidak's adjustment, P < .009) were performed to compare biomarkers modifications after fosinopril/placebo interventions. Mean age of the sample (n = 290, women 43.4%) was 66.0 years old. No significant differences were reported for C-reactive protein, interleukin 6, plasminogen activator inhibitor 1, vascular cell adhesion molecule 1, and endothelin 1 levels in the comparisons between fosinopril and placebo interventions. D-dimer was the only biomarker showing a significant difference between fosinopril intervention (median 0.32 microg/mL, interquartile range 0.22-0.52 microg/mL) and placebo (median 0.29 microg/mL, interquartile range 0.20-0.47 microg/mL, P = .007) when analyses were restricted to participants with higher compliance to treatment and receiving the maximum ACE inhibitor dosage. Angiotensin-converting enzyme inhibition does not significantly modify major biomarkers of inflammation, hemostasis, and endothelial function. Further studies should confirm the possible effect of ACE inhibitors on the fibrinolysis pathway.

Effect of protease inhibitors on angiotensin-converting enzyme activity in human T-lymphocytes.

PubMed

Petrov, V; Fagard, R; Lijnen, P

2000-05-01

The purpose of these investigations was to determine whether the aminopeptidase B and leucine aminopeptidase inhibitor bestatin, the chymase inhibitor chymostatin, the calpain inhibitor E-64, and the neutral serine protease inhibitor leupeptin affect the angiotensin converting enzyme (ACE) activity in T-lymphocytes. ACE activity in homogenates of T-lymphocytes or in intact T-lymphocytes in suspension was measured by determining fluorimetrically histidyl-leucine, formed from the conversion of hippuryl-histidyl-leucine, coupled with ophtaldialdehyde. The effect of various concentrations (10(-9) to 10(-3) mol/L) of the angiotensin-converting enzyme inhibitors lisinopril and captopril and of the various protease inhibitors on ACE activity was studied. Lisinopril and captopril reduced the ACE activity in homogenates of T-lymphocytes in a concentration-dependent manner. Lisinopril exhibited a more pronounced inhibition of ACE in T-lymphocytes than did captopril. Chymostatin and E-64 had no effect on the ACE activity in T-lymphocytes, whereas leupeptin inhibited its activity in a dose-dependent fashion. Bestatin, on the contrary, increased the ACE activity in homogenates of T-lymphocytes as well as in intact T-lymphocytes in proportion to the concentration. Our data showed that the ACE activity in T-lymphocytes was stimulated by bestatin and inhibited by leupeptin, whereas chymostatin and E-64 did not affect the ACE activity in T-lymphocytes.

Screening the Brazilian flora for antihypertensive plant species for in vitro angiotensin-I-converting enzyme inhibiting activity.

PubMed

Castro Braga, F; Wagner, H; Lombardi, J A; de Oliveira, A B

2000-06-01

The evaluation of several antihypertensive activity of Brazilian plant species was performed using in vitro inhibition of the angiotensin I-converting enzyme (ACE). Nineteen species belonging to 13 families were investigated. Plants were selected based on their use as diuretics and on a chemosystematic consideration. Extracts of the following species presented the highest ACE inhibition rate, at concentrations of 0.33 mg/ml: Ouratea semiserrata (Mart. & Nees) Engl. stems (68%), Cuphea cartagenesis (Jacq.) Macbride leaves (50%) and Mansoa hirsuta DC. leaves (54%). Some hypotheses about the nature of the compounds that may be responsible for the activity of these species are discussed in the paper.

Active peptides from skate (Okamejei kenojei) skin gelatin diminish angiotensin-I converting enzyme activity and intracellular free radical-mediated oxidation.

PubMed

Ngo, Dai-Hung; Ryu, BoMi; Kim, Se-Kwon

2014-01-15

Skin gelatin of skate (Okamejei kenojei) was hydrolyzed using Alcalase, flavourzyme, Neutrase and protamex. It was found that the Alcalase hydrolysate exhibited the highest angiotensin-I converting enzyme (ACE) inhibitory activity. Then, Alcalase hydrolysate was further hydrolyzed with protease and separated by an ultrafiltration membrane system. Finally, two peptides responsible for ACE inhibitory activity were identified to be MVGSAPGVL (829Da) and LGPLGHQ (720Da), with IC50 values of 3.09 and 4.22μM, respectively. Moreover, the free radical-scavenging activity of the purified peptides was determined in human endothelial cells. In addition, the antioxidative mechanism of the purified peptides was evaluated by protein and gene expression levels of antioxidant enzymes. The current study demonstrated that the peptides derived from skate skin gelatin could be used in the food industry as functional ingredients with potent antihypertensive and antioxidant benefits. Copyright © 2013 Elsevier Ltd. All rights reserved.

Evaluation of subacute change in RAAS activity (as indicated by urinary aldosterone:creatinine, after pharmacologic provocation) and the response to ACE inhibition.

PubMed

Ames, Marisa K; Atkins, Clarke E; Lantis, Andrea C; zum Brunnen, James

2016-01-01

The objective of this study was to evaluate subacute changes in renin-angiotensin-aldosterone system (RAAS) activity during angiotensin-converting enzyme inhibitor (ACEI) therapy in dogs with experimental RAAS activation. Analysis of data (urine aldosterone:creatinine ratio (UAldo:C) and serum angiotensin-converting enzyme activity), in 31 healthy dogs with furosemide or amlodipine-activated RAAS that received an ACEI. When furosemide or amlodipine activation of RAAS preceded ACEI administration, incomplete RAAS blockade (IRB) was defined as a UAldo:C greater than (a) the dog's 'activated' baseline value or (b) a population-derived cut-off value (mean + 2 SD (>1.0 μg/g) of pretreatment UAldo:C from our population of research dogs). In studies where RAAS activation occurred concurrently with ACEIs, IRB was defined as (a) a UAldo:C greater than either twofold the dog's prestimulation baseline value or (b) 1.0 µg/g. Dogs were followed for 7-17 days. Serum angiotensin-converting enzyme activity was measured in 19 dogs and was significantly reduced (P<0.0001) after ACEI administration. The overall incidence of IRB, when RAAS activation preceded ACEI administration, was 33% and 8% for definitions (a) and (b), respectively. The overall incidence of IRB, when ACEIs were concurrent with RAAS activation, was 65% and 61% for definitions (a) and (b), respectively. Increases in UAldo:C, despite ACEI administration, is evidence of IRB in this subacute model of experimental RAAS activation and suppression. © The Author(s) 2016.

The Nuts and Bolts of Michaelis-Menten Enzyme Kinetics: Suggestions and Clarifications

ERIC Educational Resources Information Center

Silverstein, Todd

2011-01-01

Matthew Junker's recent article describes a useful and effective enzyme kinetics application and analogy in which students simulate enzyme activity by unscrewing nut-bolt "substrate molecules", thus, converting them into separate nuts and bolts "products". A number of suggestions and corrections are presented that improve the clarity and accuracy…

EI-2128-1, a novel interleukin-1beta converting enzyme inhibitor produced by Penicillium sp. E-2128.

PubMed

Koizumi, Fumito; Agatsuma, Tsutomu; Ando, Katsuhiko; Kondo, Hidemasa; Saitoh, Yutaka; Matsuda, Yuzuru; Nakanishi, Satoshi

2003-11-01

EI-2128-1, a novel interleukin-1beta converting enzyme (ICE) inhibitor, was isolated from the culture broths of Penicillium sp. E-2128. EI-2128-1 selectively inhibited human recombinant ICE activity with IC50 value of 0.59 microM, without inhibiting elastase and cathepsin B. EI-2128-1 also inhibited mature interleukin-1beta secretion from THP-1 cells induced by LPS with IC50 value of 0.28 microM.

Soybean phenolic-rich extracts inhibit key-enzymes linked to type 2 diabetes (α-amylase and α-glucosidase) and hypertension (angiotensin I converting enzyme) in vitro.

PubMed

Ademiluyi, Adedayo O; Oboh, Ganiyu

2013-03-01

This study sought to assess the inhibitory activities of phenolic-rich extracts from soybean on α-amylase, α-glucosidase and angiotensin I converting enzyme (ACE) activities in vitro. The free phenolic extract of the soybean was obtained by extraction with 80% acetone, while that of the bound phenolic extract was done by extracting the alkaline and acid hydrolyzed residue with ethyl acetate. The inhibitory action of these extracts on the enzymes activity as well as their antioxidant properties was assessed. Both phenolic-rich extracts inhibited α-amylase, α-glucosidase and ACE enzyme activities in a dose dependent pattern. However, the bound phenolic extract exhibited significantly (P < 0.05) higher α-amylase and ACE inhibition while the free phenolic extract had significantly (P < 0.05) higher α-glucosidase inhibitory activity. Nevertheless, the free phenolic extract had higher α-glucosidase inhibitory activity when compared to that of α-amylase; this property confer an advantage on soybean phenolic-rich extracts over commercial antidiabetic drugs with little or no side effect. And inhibition of ACE suggests the antihypertension potential of soybean phenolic-rich extracts. Furthermore, the enzyme inhibitory activities of the phenolic-rich extracts were not associated with their phenolic content. Therefore, phenolic-rich extracts of soybean could inhibit key-enzyme linked to type 2 diabetes (α-amylase and α-glucosidase) and hypertension (ACE) and thus could explain in part the mechanism by which soybean renders these health promoting effect. Copyright © 2011 Elsevier GmbH. All rights reserved.

Reduction of dopaminergic degeneration and oxidative stress by inhibition of angiotensin converting enzyme in a MPTP model of parkinsonism.

PubMed

Muñoz, Ana; Rey, Pablo; Guerra, Maria J; Mendez-Alvarez, Estefania; Soto-Otero, Ramon; Labandeira-Garcia, Jose L

2006-07-01

There is growing evidence indicating that oxidative stress is a key contributor to the pathogenesis and progression of Parkinson's disease. The brain, and particularly the basal ganglia, possesses a local rennin-angiotensin system. Angiotensin activates NAD(P)H-dependent oxidases, which are a major intracellular source of superoxide, and angiotensin converting enzyme inhibitors (ACEIs) have shown antioxidant properties. We treated mice with MPTP and the ACEI captopril to study the possible neuroprotective and antioxidant effects of the latter on the dopaminergic system. Pre-treatment with captopril induced a significant reduction in the MPTP-induced loss of dopaminergic neurons in the substantia nigra and a significant reduction in the loss of dopaminergic terminals in the striatum. Furthermore, captopril reduced the MPTP-induced increase in the levels of major oxidative stress indicators (i.e. lipid peroxidation and protein oxidation) in the ventral midbrain and the striatum. Captopril did not reduce striatal MPP(+) levels, MAO-B activity or dopamine transporter activity, which may reduce MPTP neurotoxicity. Our results suggest that angiotensin-converting enzyme inhibitors may be useful for treatment of Parkinson's disease, and that further investigation should focus on the neuroprotective capacity of these compounds.

Production of angiotensin I converting enzyme inhibitory (ACE-I) peptides during milk fermentation and their role in reducing hypertension.

PubMed

Rai, Amit Kumar; Sanjukta, Samurailatpam; Jeyaram, Kumaraswamy

2017-09-02

Fermented milk is a potential source of various biologically active peptides with specific health benefits. Angiotensin converting enzyme inhibitory (ACE-I) peptides are one of the most studied bioactive peptides produced during milk fermentation. The presence of these peptides is reported in various fermented milk products such as, yoghurt, cheese, sour milk, etc., which are also available as commercial products. Many of the ACE-I peptides formed during milk fermentation are resistant to gastrointestinal digestion and inhibit angiotensin converting enzyme (ACE) in the rennin angiotension system (RAS). There are various factors, which affect the formation ACE-I peptides and their ability to reach the target tissue in active form, which includes type of starters (lactic acid bacteria (LAB), yeast, etc.), substrate composition (casein type, whey protein, etc.), composition of ACE-I peptide, pre and post-fermentation treatments, and its stability during gastrointestinal digestion. The antihypertensive effect of fermented milk products has also been proved by various in vitro and in vivo (animal and human trials) experiments. This paper reviews the literature on fermented milk products as a source of ACE-I peptides and various factors affecting the production and activity of ACE-I peptides.

Utilization of peptide carrier system to improve intestinal absorption: targeting prolidase as a prodrug-converting enzyme

NASA Technical Reports Server (NTRS)

Bai, J. P.; Hu, M.; Subramanian, P.; Mosberg, H. I.; Amidon, G. L.

1992-01-01

The feasibility of targeting prolidase as a peptide prodrug-converting enzyme has been examined. The enzymatic hydrolysis by prolidase of substrates for the peptide transporter L-alpha-methyldopa-pro and several dipeptide analogues without an N-terminal alpha-amino group (phenylpropionylproline, phenylacetylproline, N-benzoylproline, and N-acetylproline) was investigated. The Michaelis-Menten parameters Km and Vmax for L-alpha-methyldopa-pro are 0.09 +/- 0.02 mM and 3.98 +/- 0.25 mumol/min/mg protein, respectively. However, no hydrolysis of the dipeptide analogues without an N-terminal alpha-amino group is observed, suggesting that an N-terminal alpha-amino group is required for prolidase activity. These results demonstrate that prolidase may serve as a prodrug-converting enzyme for the dipeptide-type prodrugs, utilizing the peptide carrier for transport of prodrugs into the mucosal cells and prolidase, a cytosolic enzyme, to release the drug. However, a free alpha-amino group appears to be necessary for prolidase hydrolysis.

In silico prediction of potential chemical reactions mediated by human enzymes.

PubMed

Yu, Myeong-Sang; Lee, Hyang-Mi; Park, Aaron; Park, Chungoo; Ceong, Hyithaek; Rhee, Ki-Hyeong; Na, Dokyun

2018-06-13

Administered drugs are often converted into an ineffective or activated form by enzymes in our body. Conventional in silico prediction approaches focused on therapeutically important enzymes such as CYP450. However, there are more than thousands of different cellular enzymes that potentially convert administered drug into other forms. We developed an in silico model to predict which of human enzymes including metabolic enzymes as well as CYP450 family can catalyze a given chemical compound. The prediction is based on the chemical and physical similarity between known enzyme substrates and a query chemical compound. Our in silico model was developed using multiple linear regression and the model showed high performance (AUC = 0.896) despite of the large number of enzymes. When evaluated on a test dataset, it also showed significantly high performance (AUC = 0.746). Interestingly, evaluation with literature data showed that our model can be used to predict not only enzymatic reactions but also drug conversion and enzyme inhibition. Our model was able to predict enzymatic reactions of a query molecule with a high accuracy. This may foster to discover new metabolic routes and to accelerate the computational development of drug candidates by enabling the prediction of the potential conversion of administered drugs into active or inactive forms.

Screening the ToxCast Phase 1, 2, and e1k chemical libraries for inhibition of Deiodinase Types 1, 2 and 3 enzyme activity

EPA Science Inventory

Thyroid hormone (TH) homeostasis is dependent on multiple proteins for TH synthesis, transport, and peripheral metabolism and elimination. Deiodinase enzymes play an essential role in converting THs between active and inactive forms by deiodinating the pro-hormone thyroxine (T4) ...

Purification and characterization of a bacterial nitrophenol oxygenase which converts ortho-nitrophenol to catechol and nitrite.

PubMed Central

Zeyer, J; Kocher, H P

1988-01-01

A nitrophenol oxygenase which stoichiometrically converted ortho-nitrophenol (ONP) to catechol and nitrite was isolated from Pseudomonas putida B2 and purified. The substrate specificity of the enzyme was broad and included several halogen- and alkyl-substituted ONPs. The oxygenase consisted of a single polypeptide chain with a molecular weight of 58,000 (determined by gel filtration) or 65,000 (determined on a sodium dodecyl sulfate-polyacrylamide gel). The enzymatic reaction was NADPH dependent, and one molecule of oxygen was consumed per molecule of ONP converted. Enzymatic activity was stimulated by magnesium or manganese ions, whereas the addition of flavin adenine dinucleotide, flavin mononucleotide, or reducing agents had no effect. The apparent Kms for ONP and NADPH were 8 and 140 microM, respectively. 2,4-Dinitrophenol competitively (Ki = 0.5 microM) inhibited ONP turnover. The optimal pH for enzyme stability and activity was in the range of 7.5 to 8.0. At 40 degrees C, the enzyme was totally inactivated within 2 min; however, in the presence of 1 mM ONP, 40% of the activity was recovered, even after 10 min. Enzymatic activity was best preserved at -20 degrees C in the presence of 50% glycerol. Images PMID:3350791

Angiotensin-converting enzyme inhibition and angiotensin AT1 receptor blockade downregulate angiotensin-converting enzyme expression and attenuate renal injury in streptozotocin-induced diabetic rats.

PubMed

Motawi, Tarek K; El-Maraghy, Shohda A; Senousy, Mahmoud A

2013-07-01

Angiotensin-converting enzyme (ACE) is upregulated in the diabetic kidney and contributes to renal injury. This study investigates the possible beneficial effects of the ACE inhibitor (ACEI), enalapril and the AT1 receptor blocker (ARB), valsartan, on renal ACE expression, renal structure, and function in streptozotocin (STZ)-induced diabetic rats. Male Wistar rats were allocated into four groups: control, STZ-diabetic rats, and STZ-diabetic rats treated with either enalapril (10 mg/kg/day) or valsartan (50 mg/kg/day) for 8 weeks. Enalapril and valsartan reduced renal ACE mRNA and protein expression, Na(+) /K(+) -ATPase activity, oxidative stress, and serum transforming growth factor-β1 levels compared to the diabetic group. Both treatments normalized renal nitrate/nitrite levels and ameliorated the observed histopathological changes. In conclusion, ACE downregulation by ACEI and ARB indicates that angiotensin II upregulates ACE through AT1 receptor. Prevention of diabetes-induced changes in ACE expression and Na(+) /K(+) -ATPase activity could be a new explanation of the renoprotective effects of ACEIs and ARBs. © 2013 Wiley Periodicals, Inc.

Analysis and Evaluation of the Inhibitory Mechanism of a Novel Angiotensin-I-Converting Enzyme Inhibitory Peptide Derived from Casein Hydrolysate.

PubMed

Tu, Maolin; Liu, Hanxiong; Zhang, Ruyi; Chen, Hui; Mao, Fengjiao; Cheng, Shuzhen; Lu, Weihong; Du, Ming

2018-04-25

Casein hydrolysates exert various biological activities, and the responsible functional peptides are being identified from them continuously. In this study, the tryptic casein hydrolysate was fractionated by an ultrafiltration membrane (3 kDa), and the peptides were identified by capillary electrophoresis-quadrupole-time-of-flight-tandem mass spectrometry. Meanwhile, in silico methods were used to analyze the toxicity, solubility, stability, and affinity between the peptides and angiotensin-I-converting enzyme (ACE). Finally, a new angiotensin-I-converting enzyme inhibitory (ACEI) peptide, EKVNELSK, derived from α s1 -casein (fragment 35-42) was screened. The half maximal inhibitory concentration value of the peptide is 5.998 mM, which was determined by a high-performance liquid chromatography method. The Lineweaver-Burk plot indicated that this peptide is a mixed-type inhibitor against ACE. Moreover, Discovery Studio 2017 R2 software was adopted to perform molecular docking to propose the potential mechanisms underlying the ACEI activity of the peptide. These results indicated that EKVNELSK is a new ACEI peptide identified from casein hydrolysate.

Synthesis and evaluation of novel triazoles and mannich bases functionalized 1,4-dihydropyridine as angiotensin converting enzyme (ACE) inhibitors.

PubMed

Kumbhare, Ravindra M; Kosurkar, Umesh B; Bagul, Pankaj K; Kanwal, Abhinav; Appalanaidu, K; Dadmal, Tulshiram L; Banerjee, Sanjay Kumar

2014-11-01

A series of novel diethyl 2,6-dimethyl-1,4-dihydropyridine-3,5-dicarboxylate embedded triazole and mannich bases were synthesized, and evaluated for their angiotensin converting enzyme (ACE) inhibitory activity. Screening of above synthesized compounds for ACE inhibition showed that triazoles functionalized compounds have better ACE inhibitory activity compared to that of mannich bases analogues. Among all triazoles we found 6 h, 6 i and 6 j to have good ACE inhibition activity with IC50 values 0.713 μM, 0.409 μM and 0.653 μM, respectively. Among mannich bases series compounds, only 7c resulted as most active ACE inhibitor with IC50 value of 0.928 μM. Copyright © 2014. Published by Elsevier Ltd.

Abalone Protein Hydrolysates: Preparation, Angiotensin I Converting Enzyme Inhibition and Cellular Antioxidant Activity.

PubMed

Park, Soo Yeon; Je, Jae-Young; Hwang, Joung-Youl; Ahn, Chang-Bum

2015-09-01

Abalone protein was hydrolyzed by enzymatic hydrolysis and the optimal enzyme/substrate (E/S) ratios were determined. Abalone protein hydrolysates (APH) produced by Protamex at E/S ratio of 1:100 showed angiotensin I converting enzyme inhibitory activity with IC50 of 0.46 mg/mL, and APH obtained by Flavourzyme at E/S ratio of 1:100 possessed the oxygen radical absorbance capacity value of 457.6 μM trolox equivalent/mg sample. Flavourzyme abalone protein hydrolysates (FAPH) also exhibited H2O2 scavenging activity with IC50 of 0.48 mg/mL and Fe(2+) chelating activity with IC50 of 2.26 mg/mL as well as high reducing power. FAPH significantly (P<0.05) protected H2O2-induced hepatic cell damage in cultured hepatocytes, and the cell viability was restored to 90.27% in the presence of FAPH. FAPH exhibited 46.20% intracellular ROS scavenging activity and 57.89% lipid peroxidation inhibition activity in cultured hepatocytes. Overall, APH may be useful as an ingredient for functional foods.

Abalone Protein Hydrolysates: Preparation, Angiotensin I Converting Enzyme Inhibition and Cellular Antioxidant Activity

PubMed Central

Park, Soo Yeon; Je, Jae-Young; Hwang, Joung-Youl; Ahn, Chang-Bum

2015-01-01

Abalone protein was hydrolyzed by enzymatic hydrolysis and the optimal enzyme/substrate (E/S) ratios were determined. Abalone protein hydrolysates (APH) produced by Protamex at E/S ratio of 1:100 showed angiotensin I converting enzyme inhibitory activity with IC50 of 0.46 mg/mL, and APH obtained by Flavourzyme at E/S ratio of 1:100 possessed the oxygen radical absorbance capacity value of 457.6 μM trolox equivalent/mg sample. Flavourzyme abalone protein hydrolysates (FAPH) also exhibited H2O2 scavenging activity with IC50 of 0.48 mg/mL and Fe2+ chelating activity with IC50 of 2.26 mg/mL as well as high reducing power. FAPH significantly (P<0.05) protected H2O2-induced hepatic cell damage in cultured hepatocytes, and the cell viability was restored to 90.27% in the presence of FAPH. FAPH exhibited 46.20% intracellular ROS scavenging activity and 57.89% lipid peroxidation inhibition activity in cultured hepatocytes. Overall, APH may be useful as an ingredient for functional foods. PMID:26451354

Abeta-degrading enzymes in Alzheimer's disease.

PubMed

Miners, James Scott; Baig, Shabnam; Palmer, Jennifer; Palmer, Laura E; Kehoe, Patrick G; Love, Seth

2008-04-01

In Alzheimer's disease (AD) Abeta accumulates because of imbalance between the production of Abeta and its removal from the brain. There is increasing evidence that in most sporadic forms of AD, the accumulation of Abeta is partly, if not in some cases solely, because of defects in its removal--mediated through a combination of diffusion along perivascular extracellular matrix, transport across vessel walls into the blood stream and enzymatic degradation. Multiple enzymes within the central nervous system (CNS) are capable of degrading Abeta. Most are produced by neurons or glia, but some are expressed in the cerebral vasculature, where reduced Abeta-degrading activity may contribute to the development of cerebral amyloid angiopathy (CAA). Neprilysin and insulin-degrading enzyme (IDE), which have been most extensively studied, are expressed both neuronally and within the vasculature. The levels of both of these enzymes are reduced in AD although the correlation with enzyme activity is still not entirely clear. Other enzymes shown capable of degrading Abetain vitro or in animal studies include plasmin; endothelin-converting enzymes ECE-1 and -2; matrix metalloproteinases MMP-2, -3 and -9; and angiotensin-converting enzyme (ACE). The levels of plasmin and plasminogen activators (uPA and tPA) and ECE-2 are reported to be reduced in AD. Reductions in neprilysin, IDE and plasmin in AD have been associated with possession of APOEepsilon4. We found no change in the level or activity of MMP-2, -3 or -9 in AD. The level and activity of ACE are increased, the level being directly related to Abeta plaque load. Up-regulation of some Abeta-degrading enzymes may initially compensate for declining activity of others, but as age, genetic factors and diseases such as hypertension and diabetes diminish the effectiveness of other Abeta-clearance pathways, reductions in the activity of particular Abeta-degrading enzymes may become critical, leading to the development of AD and CAA.

Peptidases in dog-ileum circular and longitudinal smooth-muscle plasma membranes. Their relative contribution to the metabolism of neurotensin.

PubMed

Checler, F; Ahmad, S; Kostka, P; Barelli, H; Kitabgi, P; Fox, J A; Kwan, C Y; Daniel, E E; Vincent, J P

1987-07-15

We established the content in neuropeptide-metabolizing peptidases present in highly purified plasma membranes prepared from the circular and longitudinal muscles of dog ileum. Activities were measured by the use of fluorigenic substrates and the identities of enzymes were confirmed by the use of specific peptidase inhibitors. Endopeptidase 24.11, angiotensin-converting enzyme, post-proline dipeptidyl aminopeptidase and aminopeptidases were found in both membrane preparations. Proline endopeptidase was only detected in circular smooth muscle plasma membranes while pyroglutamyl-peptide hydrolase was not observed in either tissue. The relative contribution of these peptidases to the inactivation of neurotensin was assessed. The enzymes involved in the primary inactivating cleavages occurring on the neurotensin molecule were as follows. In both membrane preparations, endopeptidase 24.11 was responsible for the formation of neurotensin-(1-11) and contributed to the formation of neurotensin-(1-10); a recently purified neurotensin-degrading neutral metallopeptidase was also involved in the formation of neurotensin-(1-10). A carboxypeptidase-like activity hydrolysed neurotensin at the Ile12-Leu13 peptide bond, leading to the formation of neurotensin-(1-12). Proline endopeptidase and endopeptidase 24.15 only occurred in circular muscle plasma membranes, yielding neurotensin-(1-7) and neurotensin-(1-8), respectively. In addition, the secondary processing of neurotensin degradation products was catalyzed by the following peptidases. In circular and longitudinal muscle membranes, angiotensin-converting enzyme converted neurotensin-(1-10) into neurotensin-(1-8) and tyrosine resulted from the rapid hydrolysis of neurotensin-(11-13) by bestatin-sensitive aminopeptidases. A post-proline dipeptidyl aminopeptidase activity converted neurotensin-(9-13) into neurotensin-(11-13) in circular muscle plasma membranes. The mechanism of neurotensin inactivation occurring in these membranes will be compared to that previously established for membranes from central origin.

Genetics Home Reference: combined malonic and methylmalonic aciduria

MedlinePlus

... acids are building blocks used to make fats (lipids). The ACSF3 enzyme performs a chemical reaction that converts malonic acid to malonyl-CoA, which is the first step of fatty acid synthesis in cellular structures called mitochondria . Based on this activity, the enzyme ...

Serum angiotensin-1 converting enzyme activity processes a human immunodeficiency virus 1 gp160 peptide for presentation by major histocompatibility complex class I molecules

PubMed Central

1992-01-01

T cell stimulation by the human immunodeficiency virus 1 gp160-derived peptide p18 presented by H-2Dd class I major histocompatibility complex molecules in a cell-free system was found to require proteolytic cleavage. This extracellular processing was mediated by peptidases present in fetal calf serum. In vitro processing of p18 resulted in a distinct reverse phase high performance liquid chromatography profile, from which a biologically active product was isolated and sequenced. This peptide processing can be specifically blocked by the angiotensin- 1 converting enzyme (ACE) inhibitor captopril, and can occur by exposing p18 to purified ACE. The ability of naturally occurring extracellular proteases to convert inactive peptides to T cell antigens has important implications for understanding cytotoxic T lymphocyte responses in vivo, and for rational peptide vaccine design. PMID:1316930

Evaluation of subacute change in RAAS activity (as indicated by urinary aldosterone:creatinine, after pharmacologic provocation) and the response to ACE inhibition

PubMed Central

Ames, Marisa K; Atkins, Clarke E; Lantis, Andrea C; zum Brunnen, James

2016-01-01

Objective: The objective of this study was to evaluate subacute changes in renin–angiotensin–aldosterone system (RAAS) activity during angiotensin-converting enzyme inhibitor (ACEI) therapy in dogs with experimental RAAS activation. Methods: Analysis of data (urine aldosterone:creatinine ratio (UAldo:C) and serum angiotensin-converting enzyme activity), in 31 healthy dogs with furosemide or amlodipine-activated RAAS that received an ACEI. When furosemide or amlodipine activation of RAAS preceded ACEI administration, incomplete RAAS blockade (IRB) was defined as a UAldo:C greater than (a) the dog’s ‘activated’ baseline value or (b) a population-derived cut-off value (mean + 2 SD (>1.0 μg/g) of pretreatment UAldo:C from our population of research dogs). In studies where RAAS activation occurred concurrently with ACEIs, IRB was defined as (a) a UAldo:C greater than either twofold the dog’s prestimulation baseline value or (b) 1.0 µg/g. Dogs were followed for 7–17 days. Results: Serum angiotensin-converting enzyme activity was measured in 19 dogs and was significantly reduced (P<0.0001) after ACEI administration. The overall incidence of IRB, when RAAS activation preceded ACEI administration, was 33% and 8% for definitions (a) and (b), respectively. The overall incidence of IRB, when ACEIs were concurrent with RAAS activation, was 65% and 61% for definitions (a) and (b), respectively. Conclusion: Increases in UAldo:C, despite ACEI administration, is evidence of IRB in this subacute model of experimental RAAS activation and suppression. PMID:27009288

Aβ degradation or cerebral perfusion? Divergent effects of multifunctional enzymes.

PubMed

Miners, J Scott; Palmer, Jennifer C; Tayler, Hannah; Palmer, Laura E; Ashby, Emma; Kehoe, Patrick G; Love, Seth

2014-01-01

There is increasing evidence that deficient clearance of β-amyloid (Aβ) contributes to its accumulation in late-onset Alzheimer disease (AD). Several Aβ-degrading enzymes, including neprilysin (NEP), endothelin-converting enzyme (ECE), and angiotensin-converting enzyme (ACE) reduce Aβ levels and protect against cognitive impairment in mouse models of AD. In post-mortem human brain tissue we have found that the activity of these Aβ-degrading enzymes rise with age and increases still further in AD, perhaps as a physiological response that helps to minimize the build-up of Aβ. ECE-1/-2 and ACE are also rate-limiting enzymes in the production of endothelin-1 (ET-1) and angiotensin II (Ang II), two potent vasoconstrictors, increases in the levels of which are likely to contribute to reduced blood flow in AD. This review considers the possible interdependence between Aβ-degrading enzymes, ischemia and Aβ in AD: ischemia has been shown to increase Aβ production both in vitro and in vivo, whereas increased Aβ probably enhances ischemia by vasoconstriction, mediated at least in part by increased ECE and ACE activity. In contrast, NEP activity may help to maintain cerebral perfusion, by reducing the accumulation of Aβ in cerebral blood vessels and lessening its toxicity to vascular smooth muscle cells. In assessing the role of Aβ-degrading proteases in the pathogenesis of AD and, particularly, their potential as therapeutic agents, it is important to bear in mind the multifunctional nature of these enzymes and to consider their effects on other substrates and pathways.

Joseph Rudinger memorial lecture: Unexpected functions of angiotensin converting enzyme, beyond its enzymatic activity.

PubMed

Martinez, Jean

2017-10-01

Angiotensin converting enzyme (ACE) is a well-known enzyme, largely studied for its action on hypertension, as it produces angiotensin II from angiotensin I. This paper describes two original behaviours of ACE. We showed that ACE could hydrolyse gastrin, a neuropeptide from the gastrointestinal tract, releasing the C-terminal amidated dipeptide H-Asp-Phe-NH 2 . This dipeptide is believed to be involved in the gastrin-induced acid secretion in the stomach. This hypothetic mechanism of action of gastrin resulted in a strategy to rationally design gastrin receptor antagonists. Beyond, we showed that the brain renin angiotensin system (RAS) could be activated by a new characterized peptide named acein, resulting in stimulation of dopamine release within the striatum. This new and original 'receptor-like' activity for brain membrane-bound ACE is quite significant taking into account the role of dopamine in the brain, particularly in neurodegenerative diseases. Copyright © 2017 European Peptide Society and John Wiley & Sons, Ltd. Copyright © 2017 European Peptide Society and John Wiley & Sons, Ltd.

Porcine pulmonary angiotensin I-converting enzyme--biochemical characterization and spatial arrangement of the N- and C-domains by three-dimensional electron microscopic reconstruction.

PubMed

Chen, Hui-Ling; Lünsdorf, Heinrich; Hecht, Hans-Jürgen; Tsai, Hsin

2010-08-01

The somatic angiotensin I-converting enzyme (sACE; peptidyl-dipeptidase A; EC 3.4.15.1) was isolated from pig lung and purified to homogeneity. The purified enzyme has a molecular mass of about 180 kDa. Upon proteolytic cleavage, two approximately 90 kDa fragments were obtained and identified by amino-terminal sequence analysis as the N- and C-domains of sACE. Both purified domains were shown to be catalytically active. A 2.3 nm resolution model of sACE was obtained by three-dimensional electron microscopic reconstruction of negatively stained sACE particles, based on atomic X-ray data fitting. Our model shows for the first time the relative orientation of the sACE catalytically active domains and their spatial distance. (c) 2010 Elsevier Ltd. All rights reserved.

Production of high concentration of L-lactic acid from cellobiose by thermophilic Bacillus coagulans WCP10-4.

PubMed

Ong, Shufen Angeline; Ng, Zhi Jian; Wu, Jin Chuan

2016-07-01

Thermophilic Bacillus coagulans WCP10-4 is found to be able to convert cellobiose to optically pure L-lactic acid. Its β-glucosidase activity is detected in whole cells (7.3 U/g dry cells) but not in culture medium, indicating the intracellular location of the enzyme. Its β-glucosidase activity is observed only when cultured using cellobiose as the sole carbon source, indicating that the expression of this enzyme is tightly regulated in cells. The enzyme is most active at 50 °C and pH 7.0. The supplement of external β-glucosidase during fermentation of cellobiose (106 g/l) by B. coagulans WCP10-4 increased the fermentation time from 21 to 23 h and decreased the lactic acid yield from 96.1 to 92.9 % compared to the control without β-glucosidase supplementation. B. coagulans WCP10-4 converted 200 g/l of cellobiose to 196.3 g/l of L-lactic acid at a yield of 97.8 % and a productivity of 7.01 g/l/h. This result shows that B. coagulans WCP10-4 is a highly efficient strain for converting cellobiose to L-lactic acid without the need of supplementing external β-glucosidases.

Interaction of angiotensin-converting enzyme (ACE) with membrane-bound carboxypeptidase M (CPM) - a new function of ACE.

PubMed

Sun, Xiaoou; Wiesner, Burkhard; Lorenz, Dorothea; Papsdorf, Gisela; Pankow, Kristin; Wang, Po; Dietrich, Nils; Siems, Wolf-Eberhard; Maul, Björn

2008-12-01

Angiotensin-converting enzyme (ACE) demonstrates, besides its typical dipeptidyl-carboxypeptidase activity, several unusual functions. Here, we demonstrate with molecular, biochemical, and cellular techniques that the somatic wild-type murine ACE (mACE), stably transfected in Chinese Hamster Ovary (CHO) or Madin-Darby Canine Kidney (MDCK) cells, interacts with endogenous membranal co-localized carboxypeptidase M (CPM). CPM belongs to the group of glycosylphosphatidylinositol (GPI)-anchored proteins. Here we report that ACE, completely independent of its known dipeptidase activities, has GPI-targeted properties. Our results indicate that the spatial proximity between mACE and the endogenous CPM enables an ACE-evoked release of CPM. These results are discussed with respect to the recently proposed GPI-ase activity and function of sperm-bound ACE.

In vitro angiotensin I converting enzyme inhibition by a peptide isolated from Chiropsalmus quadrigatus Haeckel (box jellyfish) venom hydrolysate.

PubMed

So, Pamela Berilyn T; Rubio, Peter; Lirio, Stephen; Macabeo, Allan Patrick; Huang, Hsi-Ya; Corpuz, Mary Jho-Anne T; Villaflores, Oliver B

2016-09-01

The anti-angiotensin I converting enzyme activity of box jellyfish, Chiropsalmus quadrigatus Haeckel venom hydrolysate was studied. The venom extract was obtained by centrifugation and ultrasonication. Protein concentration of 12.99 μg/mL was determined using Bradford assay. The pepsin and papain hydrolysate was tested for its toxicity by Limit test following the OECD Guideline 425 using 5 female Sprague-Dawley rats. Results showed that the hydrolysate is nontoxic with an LD50 above 2000 mg/kg. In vitro angiotensin I converting enzyme (ACE) inhibitory activity was determined using ACE kit-WST. Isolation of ACE inhibitory peptides using column chromatography with SP-Sephadex G-25 yielded 8 pooled fractions with fraction 3 (86.5%) exhibiting the highest activity. This was followed by reverse phase - high performance liquid chromatography (RP-HPLC) with an octadecyl silica column (Inertsil ODS-3) using methanol:water 15:85 at a flow rate of 1.0 mL/min. Among the 13 fractions separated with the RP-HPLC, fraction 3.5 exhibited the highest ACE inhibitory activity (84.1%). The peptide sequence ACPGPNPGRP (IC50 2.03 μM) from fraction 3.5 was identified using Matrix-assisted laser desorption/ionization with time-of-flight tandem mass spectroscopy analysis (MALDI-TOF/MS). Copyright © 2016 Elsevier Ltd. All rights reserved.

Hydrolysis by somatic angiotensin-I converting enzyme of basic dipeptides from a cholecystokinin/gastrin and a LH-RH peptide extended at the C-terminus with gly-Arg/Lys-arg, but not from diarginyl insulin.

PubMed

Isaac, R E; Michaud, A; Keen, J N; Williams, T A; Coates, D; Wetsel, W C; Corvol, P

1999-06-01

Endoproteolytic cleavage of protein prohormones often generates intermediates extended at the C-terminus by Arg-Arg or Lys-Arg, the removal of which by a carboxypeptidase (CPE) is normally an important step in the maturation of many peptide hormones. Recent studies in mice that lack CP activity indicate the existence of alternative tissue or plasma enzymes capable of removing C-terminal basic residues from prohormone intermediates. Using inhibitors of angiotensin I-converting enzyme (ACE) and CP, we show that both these enzymes in mouse serum can remove the basic amino acids from the C-terminus of CCK5-GRR and LH-RH-GKR, but only CP is responsible for converting diarginyl insulin to insulin. ACE activity removes C-terminal dipeptides to generate the Gly-extended peptides, whereas CP hydrolysis gives rise to CCK5-GR and LH-RH-GK, both of which are susceptible to the dipeptidyl carboxypeptidase activity of ACE. Somatic ACE has two similar protein domains (the N-domain and the C-domain), each with an active site that can display different substrate specificities. CCK5-GRR is a high-affinity substrate for both the N-domain and C-domain active sites of human sACE (Km of 9.4 microm and 9.0 microm, respectively) with the N-domain showing greater efficiency (kcat : Km ratio of 2.6 in favour of the N-domain). We conclude that somatic forms of ACE should be considered as alternatives to CPs for the removal of basic residues from some Arg/Lys-extended peptides.

Preparation of progenin III from total steroidal saponins of Dioscorea nipponica Makino using a crude enzyme from Aspergillus oryzae strain.

PubMed

Liu, Tingqiang; Yu, Hongshan; Liu, Chunying; Bao, Yongming; Hu, Xiangchun; Wang, Yuanhao; Liu, Bing; Fu, Yaoyao; Tang, Sihui; Jin, Fengxie

2013-05-01

Progenin III, one of the most active spirostanol saponins, is a potential candidate for anti-cancer therapy due to its strong antitumor activity and low hemolytic activity. However, the concentration of progenin III is extremely low in natural Dioscorea plants. In this paper, the progenin III production from total steroidal saponins of Dioscorea nipponica Makino was studied using the crude enzyme from Aspergillus oryzae DLFCC-38. The crude enzyme converting total steroidal saponins into progenin III was obtained from the A. oryzae DLFCC-38 culture. For enzyme production, the strain was cultured for 72 h at 30 °C with shaking at 150 rpm in 5 % (w/v) malt extract medium containing 2 % (v/v) extract of D. nipponica as the enzyme inducer. The crude enzyme converted total steroidal saponins into major progenin III with a high yield when the reaction was carried out for 9 h at 50 °C and pH 5.0 with the 20 mg/ml of substrate. In the preparation of progenin III, 117 g of crude progenin III was obtained from 160 g of substrate, and the crude product was purified with silica gel column to obtain 60.3 g progenin III of 93.4 % purity.

Soy Pulp Extract Inhibits Angiotensin I-Converting Enzyme (ACE) Activity In Vitro: Evidence for Its Potential Hypertension-Improving Action.

PubMed

Nishibori, Naoyoshi; Kishibuchi, Reina; Morita, Kyoji

2017-05-04

Soy pulp, called "okara" in Japanese, is known as a by-product of the production of bean curd (tofu), and expected to contain a variety of biologically active substances derived from soybean. However, the biological activities of okara ingredients have not yet been fully understood, and the effectiveness of okara as a functional food seems necessary to be further evaluated. Then the effect of okara extract on angiotensin I-converting enzyme (ACE) activity was examined in vitro, and the extract was shown to cause the inhibition of ACE activity in a manner depending on its concentration. Kinetic analysis indicated that this enzyme inhibition was accompanied by an increase in the Km value without any change in Vmax. Further studies suggested that putative inhibitory substances contained in the extract might be heat stable and dialyzable, and recovered mostly in the peptide fraction obtained by a spin-column separation and a high performance liquid chromatography (HPLC) fractionation. Therefore, the extract was speculated to contain small-size peptides responsible for the inhibitory effect of okara extract on ACE activity, and could be expected to improve the hypertensive conditions by reducing the production of hypertensive peptide.

Purification and characterization of a tuliposide-converting enzyme from bulbs of Tulipa gesneriana.

PubMed

Kato, Yasuo; Shoji, Kazuaki; Ubukata, Makoto; Shigetomi, Kengo; Sato, Yukio; Nakajima, Noriyuki; Ogita, Shinjiro

2009-08-01

An enzyme that catalyzes the stoichiometric conversion of 6-tuliposide into tulipalin was purified and characterized from bulbs of Tulipa gesneriana. The enzyme appeared to be a dimer, the relative molecular mass (Mr) of each subunit being 34,900; it had maximum activity and stability at neutral pH and moderate temperature. The enzyme preferentially acted on such glucose esters as 6-tuliposides, and to a lesser extent on p-nitrophenylacetate.

Adenine formation from adenosine by mycoplasmas: adenosine phosphorylase activity.

PubMed Central

Hatanaka, M; Del Giudice, R; Long, C

1975-01-01

Mammalian cells have enzymes to convert adenosine to inosine by deamination and inosine to hypoxanthine by phosphorolysis, but they do not possess the enzymes necessary to form the free base, adenine, from adenosine. Mycoplasmas grown in broth or in cell cultures can produce adenine from adenosine. This activity was detected in a variety of mycoplasmatales, and the enzyme was shown to be adenosine phosphorylase. Adenosine formation from adenine and ribose 1-phosphate, the reverse reaction of adenine formation from adenosine, was also observed with the mycoplasma enzyme. Adenosine phosphorylase is apparently common to the mycoplasmatales but it is not universal, and the organisms can be divided into three groups on the basis of their use of adenosine as substrate. Thirteen of 16 Mycoplasma, Acholeplasma, and Siroplasma species tested exhibit adenosine phosphorylase activity. M. lipophilium differed from the other mycoplasmas and shared with mammalian cells the ability to convert adenosine to inosine by deamination. M. pneumoniae and the unclassified M. sp. 70-159 showed no reaction with adenosine. Adenosine phosphorylase activity offers an additional method for the detection of mycoplasma contamination of cells. The patterns of nucleoside metabolism will provide additional characteristics for identification of mycoplasmas and also may provide new insight into the classification of mycoplasmas. PMID:236559

Heterologous Acidothermus cellulolyticus 1,4-β-Endoglucanase E1 Produced Within the Corn Biomass Converts Corn Stover Into Glucose

NASA Astrophysics Data System (ADS)

Ransom, Callista; Balan, Venkatesh; Biswas, Gadab; Dale, Bruce; Crockett, Elaine; Sticklen, Mariam

Commercial conversion of lignocellulosic biomass to fermentable sugars requires inexpensive bulk production of biologically active cellulase enzymes, which might be achieved through direct production of these enzymes within the biomass crops. Transgenic corn plants containing the catalytic domain of Acidothermus cellulolyticus E1 endo-1,4-β glucanase and the bar bialaphos resistance coding sequences were generated after Biolistic® (BioRad Hercules, CA) bombardment of immature embryo-derived cells. E1 sequences were regulated under the control of the cauliflower mosaic virus 35S promoter and tobacco mosaic virus translational enhancer, and E1 protein was targeted to the apoplast using the signal peptide of tobacco pathogenesis-related protein to achieve accumulation of this enzyme. The integration, expression, and segregation of E1 and bar transgenes were demonstrated, respectively, through Southern and Western blotting, and progeny analyses. Accumulation of up to 1.13% of transgenic plant total soluble proteins was detected as biologically active E1 by enzymatic activity assay. The corn-produced, heterologous E1 could successfully convert ammonia fiber explosion-pretreated corn stover polysaccharides into glucose as a fermentable sugar for ethanol production, confirming that the E1 enzyme is produced in its active from.

Withholding versus Continuing Angiotensin-converting Enzyme Inhibitors or Angiotensin II Receptor Blockers before Noncardiac Surgery: An Analysis of the Vascular events In noncardiac Surgery patIents cOhort evaluatioN Prospective Cohort.

PubMed

Roshanov, Pavel S; Rochwerg, Bram; Patel, Ameen; Salehian, Omid; Duceppe, Emmanuelle; Belley-Côté, Emilie P; Guyatt, Gordon H; Sessler, Daniel I; Le Manach, Yannick; Borges, Flavia K; Tandon, Vikas; Worster, Andrew; Thompson, Alexandra; Koshy, Mithin; Devereaux, Breagh; Spencer, Frederick A; Sanders, Robert D; Sloan, Erin N; Morley, Erin E; Paul, James; Raymer, Karen E; Punthakee, Zubin; Devereaux, P J

2017-01-01

The effect on cardiovascular outcomes of withholding angiotensin-converting enzyme inhibitors or angiotensin II receptor blockers in chronic users before noncardiac surgery is unknown. In this international prospective cohort study, the authors analyzed data from 14,687 patients (including 4,802 angiotensin-converting enzyme inhibitor/angiotensin II receptor blocker users) at least 45 yr old who had in-patient noncardiac surgery from 2007 to 2011. Using multivariable regression models, the authors studied the relationship between withholding angiotensin-converting enzyme inhibitors/angiotensin II receptor blockers and a primary composite outcome of all-cause death, stroke, or myocardial injury after noncardiac surgery at 30 days, with intraoperative and postoperative clinically important hypotension as secondary outcomes. Compared to patients who continued their angiotensin-converting enzyme inhibitors/angiotensin II receptor blockers, the 1,245 (26%) angiotensin-converting enzyme inhibitor/angiotensin II receptor blocker users who withheld their angiotensin-converting enzyme inhibitors/angiotensin II receptor blockers in the 24 h before surgery were less likely to suffer the primary composite outcome of all-cause death, stroke, or myocardial injury (150/1,245 [12.0%] vs. 459/3,557 [12.9%]; adjusted relative risk, 0.82; 95% CI, 0.70 to 0.96; P = 0.01) and intraoperative hypotension (adjusted relative risk, 0.80; 95% CI, 0.72 to 0.93; P < 0.001). The risk of postoperative hypotension was similar between the two groups (adjusted relative risk, 0.92; 95% CI, 0.77 to 1.10; P = 0.36). Results were consistent across the range of preoperative blood pressures. The practice of withholding angiotensin-converting enzyme inhibitors/angiotensin II receptor blockers was only modestly correlated with patient characteristics and the type and timing of surgery. Withholding angiotensin-converting enzyme inhibitors/angiotensin II receptor blockers before major noncardiac surgery was associated with a lower risk of death and postoperative vascular events. A large randomized trial is needed to confirm this finding. In the interim, clinicians should consider recommending that patients withhold angiotensin-converting enzyme inhibitors/angiotensin II receptor blockers 24 h before surgery.

Screening the ToxCast Phase I, II, and e1K Chemical Libraries for Inhibition of Deiodinase Type 1,2 and 3 Enzyme Activity

EPA Science Inventory

Thyroid hormone (TH) signaling and homeostasis is dependent upon coordination of multiple key events including thyroidal iodide uptake and hormone synthesis, and peripheral metabolism and elimination. Deiodinase enzymes play an essential role in converting the pro-hormone thyroxi...

3D-quantitative structure-activity relationship studies on benzothiadiazepine hydroxamates as inhibitors of tumor necrosis factor-alpha converting enzyme.

PubMed

Murumkar, Prashant R; Giridhar, Rajani; Yadav, Mange Ram

2008-04-01

A set of 29 benzothiadiazepine hydroxamates having selective tumor necrosis factor-alpha converting enzyme inhibitory activity were used to compare the quality and predictive power of 3D-quantitative structure-activity relationship, comparative molecular field analysis, and comparative molecular similarity indices models for the atom-based, centroid/atom-based, data-based, and docked conformer-based alignment. Removal of two outliers from the initial training set of molecules improved the predictivity of models. Among the 3D-quantitative structure-activity relationship models developed using the above four alignments, the database alignment provided the optimal predictive comparative molecular field analysis model for the training set with cross-validated r(2) (q(2)) = 0.510, non-cross-validated r(2) = 0.972, standard error of estimates (s) = 0.098, and F = 215.44 and the optimal comparative molecular similarity indices model with cross-validated r(2) (q(2)) = 0.556, non-cross-validated r(2) = 0.946, standard error of estimates (s) = 0.163, and F = 99.785. These models also showed the best test set prediction for six compounds with predictive r(2) values of 0.460 and 0.535, respectively. The contour maps obtained from 3D-quantitative structure-activity relationship studies were appraised for activity trends for the molecules analyzed. The comparative molecular similarity indices models exhibited good external predictivity as compared with that of comparative molecular field analysis models. The data generated from the present study helped us to further design and report some novel and potent tumor necrosis factor-alpha converting enzyme inhibitors.

In vitro autoradiographic localization of angiotensin-converting enzyme in sarcoid lymph nodes

DOE Office of Scientific and Technical Information (OSTI.GOV)

Allen, R.K.; Chai, S.Y.; Dunbar, M.S.

1986-09-01

Angiotensin-converting enzyme (ACE) was localized in sarcoid lymph nodes by an in vitro autoradiographic technique using a synthetic ACE inhibitor of high affinity, /sup 125/I-labelled 351A. The lymph nodes were from seven patients with active sarcoidosis who underwent mediastinoscopy and from six control subjects who had nodes resected at either mediastinoscopy or laparotomy. Angiotensin-converting enzyme was localized in the epithelioid cells of sarcoid granulomata in markedly increased amounts compared with control nodes, where it was restricted to vessels and some histiocytes. In sarcoid lymph nodes, there was little ACE present in lymphocytes or fibrous tissue. Sarcoid nodes with considerable fibrosismore » had much less intense ACE activity than the nonfibrotic nodes. The specific activity of ACE measured by an enzymatic assay in both the control and sarcoid lymph nodes closely reflected the ACE activity demonstrated by autoradiography. Sarcoid lymph nodes with fibrosis had an ACE specific activity of half that of nonfibrotic nodes (p less than 0.05). There was a 15-fold increase in specific ACE activity in sarcoid nodes (p less than 0.05) compared to normal. Serum ACE was significantly higher in those sarcoid patients whose lymph nodes were not fibrosed compared with those with fibrosis (p less than 0.01). This technique offers many advantages over the use of polyclonal antibodies. The 351A is a highly specific ACE inhibitor, chemically defined and in limitless supply. This method enables the quantitation of results, and autoradiographs may be stored indefinitely for later comparison.« less

Serum angiotensin-converting enzyme (SACE) in sarcoidosis and other granulomatous disorders.

PubMed

Studdy, P; Bird, R; James, D G

Serum angiotensin-converting enzyme (SACE) activity was significantly higher in 90 patients with sarcoidosis (55 +/- [S.D.] 23 nmol min-1 ml-1) than in 80 healthy controls (34 +/- 9 nmol min-1 ml-1). Steroid therapy modified SACE activity; 60 sarcoidosis patients who were not being treated with steroids had significantly higher enzyme activities (58 +/- 24 nmol min-1 ml-1) than 30 steroid-treated sarcoidosis patients (40 +/- 19 nmol min-1 ml-1). In 50% of the non-steroid treated sarcoidosis patients SACE activity was more than 2 S.D. above the mean value for the controls. SACE activity was measured in 22 tuberculous patients (38 +/- 14 nmol min-1 ml-1), 20 leprosy patients (34 +/- 9 nmol min-1 ml-1), 31 with primary biliary cirrhosis (44 +/- 20 nmol min-1 ml-1), 26 with inflammatory bowel disease (31 +/- 9 nmol min-1 ml-1), 8 with hepatic granulomatous disease, 5 with Hodgkin's disease, and 2 with schistosomiasis. The combined false-positive rate for these non-sarcoidosis patients was 10%. Serial SACE assays provide useful information on the course of sarcoidosis and response to steroid treatment.

Serum angiotensin--converting enzyme (SACE) in sarcoidosis and other granulomatous disorders.

PubMed

Studdy, P; Bird; James, D G; Sherlock, S

Serum angiotensin-converting enzyme (SACE) activity was significantly higher in 90 patients with sarcoidosis (55 +/- [S.D.] 23 nmol min-1 ml-1) than in 80 healthy controls (34 +/- 9 nmol min-1 ml-1). Steroid therapy modified SACE activity; 60 sarcoidosis patients who were not being treated with steroids had significantly higher enzyme activities (58 +/- 24 nmol min-1 ml-1) than 30 steroid-treated sarcoidosis patients (40 +/- 19 nmol min-1 ml-1). In 50% of the non-steroid treated sarcoidosis patients SACE activity was more than than 2 S.D. above the mean value for the controls. SACE activity was measured in 22 tuberculous patients (38 +/- 14 nmol min-1 ml-1), 20 leprosy patients (34 +/- 9 nmol min-1 ml-1), 31 with primary biliary cirrhosis (44 +/- 20 nmol min-1 ml-1), 26 with inflammatory bowel disease (31 +/- 9 nmol min-1 ml-1), eight with hepatic granulomatous disease, five with Hodgkin's disease, and two with schistosomiasis. The combined false-positive rate for these non-sarcoidosis patients was 10%. Serial SACE assays provide useful information on the course of sarcoidosis and response to steroid treatment.

Angiotensin converting enzyme (ACE) inhibitory and antihypertensive activities of protein hydrolysate from meat of Kacang goat (Capra aegagrus hircus).

PubMed

Mirdhayati, Irdha; Hermanianto, Joko; Wijaya, Christofora H; Sajuthi, Dondin; Arihara, Keizo

2016-08-01

The meat of Kacang goat has potential for production of a protein hydrolysate. Functional ingredients from protein hydrolysate of Kacang goat meat were determined by the consistency of angiotensin-converting enzyme (ACE) inhibitory activity and antihypertensive effect. This study examined the potency of Kacang goat protein hydrolysate in ACE inhibition and antihypertensive activity. Protein hydrolysates of Kacang goat meat were prepared using sequential digestion of endo-proteinase and protease complex at several concentrations and hydrolysis times. The highest ACE inhibitory activity resulted from a hydrolysate that was digested for 4 h with 5 g kg(-1) of both enzymes. An ACE inhibitory peptide was purified and a novel peptide found with a sequence of Phe-Gln-Pro-Ser (IC50 value of 27.0 µmol L(-1) ). Both protein hydrolysates and a synthesised peptide (Phe-Gln-Pro-Ser) demonstrated potent antihypertensive activities in spontaneously hypertensive rats. Protein hydrolysate of Kacang goat meat produced by sequential digestion with endo-proteinase and protease complex has great potential as a functional ingredient, particularly as an antihypertensive agent. © 2015 Society of Chemical Industry. © 2015 Society of Chemical Industry.

HPLC-DAD Analysis and In-Vitro Property of Polyphenols Extracts from (Solanum Aethiopium) Fruits on α -Amylase, α -Glucosidase and Angiotensin - 1- Converting Enzyme Activities

PubMed Central

Nwanna, E. E; Ibukun, E. O; Oboh, G.; Ademosun, A. O.; Boligon, A. A.; Athayde, M.

2014-01-01

AIM: Garden egg (Solanum aethiopium) is an edible fruits vegetable with  different species.This study investigated characterisation and the effect of the phenolics extracts from S. aethiopium species with enzymes linked with type -2-diabetes (α-amylase and α-glucosidase) and hypertension [Angiotensin-1-converting enzyme (ACE)]. METHODS: Fresh samples of the 5 species of the garden egg namely, [Solanum gilo (PW), Solanum torvum (TWS), Solanum kumba (PGR), Solanum incanum (GSB), and Solanum indicum (WSB)] were oven-dried at 50°C and milled into flour. The aqueous extracts were prepared (1:50 w/v). The phenolic contents (total phenol and total flavonoid), vitamin C and 1,1-diphenyl–2-picrylhydrazyl (DPPH), the antioxidant activities of the extracts were evaluated. The ability of the extracts to inhibit diabetes enzymes in rat pancreas as well as the inhibition of angiotensin-1-converting (ACE) enzyme in lungs homogenates in vitro were investigated. Furthermore, the fruits polyphenols were identified and quantified using HPLC-DAD. RESULTS: The phenolic contents ranged from 2.70-3.76 mgGAE/g, while there were no significant (P>0.05) differences in their flavonoid content and ability to reduce Fe3+ to Fe2+. The vitamin C contents of the species ranged from 4.01-6.52 mg/ml. The extracts scavenged DPPH in a dose dependent manner with the IC50 values ranging from 3.23-4.20 mg/ml. Furthermore, the extracts showed strong inhibition of α-glucosidase, mild inhibition of α-amylase and strong inhibition of ACE activities. CONCLUSION: This study showed that the inhibition of the key enzymes relevant to type-2 diabetes and hypertension could be part of the mechanisms by which garden egg manage/prevent the degenerative conditions. PMID:25598760

Peptides derived from Rhopilema esculentum hydrolysate exhibit angiotensin converting enzyme (ACE) inhibitory and antioxidant abilities.

PubMed

Li, Jun; Li, Qian; Li, Jingyun; Zhou, Bei

2014-09-02

Jellyfish (Rhopilema esculentum) was hydrolyzed using alcalase, and two peptides with angiotensin-I-converting enzyme (ACE) inhibitory and antioxidant activities were purified by ultrafiltration and consecutive chromatographic methods. The amino acid sequences of the two peptides were identified as VKP (342 Da) and VKCFR (651 Da) by electrospray ionization tandem mass spectrometry. The IC50 values of ACE inhibitory activities of the two peptides were 1.3 μM and 34.5 μM, respectively. Molecular docking results suggested that VKP and VKCFR bind to ACE through coordinating with the active site Zn(II) atom. Free radical scavenging activity and protection against hydrogen peroxide (H2O2)-induced rat cerebral microvascular endothelial cell (RCMEC) injury were used to evaluate the antioxidant activities of the two peptides. As the results clearly showed that the peptides increased the superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GSH-px) activities in RCMEC cells), it is proposed that the R. esculentum peptides exert significant antioxidant effects.

Physiological effects of formulation containing tannase-converted green tea extract on skin care: physical stability, collagenase, elastase, and tyrosinase activities.

PubMed

Hong, Yang-Hee; Jung, Eun Young; Noh, Dong Ouk; Suh, Hyung Joo

2014-03-01

Green tea contains numerous polyphenols, which have health-promoting effects. The purpose of this study was to evaluate the effect of tannase-converted green tea extract (TGE) formulation on the physical stability and activities of skin-related enzymes. Physical stability was evaluated by measuring the pH, precipitation, and colors at 25 ± 2 °C/ambient humidity and at 40 ± 2 °C/70% ± 5% relative humidity for 4 months. Activities of collagenase, elastase, and tyrosinase as skin-related enzymes were assessed on TGE formulation. The concentrations of epigallocatechin-3-gallate and epicatechin-3-gallate in green tea extract were greatly decreased to the extent of negligible level when treated with tannase. The formulation containing 5% tannase-converted green tea extract showed relatively stable pH, precipitation, and color features for 16 weeks. When TGE was added to the formulation, there was a significant increase in the inhibition of elastase and tyrosinase activities ( p  < 0.05) compared with the formulation containing 5% normal green tea extract. The TGE could be used in cosmetics as skin antiwrinkling or depigmenting agent.

Bioequivalence of azathioprine products.

PubMed

Baker, Daniel E

2003-01-01

All azathioprine oral tablets are considered bioequivalent by the Food and Drug Administration based on traditional testing. However, since these tests were conducted, it has been determined that some patients have a deficiency of the enzyme most responsible for the metabolism of 6-mercaptopurine-thiopurine methyltransferase (TPMT). Azathioprine is rapidly converted to 6-mercaptopurine, its active metabolite. So it is possible that differences in TPMT activity may influence the bioequivalence of azathioprine products among individuals, especially those patients deficient in TPMT enzyme activity. However, this possibility has not been evaluated.

Cellulolytic enzymes, nucleic acids encoding them and methods for making and using them

DOEpatents

Gray, Kevin A [San Diego, CA; Zhao, Lishan [Emeryville, CA; Cayouette, Michelle H [San Diego, CA

2012-01-24

The invention provides polypeptides having any cellulolytic activity, e.g., a cellulase activity, a endoglucanase, a cellobiohydrolase, a beta-glucosidase, a xylanase, a mannanse, a .beta.-xylosidase, an arabinofuranosidase, and/or an oligomerase activity, polynucleotides encoding these polypeptides, and methods of making and using these polynucleotides and polypeptides. In one aspect, the invention is directed to polypeptides having any cellulolytic activity, e.g., a cellulase activity, e.g., endoglucanase, cellobiohydrolase, beta-glucosidase, xylanase, mannanse, .beta.-xylosidase, arabinofuranosidase, and/or oligomerase activity, including thermostable and thermotolerant activity, and polynucleotides encoding these enzymes, and making and using these polynucleotides and polypeptides. In one aspect, the invention provides polypeptides having an oligomerase activity, e.g., enzymes that convert recalcitrant soluble oligomers to fermentable sugars in the saccharification of biomass. The polypeptides of the invention can be used in a variety of pharmaceutical, agricultural, food and feed processing and industrial contexts. The invention also provides compositions or products of manufacture comprising mixtures of enzymes comprising at least one enzyme of this invention.

Utility of Adenosine Monophosphate Detection System for Monitoring the Activities of Diverse Enzyme Reactions.

PubMed

Mondal, Subhanjan; Hsiao, Kevin; Goueli, Said A

Adenosine monophosphate (AMP) is a key cellular metabolite regulating energy homeostasis and signal transduction. AMP is also a product of various enzymatic reactions, many of which are dysregulated during disease conditions. Thus, monitoring the activities of these enzymes is a primary goal for developing modulators for these enzymes. In this study, we demonstrate the versatility of an enzyme-coupled assay that quantifies the amount of AMP produced by any enzymatic reaction regardless of its substrates. We successfully implemented it to enzyme reactions that use adenosine triphosphate (ATP) as a substrate (aminoacyl tRNA synthetase and DNA ligase) by an elaborate strategy of removing residual ATP and converting AMP produced into ATP; so it can be detected using luciferase/luciferin and generating light. We also tested this assay to measure the activities of AMP-generating enzymes that do not require ATP as substrate, including phosphodiesterases (cyclic adenosine monophosphate) and Escherichia coli DNA ligases (nicotinamide adenine dinucleotide [NAD + ]). In a further elaboration of the AMP-Glo platform, we coupled it to E. coli DNA ligase, enabling measurement of NAD + and enzymes that use NAD + like monoadenosine and polyadenosine diphosphate-ribosyltransferases. Sulfotransferases use 3'-phosphoadenosine-5'-phosphosulfate as the universal sulfo-group donor and phosphoadenosine-5'-phosphate (PAP) is the universal product. PAP can be quantified by converting PAP to AMP by a Golgi-resident PAP-specific phosphatase, IMPAD1. By coupling IMPAD1 to the AMP-Glo system, we can measure the activities of sulfotransferases. Thus, by utilizing the combinations of biochemical enzymatic conversion of various cellular metabolites to AMP, we were able to demonstrate the versatility of the AMP-Glo assay.

The 46 kDa dimeric protein from Variovorax paradoxus shows faster methotrexate degrading activity in its nanoform compare to the native enzyme.

PubMed

Bayineni, Venkata Krishna; Venkatesh, Krishna; Sahu, Chandan Kumar; Kadeppagari, Ravi-Kumar

2016-04-01

Methotrexate degrading enzymes are required to overcome the toxicity of the methotrexate while treating the cancer. The enzyme from Variovorax paradoxus converts the methotrexate in to non toxic products. Methotrexate degrading enzyme from V. paradoxus is a dimeric protein with a molecular mass of 46 kDa and it acts on casein and gelatin. This enzyme is optimally active at pH 7.5 and 40°C and nanoparticles of this enzyme were prepared by desolvation-crosslinking method. Enzyme nanoparticles could degrade methotrexate faster than the native enzyme and they show lower Km compare to the native enzyme. Enzyme nanoparticles show better thermostability and they were stable for much longer time in the serum compare to the native enzyme. Enzyme nanoparticles show better functionality than the native enzyme while clearing the methotrexate added to the serum suggesting their advantage over the native enzyme for the therapeutic and biotechnological applications. Copyright © 2016 Elsevier Inc. All rights reserved.

Purification of angiotensin I-converting enzyme (ACE) inhibitory peptides from casein hydrolysate by IMAC-Ni2.

PubMed

Wu, Shanguang; Feng, Xuezhen; Lu, Yuan; Lu, Yuting; Liu, Saisai; Tian, Yuhong

2017-10-01

Casein proteins were hydrolyzed by papain to identify inhibitory peptides of angiotensin I-converting enzyme (ACE). The hydrolysate was fractionized by immobilized metal affinity chromatography (IMAC-Ni 2+ ). The fraction with high ACE inhibitory activity was enriched and further chromatographed on a reverse-phase column to yield four fractions. Among the fractions, the L4 fraction exhibited the highest ACE inhibitory activity and was identified by sequence analysis as Trp-Tyr-Leu-His-Tyr-Ala (WYLHYA), with IC 50 value of 16.22 ± 0.83 µM in vitro. This peptide was expected to be applied as an ingredient for preventing hypertension and IMAC-Ni 2+ may provide a simple method for purification of ACE inhibitory peptides.

Spätzle-Processing Enzyme-independent Activation of the Toll Pathway in Drosophila Innate Immunity.

PubMed

Yamamoto-Hino, Miki; Goto, Satoshi

2016-05-07

The Toll pathway regulates innate immunity in insects and vertebrates. The Drosophila Toll receptor is activated by a processed form of a ligand, Spätzle. Spätzle-processing enzyme (SPE) is the only enzyme identified to date that functions in converting Spätzle to an active form during the immune response. In the present study, Toll activation induced by immune challenge was almost suppressed in spätzle mutant larvae and adults, whereas it was present in SPE mutant larvae challenged with Micrococcus luteus and adults challenged with Bacillus subtilis. Our data suggest that an unidentified protease besides SPE processes Spätzle under conditions of microbial challenge.

Remote enzyme activation using gold coated magnetite as antennae for radio frequency fields

NASA Astrophysics Data System (ADS)

Collins, Christian B.; Ackerson, Christopher J.

2018-02-01

The emerging field of remote enzyme activation, or the ability to remotely turn thermophilic increase enzyme activity, could be a valuable tool for understanding cellular processes. Through exploitation of the temperature dependence of enzymatic processes and high thermal stability of thermophilic enzymes these experiments utilize nanoparticles as `antennae' that convert radiofrequency (RF) radiation into local heat, increasing activity of the enzymes without increasing the temperature of the surrounding bulk solution. To investigate this possible tool, thermolysin, a metalloprotease was covalently conjugated to 4nm gold coated magnetite particles via peptide bond formation with the protecting ligand shell. RF stimulated protease activity at 17.76 MHz in a solenoid shaped antenna, utilizing both electric and magnetic field interactions was investigated. On average 40 percent higher protease activity was observed in the radio frequency fields then when bulk heating the sample to the same temperature. This is attributed to electrophoretic motion of the nanoparticle enzyme conjugates and local regions of heat generated by the relaxation of the magnetite cores with the oscillating field. Radio frequency local heating of nanoparticles conjugated to enzymes as demonstrated could be useful in the activation of specific enzymes in complex cellular environments.

Angiotensin-converting enzyme activity in Cavalier King Charles Spaniels with an ACE gene polymorphism and myxomatous mitral valve disease.

PubMed

Meurs, Kathryn M; Olsen, Lisbeth H; Reimann, Maria J; Keene, Bruce W; Atkins, Clarke E; Adin, Darcy; Aona, Brent; Condit, Julia; DeFrancesco, Teresa; Reina-Doreste, Yamir; Stern, Joshua A; Tou, Sandra; Ward, Jessica; Woodruff, Kathleen

2018-02-01

Myxomatous mitral valve disease (MMVD) is the most common heart disease in the dog. It is particularly common in the Cavalier King Charles Spaniel (CKCS) breed and affected dogs are frequently managed with angiotensin-converting enzyme inhibitors (ACE-I). We have previously identified a canine ACE gene polymorphism associated with a decrease in angiotensin-converting enzyme (ACE) activity. The aim of this study was to evaluate for the prevalence of the ACE polymorphism in CKCS with mitral valve disease and to determine whether the presence of the polymorphism is associated with alterations in ACE activity at different stages of cardiac disease. Seventy-three dogs with a diagnosis of mitral valve disease were evaluated and a blood sample was drawn for ACE polymorphism genotyping and ACE activity measurement. Forty-three dogs were homozygous for the ACE polymorphism; five were heterozygous and 25 were homozygous wild type. The mean age and the median severity of disease were not different for dogs with the polymorphism and dogs with the wild-type sequence. The median baseline ACE activity was significantly lower for the ACE polymorphism (27.0 U/l) than the wild-type sequence dogs (31.0 U/l) (P=0.02). Dogs with more severe disease and the ACE polymorphism had significantly lower levels of ACE activity than dogs with the wild-type sequence (P=0.03). The CKCS appears to have a high prevalence of the ACE variant. Dogs with the ACE variant had lower levels of ACE activity even in more advanced mitral valve disease than dogs without the variant. The clinical significance of this finding and its impact on the need for ACE-I in dogs with the polymorphism and heart disease deserves further study.

Efficient conversion of mannitol derived from brown seaweed to fructose for fermentation with a thraustochytrid.

PubMed

Tajima, Takahisa; Tomita, Kousuke; Miyahara, Hiroyuki; Watanabe, Kenshi; Aki, Tsunehiro; Okamura, Yoshiko; Matsumura, Yukihiko; Nakashimada, Yutaka; Kato, Junichi

2018-02-01

Macroalgae are a promising biomass feedstock for energy and valuable chemicals. Mannitol and alginate are the major carbohydrates found in the microalga Laminaria japonica (Konbu). To convert mannitol to fructose for its utilization as a carbon source in mannitol non-assimilating bacteria, a psychrophile-based simple biocatalyst (PSCat) was constructed using a psychrophile as a host by expressing mesophilic enzymes, including mannitol 2-dehydrogenase for mannitol oxidation, and NADH oxidase and alkyl hydroxyperoxide reductase for NAD + regeneration. PSCat was treated at 40 °C to inactivate the psychrophilic enzymes responsible for byproduct formation and to increase the membrane permeability of the substrate. PSCat efficiently converted mannitol to fructose with high conversion yield without additional input of NAD + . Konbu extract containing mannitol was converted to fructose with hydroperoxide scavenging, inhibiting the mannitol dehydrogenase activity. Auranthiochytrium sp. could grow well in the presence of fructose converted by PSCat. Thus, PSCat is a potential carbohydrate converter for mannitol non-assimilating microorganism. Copyright © 2017 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

Angiotensin-I-Converting Enzyme (ACE)-Inhibitory Peptides from Plants

PubMed Central

Daskaya-Dikmen, Ceren; Yucetepe, Aysun; Karbancioglu-Guler, Funda; Daskaya, Hayrettin; Ozcelik, Beraat

2017-01-01

Hypertension is an important factor in cardiovascular diseases. Angiotensin-I-converting enzyme (ACE) inhibitors like synthetic drugs are widely used to control hypertension. ACE-inhibitory peptides from food origins could be a good alternative to synthetic drugs. A number of plant-based peptides have been investigated for their potential ACE inhibitor activities by using in vitro and in vivo assays. These plant-based peptides can be obtained by solvent extraction, enzymatic hydrolysis with or without novel food processing methods, and fermentation. ACE-inhibitory activities of peptides can be affected by their structural characteristics such as chain length, composition and sequence. ACE-inhibitory peptides should have gastrointestinal stability and reach the cardiovascular system to show their bioactivity. This paper reviews the current literature on plant-derived ACE-inhibitory peptides including their sources, production and structure, as well as their activity by in vitro and in vivo studies and their bioavailability. PMID:28333109

The angiotensin converting enzyme inhibitor captopril protects nigrostriatal dopamine neurons in animal models of parkinsonism

PubMed Central

Sonsalla, Patricia K.; Coleman, Christal; Wong, Lai-Yoong; Harris, Suzan L.; Richardson, Jason R.; Gadad, Bharathi S.; Li, Wenhao; German, Dwight C.

2013-01-01

Parkinson’s disease (PD) is a progressive neurodegenerative disorder characterized by a prominent loss of nigrostriatal dopamine (DA) neurons with an accompanying neuroinflammation. The peptide angiotensin II (AngII) plays a role in oxidative-stress induced disorders and is thought to mediate its detrimental actions via activation of AngII AT1 receptors. The brain renin-angiotensin system is implicated in neurodegenerative disorders including PD. Blockade of the angiotensin converting enzyme or AT1 receptors provides protection in acute animal models of parkinsonism. We demonstrate here that treatment of mice with the angiotensin converting enzyme inhibitor captopril protects the striatum from acutely administered 1-methyl-4-phenyl-1,2,3,6-tetrahydropyrine (MPTP), and that chronic captopril protects the nigral DA cell bodies from degeneration in a progressive rat model of parkinsonism created by the chronic intracerebral infusion of 1-methyl-4-phenylpyridinium (MPP+). The accompanying activation of microglia in the substantia nigra of MPP+-treated rats was reduced by the chronic captopril treatment. These findings indicate that captopril is neuroprotective for nigrostriatal DA neurons in both acute and chronic rodent PD models. Targeting the brain AngII pathway may be a feasible approach to slowing neurodegeneration in PD. PMID:24184050

Application of carbohydrate arrays coupled with mass spectrometry to detect activity of plant-polysaccharide degradative enzymes from the fungus Aspergillus niger.

PubMed

van Munster, Jolanda M; Thomas, Baptiste; Riese, Michel; Davis, Adrienne L; Gray, Christopher J; Archer, David B; Flitsch, Sabine L

2017-02-21

Renewables-based biotechnology depends on enzymes to degrade plant lignocellulose to simple sugars that are converted to fuels or high-value products. Identification and characterization of such lignocellulose degradative enzymes could be fast-tracked by availability of an enzyme activity measurement method that is fast, label-free, uses minimal resources and allows direct identification of generated products. We developed such a method by applying carbohydrate arrays coupled with MALDI-ToF mass spectrometry to identify reaction products of carbohydrate active enzymes (CAZymes) of the filamentous fungus Aspergillus niger. We describe the production and characterization of plant polysaccharide-derived oligosaccharides and their attachment to hydrophobic self-assembling monolayers on a gold target. We verify effectiveness of this array for detecting exo- and endo-acting glycoside hydrolase activity using commercial enzymes, and demonstrate how this platform is suitable for detection of enzyme activity in relevant biological samples, the culture filtrate of A. niger grown on wheat straw. In conclusion, this versatile method is broadly applicable in screening and characterisation of activity of CAZymes, such as fungal enzymes for plant lignocellulose degradation with relevance to biotechnological applications as biofuel production, the food and animal feed industry.

STRUCTURAL CHARACTERISTICS AND ANTIHYPERTENSIVE EFFECTS OF ANGIOTENSIN-I-CONVERTING ENZYME INHIBITORY PEPTIDES IN THE RENIN-ANGIOTENSIN AND KALLIKREIN KININ SYSTEMS

PubMed Central

Manoharan, Sivananthan; Shuib, Adawiyah Suriza; Abdullah, Noorlidah

2017-01-01

Background: The commercially available synthetic angiotensin-I-converting enzyme (ACE) inhibitors are known to exert negative side effects which have driven many research groups globally to discover the novel ACE inhibitors. Method: Literature search was performed within the PubMed, ScienceDirect.com and Google Scholar. Results: The presence of proline at the C-terminal tripeptide of ACE inhibitor can competitively inhibit the ACE activity. The effects of other amino acids are less studied leading to difficulties in predicting potent peptide sequences. The broad specificity of the enzyme may be due to the dual active sites observed on the somatic ACE. The inhibitors may not necessarily competitively inhibit the enzyme which explains why some reported inhibitors do not have the common ACE inhibitor characteristics. Finally, the in vivo assay has to be carried out before the peptides as the antihypertensive agents can be claimed. The peptides must be absorbed into circulation without being degraded, which will affect their bioavailability and potency. Thus, peptides with strong in vitro IC50 values do not necessarily have the same effect in vivo and vice versa. Conclusion: The relationship between peptide amino acid sequence and inhibitory activity, in vivo studies of the active peptides and bioavailability must be studied before the peptides as antihypertensive agents can be claimed. PMID:28573254

Targeted enzyme prodrug therapies.

PubMed

Schellmann, N; Deckert, P M; Bachran, D; Fuchs, H; Bachran, C

2010-09-01

The cure of cancer is still a formidable challenge in medical science. Long-known modalities including surgery, chemotherapy and radiotherapy are successful in a number of cases; however, invasive, metastasized and inaccessible tumors still pose an unresolved and ongoing problem. Targeted therapies designed to locate, detect and specifically kill tumor cells have been developed in the past three decades as an alternative to treat troublesome cancers. Most of these therapies are either based on antibody-dependent cellular cytotoxicity, targeted delivery of cytotoxic drugs or tumor site-specific activation of prodrugs. The latter is a two-step procedure. In the first step, a selected enzyme is accumulated in the tumor by guiding the enzyme or its gene to the neoplastic cells. In the second step, a harmless prodrug is applied and specifically converted by this enzyme into a cytotoxic drug only at the tumor site. A number of targeting systems, enzymes and prodrugs were investigated and improved since the concept was first envisioned in 1974. This review presents a concise overview on the history and latest developments in targeted therapies for cancer treatment. We cover the relevant technologies such as antibody-directed enzyme prodrug therapy (ADEPT), gene-directed enzyme prodrug therapy (GDEPT) as well as related therapies such as clostridial- (CDEPT) and polymer-directed enzyme prodrug therapy (PDEPT) with emphasis on prodrug-converting enzymes, prodrugs and drugs.

Discovery of novel hydroxamates as highly potent tumor necrosis factor-[alpha] converting enzyme inhibitors. Part II: Optimization of the S3′ pocket

DOE Office of Scientific and Technical Information (OSTI.GOV)

Mazzola Jr., Robert D.; Zhu, Zhaoning; Sinning, Lisa

2010-10-01

A series of cyclopropyl hydroxamic acids were prepared. Many of the compounds displayed picomolar affinity for the TACE enzyme while maintaining good to excellent selectivity profiles versus MMP-1, -2, -3, -7, -14, and ADAM-10. X-ray analysis of an inhibitor in the TACE active site indicated that the molecules bound to the enzyme in the S1{prime}-S3{prime} pocket.

Effect of SQ29,852, a new angiotensin converting enzyme (ACE) inhibitor with a phosphonic acid group, on the activity of angiotensin converting enzyme from human kidney.

PubMed

Hiwada, K; Inoue, Y; Kokubu, T

1990-01-01

1. An in vitro experiment was carried out to compare the inhibitory effect of SQ29,852 on human renal angiotensin converting enzyme (ACE) with those of captopril, enalapril and enalaprilat. 2. SQ29,852 strongly inhibited human renal ACE; its IC50 value was 1.5 x 10(-8) M. In terms of the IC50, SQ29,852's efficacy was about 1/10 of that of captopril and 1/28 of that of enalaprilat, but it was about 14 times more potent than enalapril. 3. SQ29,852 showed no inhibitory effects on cathepsin D, urinary kallikrein, renal renin, pepsin, trypsin and chymotrypsin. Its ACE-specificity was higher than that of captopril. 4. ACE inhibition by SQ29,852 was shown to be competitive, as revealed by Lineweaver-Burk plots. The affinity of SQ29,852 to ACE was shown to be high by a Ki value of 1.2 x 10(-8) M.

Separation and Characterization of Angiotensin I Converting Enzyme (ACE) Inhibitory Peptides from Saurida elongata Proteins Hydrolysate by IMAC-Ni2.

PubMed

Sun, Lixia; Wu, Shanguang; Zhou, Liqin; Wang, Feng; Lan, Xiongdiao; Sun, Jianhua; Tong, Zhangfa; Liao, Dankui

2017-02-15

Lizard fish protein hydrolysates (LFPH) were prepared from Lizard fish ( Saurida elongata ) proteins possessing powerful angiotensin I converting enzyme (ACE) inhibitory activity and the fraction (LFPH-I) with high ACE inhibitory activity was obtained through ultrafiltration. The active Fraction (F2) was isolated from LFPH-I using immobilized metal affinity chromatography (IMAC - Ni 2+ ). Analysis of amino acid levels revealed that F2 eluted from IMAC was enriched in Met, His, Tyr, Pro, Ile, and Leu compared to the crude peptide LFPH-I. F2 with the high ACE inhibitory activity (IC 50 of 0.116 mg·mL -1 ) was further separated by a reverse-phase column to yield a novel ACE inhibitory peptide with IC 50 value of 52 μM. The ACE inhibitory peptide was identified as Arg-Tyr-Arg-Pro, RYRP. The present study demonstrated that IMAC may be a useful tool for the separation of ACE inhibitory peptides from protein hydrolysate.

Isolation and structural analysis of antihypertensive peptides that exist naturally in Gouda cheese.

PubMed

Saito, T; Nakamura, T; Kitazawa, H; Kawai, Y; Itoh, T

2000-07-01

Seven kinds of ripened cheeses (8-mo-aged and 24-mo-aged Gouda, Emmental, Blue, Camembert, Edam, and Havarti) were homogenized with distilled water, and water-soluble peptides were prepared by C-18 hydrophobic chromatography. The inhibitory activity to angiotensin I-converting enzyme and decrease in the systolic blood pressure in spontaneously hypertensive rats were measured before and after oral administration of each peptide sample. The strongest depressive effect in the systolic blood pressure (-24.7 mm Hg) and intensive inhibitory activity to angiotensin I-converting enzyme (75.7%) were detected in the peptides from 8-mo-aged Gouda cheese. Four peptides were isolated by HPLC with reverse-phase and gel filtration modes. Their chemical structures and origins, clarified by combination analyses of protein sequencing, amino acid composition, and mass spectrometry, were as follows: peptide A, Arg-Pro-Lys-His-Pro-Ile-Lys-His-Gln [alpha(s1)-casein (CN), B-8P; f 1-9]; peptide B, Arg-Pro-Lys-His-Pro-Ile-Lys-His-Gln-Gly-Leu-Pro-Gln (alpha(s1)-CN, B-8P; f 1-13); peptide F, Tyr-Pro-Phe-Pro-Gly-Pro-Ile-Pro-Asn (beta-CN, A2-5P; f 60-68); and peptide G, Met-Pro-Phe-Pro-Lys-Tyr-Pro-Val-Gln-Pro-Phe (beta-CN, A2-5P; f 109-119). Peptides A and F, which were chemically synthesized, showed potent angiotensin I-converting enzyme inhibitory activity with little antihypertensive effects.

Characterization of Paenibacillus sp. MBT213 Isolated from Raw Milk and Its Ability to Convert Ginsenoside Rb1 into Ginsenoside Rd from Panax ginseng

PubMed Central

Cho, Soo Hyun; Park, Young W; Song, Gyu Yong

2017-01-01

This study was conducted to isolate and characterize Paenibacillus sp. MBT213 possessing β-glucosidase activity from raw milk, and examine the enzymatic capacity on the hydrolysis of a major ginsenoside (Rb1). Strain MBT213 was found to have a high hydrolytic ability on ginsenoside Rb1 by Esculin Iron Agar test. 16S rDNA analysis revealed that MBT213 was Paenibacillu sp. Crude enzyme of MBT213 strain exhibited high conversion capacity on ginsenoside Rb1 into ginsenoside Rd proven by TLC and HPLC analyses. The API ZYM kit confirmed that Paenibacillu sp. MBT213 exerted higher β-glucosidase and β-galactosidase activity than other strains. Optimum pH and temperature for crude enzyme were found at 7.0 and 35°C in hydrolysis of ginsenoside Rb1. After 10 d of optimal reaction conditions for the crude enzyme, ginsenoside Rb1 fully converted to ginsenoside Rd. Ginseng roots (20%) were fermented for 14 d, and analyzed by HPLC showed that amount of ginsenoside Rb1 significantly decreased, while that of ginsenoside Rd was significantly increased. The study confirmed that the β-glucosidase produced by Paenibacillus sp. MBT213 can hydrolyze the major ginsenoside Rb1 and convert to Rd during fermentation of the ginseng. The β-glucosidase activity of this novel Paenibacillus sp. MBT213 strain may be utilized in development of variety of health foods, dairy foods and pharmaceutical products. PMID:29147097

Angiotensin converting enzyme 2 activity and human atrial fibrillation: increased plasma angiotensin converting enzyme 2 activity is associated with atrial fibrillation and more advanced left atrial structural remodelling.

PubMed

Walters, Tomos E; Kalman, Jonathan M; Patel, Sheila K; Mearns, Megan; Velkoska, Elena; Burrell, Louise M

2017-08-01

Angiotensin converting enzyme 2 (ACE2) is an integral membrane protein whose main action is to degrade angiotensin II. Plasma ACE2 activity is increased in various cardiovascular diseases. We aimed to determine the relationship between plasma ACE2 activity and human atrial fibrillation (AF), and in particular its relationship to left atrial (LA) structural remodelling. One hundred and three participants from a tertiary arrhythmia centre, including 58 with paroxysmal AF (PAF), 20 with persistent AF (PersAF), and 25 controls, underwent clinical evaluation, echocardiographic analysis, and measurement of plasma ACE2 activity. A subgroup of 20 participants underwent invasive LA electroanatomic mapping. Plasma ACE2 activity levels were increased in AF [control 13.3 (9.5-22.3) pmol/min/mL; PAF 16.9 (9.7-27.3) pmol/min/mL; PersAF 22.8 (13.7-33.4) pmol/min/mL, P = 0.006]. Elevated plasma ACE2 was associated with older age, male gender, hypertension and vascular disease, elevated left ventricular (LV) mass, impaired LV diastolic function and advanced atrial disease (P < 0.05 for all). Independent predictors of elevated plasma ACE2 activity were AF (P = 0.04) and vascular disease (P < 0.01). There was a significant relationship between elevated ACE2 activity and low mean LA bipolar voltage (adjusted R2 = 0.22, P = 0.03), a high proportion of complex fractionated electrograms (R2 = 0.32, P = 0.009) and a long LA activation time (R2 = 0.20, P = 0.04). Plasma ACE2 activity is elevated in human AF. Both AF and vascular disease predict elevated plasma ACE2 activity, and elevated plasma ACE2 is significantly associated with more advanced LA structural remodelling. Published on behalf of the European Society of Cardiology. All rights reserved. © The Author 2016. For permissions please email: journals.permissions@oup.com.

Printable enzyme-embedded materials for methane to methanol conversion

DOE PAGES

Blanchette, Craig D.; Knipe, Jennifer M.; Stolaroff, Joshuah K.; ...

2016-06-15

An industrial process for the selective activation of methane under mild conditions would be highly valuable for controlling emissions to the environment and for utilizing vast new sources of natural gas. The only selective catalysts for methane activation and conversion to methanol under mild conditions are methane monooxygenases (MMOs) found in methanotrophic bacteria; however, these enzymes are not amenable to standard enzyme immobilization approaches. Using particulate methane monooxygenase (pMMO), we create a biocatalytic polymer material that converts methane to methanol. We demonstrate embedding the material within a silicone lattice to create mechanically robust, gas-permeable membranes, and direct printing of micron-scalemore » structures with controlled geometry. Remarkably, the enzymes retain up to 100% activity in the polymer construct. The printed enzyme-embedded polymer motif is highly flexible for future development and should be useful in a wide range of applications, especially those involving gas–liquid reactions.« less

Printable enzyme-embedded materials for methane to methanol conversion

DOE Office of Scientific and Technical Information (OSTI.GOV)

Blanchette, Craig D.; Knipe, Jennifer M.; Stolaroff, Joshuah K.

An industrial process for the selective activation of methane under mild conditions would be highly valuable for controlling emissions to the environment and for utilizing vast new sources of natural gas. The only selective catalysts for methane activation and conversion to methanol under mild conditions are methane monooxygenases (MMOs) found in methanotrophic bacteria; however, these enzymes are not amenable to standard enzyme immobilization approaches. Using particulate methane monooxygenase (pMMO), we create a biocatalytic polymer material that converts methane to methanol. We demonstrate embedding the material within a silicone lattice to create mechanically robust, gas-permeable membranes, and direct printing of micron-scalemore » structures with controlled geometry. Remarkably, the enzymes retain up to 100% activity in the polymer construct. The printed enzyme-embedded polymer motif is highly flexible for future development and should be useful in a wide range of applications, especially those involving gas–liquid reactions.« less

Printable enzyme-embedded materials for methane to methanol conversion

PubMed Central

Blanchette, Craig D.; Knipe, Jennifer M.; Stolaroff, Joshuah K.; DeOtte, Joshua R.; Oakdale, James S.; Maiti, Amitesh; Lenhardt, Jeremy M.; Sirajuddin, Sarah; Rosenzweig, Amy C.; Baker, Sarah E.

2016-01-01

An industrial process for the selective activation of methane under mild conditions would be highly valuable for controlling emissions to the environment and for utilizing vast new sources of natural gas. The only selective catalysts for methane activation and conversion to methanol under mild conditions are methane monooxygenases (MMOs) found in methanotrophic bacteria; however, these enzymes are not amenable to standard enzyme immobilization approaches. Using particulate methane monooxygenase (pMMO), we create a biocatalytic polymer material that converts methane to methanol. We demonstrate embedding the material within a silicone lattice to create mechanically robust, gas-permeable membranes, and direct printing of micron-scale structures with controlled geometry. Remarkably, the enzymes retain up to 100% activity in the polymer construct. The printed enzyme-embedded polymer motif is highly flexible for future development and should be useful in a wide range of applications, especially those involving gas–liquid reactions. PMID:27301270

Helodermatine, a kallikrein-like, hypotensive enzyme from the venom of Heloderma horridum horridum (Mexican beaded lizard)

PubMed Central

1986-01-01

We have purified and characterized the major N-benzoyl-L-arginine ethyl ester hydrolase from the venom of Heloderma horridum horridum. The enzyme belongs to the serine proteinase family, and its activity vs. peptide amide substrates and human high-molecular-weight kininogen suggests a similarity to the family of kallikreins. This interpretation is corroborated by its reactivity with the natural inhibitors soybean trypsin inhibitor and Kunitz-type bovine pancreatic trypsin inhibitor (aprotinin). Injection of the enzyme (2-16 micrograms/kg) into anesthetized rabbits leads to a rapid dose-dependent transient decrease of the arterial blood pressure. Like glandular kallikrein it specifically converts single-chain tissue type plasminogen activator into its double chain form. In contrast to other kallikrein-like enzymes from snake venoms it shows no thrombin-like or plasminogen activator activity. The enzyme is a single-chain glycoprotein (Mr 63,000). The N-terminal sequence revealed significant homology to pig pancreatic kallikrein and to kallikrein like enzymes from Crotalus atrox and Crotalus adamanteus venom. This enzyme, which we name Helodermatine, is the first purified from Sauria with kallikrein-like properties. PMID:3537191

Molecular diversity of tuliposide B-converting enzyme in tulip (Tulipa gesneriana): identification of the root-specific isozyme.

PubMed

Nomura, Taiji; Ueno, Ayaka; Ogita, Shinjiro; Kato, Yasuo

2017-06-01

6-Tuliposide B (PosB) is a glucose ester accumulated in tulip (Tulipa gesneriana) as a major secondary metabolite. PosB serves as the precursor of the antimicrobial lactone tulipalin B (PaB), which is formed by PosB-converting enzyme (TCEB). The gene TgTCEB1, encoding a TCEB, is transcribed in tulip pollen but scarcely transcribed in other tissues (e.g. roots) even though those tissues show high TCEB activity. This led to the prediction of the presence of a TCEB isozyme with distinct tissue specificity. Herein, we describe the identification of the TgTCEB-R gene from roots via native enzyme purification; this gene is a paralog of TgTCEB1. Recombinant enzyme characterization verified that TgTCEB-R encodes a TCEB. Moreover, TgTCEB-R was localized in tulip plastids, as found for pollen TgTCEB1. TgTCEB-R is transcribed almost exclusively in roots, indicating a tissue preference for the transcription of TCEB isozyme genes.

Redirection of pyruvate flux toward desired metabolic pathways through substrate channeling between pyruvate kinase and pyruvate-converting enzymes in Saccharomyces cerevisiae.

PubMed

Kim, Sujin; Bae, Sang-Jeong; Hahn, Ji-Sook

2016-04-07

Spatial organization of metabolic enzymes allows substrate channeling, which accelerates processing of intermediates. Here, we investigated the effect of substrate channeling on the flux partitioning at a metabolic branch point, focusing on pyruvate metabolism in Saccharomyces cerevisiae. As a platform strain for the channeling of pyruvate flux, PYK1-Coh-Myc strain was constructed in which PYK1 gene encoding pyruvate kinase is tagged with cohesin domain. By using high-affinity cohesin-dockerin interaction, the pyruvate-forming enzyme Pyk1 was tethered to heterologous pyruvate-converting enzymes, lactate dehydrogenase and α-acetolactate synthase, to produce lactic acid and 2,3-butanediol, respectively. Pyruvate flux was successfully redirected toward desired pathways, with a concomitant decrease in ethanol production even without genetic attenuation of the ethanol-producing pathway. This pyruvate channeling strategy led to an improvement of 2,3-butanediol production by 38%, while showing a limitation in improving lactic acid production due to a reduced activity of lactate dehydrogenase by dockerin tagging.

Gallium 67 citrate scanning and serum angiotensin converting enzyme levels in sarcoidosis

DOE Office of Scientific and Technical Information (OSTI.GOV)

Gupta, R.G.; Bekerman, C.; Sicilian, L.

1982-09-01

Gallium 67 citrate scans and serum angiotension converting enzyme (ACE) levels were obtained in 54 patients with sarcoidosis and analyzed in relation to clinical manifestation. /sup 67/Ga scans were abnormal in 97% of patients with clinically active disease (n = 30) and in 71% of patients with inactive disease (n = 24). Serum ACE levels were abnormally high (2 standard deviations above the control mean) in 73% of patients with clinically active disease and in 54% of patients with inactive disease. Serum ACE levels correlated significantly with /sup 67/Ga uptake score (r = .436; p < .005). The frequency ofmore » abnormal /sup 67/Ga scans and elevated serum ACE levels suggests that inflammatory activity with little or no clinical expression is common in sarcoidosis. Abnormal /sup 67/Ga scans were highly sensitive (97%) but had poor specificity (29%) to clinical disease activity. The accuracy of negative prediction of clinical activity by normal scans (87%) was better than the accuracy of positive prediction of clinical activity by abnormal scans (63%). /sup 67/Ga scans can be used to support the clinical identification of inactive sarcoidosis.« less

Investigation into the Mechanism of Homo- and Heterodimerization of Angiotensin-Converting Enzyme.

PubMed

Abrie, J Albert; Moolman, Wessel J A; Cozier, Gyles E; Schwager, Sylva L; Acharya, K Ravi; Sturrock, Edward D

2018-04-01

Angiotensin-converting enzyme (ACE) plays a central role in the renin-angiotensin system (RAS), which is primarily responsible for blood pressure homeostasis. Studies have shown that ACE inhibitors yield cardiovascular benefits that cannot be entirely attributed to the inhibition of ACE catalytic activity. It is possible that these benefits are due to interactions between ACE and RAS receptors that mediate the protective arm of the RAS, such as angiotensin II receptor type 2 (AT 2 R) and the receptor MAS. Therefore, in this study, we investigated the molecular interactions of ACE, including ACE homodimerization and heterodimerization with AT 2 R and MAS, respectively. Molecular interactions were assessed by fluorescence resonance energy transfer and bimolecular fluorescence complementation in human embryonic kidney 293 cells and Chinese hamster ovary-K1 cells transfected with vectors encoding fluorophore-tagged proteins. The specificity of dimerization was verified by competition experiments using untagged proteins. These techniques were used to study several potential requirements for the germinal isoform of angiotensin-converting enzyme expressed in the testes (tACE) dimerization as well as the effect of ACE inhibitors on both somatic isoforms of angiotensin-converting enzyme expressed in the testes (sACE) and tACE dimerization. We demonstrated constitutive homodimerization of sACE and of both of its domains separately, as well as heterodimerization of both sACE and tACE with AT 2 R, but not MAS. In addition, we investigated both soluble sACE and the sACE N domain using size-exclusion chromatography-coupled small-angle X-ray scattering and we observed dimers in solution for both forms of the enzyme. Our results suggest that ACE homo- and heterodimerization does occur under physiologic conditions. Copyright © 2018 by The American Society for Pharmacology and Experimental Therapeutics.

The Evaluation of Dipeptidyl Peptidase (DPP)-IV, α-Glucosidase and Angiotensin Converting Enzyme (ACE) Inhibitory Activities of Whey Proteins Hydrolyzed with Serine Protease Isolated from Asian Pumpkin (Cucurbita ficifolia).

PubMed

Konrad, Babij; Anna, Dąbrowska; Marek, Szołtysik; Marta, Pokora; Aleksandra, Zambrowicz; Józefa, Chrzanowska

2014-01-01

In the present study, whey protein concentrate (WPC-80) and β-lactoglobulin were hydrolyzed with a noncommercial serine protease isolated from Asian pumpkin ( Cucurbita ficifolia ). Hydrolysates were further fractionated by ultrafiltration using membranes with cut-offs equal 3 and 10 kDa. Peptide fractions of molecular weight lower than 3 and 3-10 kDa were further subjected to the RP-HPLC. Separated preparations were investigated for their potential as the natural inhibitors of dipeptidyl peptidase (DPP-IV), α-glucosidase and angiotensin converting enzyme (ACE). WPC-80 hydrolysate showed higher inhibitory activities against the three tested enzymes than β-lactoglobulin hydrolysate. Especially high biological activities were exhibited by peptide fractions of molecular weight lower than 3 kDa, with ACE IC50 <0.64 mg/mL and DPP-IV IC50 <0.55 mg/mL. This study suggests that peptides generated from whey proteins may support postprandial glycemia regulation and blood pressure maintenance, and could be used as functional food ingredients in the diet of patients with type 2 diabetes.

Antioxidation, angiotensin converting enzyme inhibition activity, nattokinase, and antihypertension of Bacillus subtilis (natto)-fermented pigeon pea.

PubMed

Lee, Bao-Hong; Lai, Yi-Syuan; Wu, She-Ching

2015-12-01

Because of the high incidence of cardiovascular diseases in Asian countries, traditional fermented foods from Asia have been increasingly investigated for antiatherosclerotic effects. This study investigated the production of nattokinase, a serine fibrinolytic enzyme, in pigeon pea by Bacillus subtilis fermentation. B. subtilis 14714, B. subtilis 14715, B. subtilis 14716, and B. subtilis 14718 were employed to produce nattokinase. The highest nattokinase activity in pigeon pea was obtained using B. subtilis 14715 fermentation for 32 hours. In addition, the levels of antioxidants (phenolics and flavonoids) and angiotensin converting enzyme inhibitory activity were increased in B. subtilis 14715-fermented pigeon pea, compared with those in nonfermented pigeon pea. In an animal model, we found that both water extracts of pigeon pea (100 mg/kg body weight) and water extracts of B. subtilis-fermented pigeon pea (100 mg/kg body weight) significantly improved systolic blood pressure (21 mmHg) and diastolic blood pressure (30 mmHg) in spontaneously hypertensive rats. These results suggest that Bacillus-fermented pigeon pea has benefits for cardiovascular health and can be developed as a new dietary supplement or functional food that prevents hypertension. Copyright © 2015. Published by Elsevier B.V.

Fructose 1,6-diphosphatase in striated muscle

PubMed Central

Krebs, H. A.; Woodford, Muriel

1965-01-01

1. The occurrence of fructose diphosphatase in muscle tissue was investigated with reference to the question whether lactate can be converted into glycogen in muscle, as postulated by Meyerhof (1930), fructose diphosphatase being one of the enzymes required for this conversion. 2. Fructose diphosphatase was found in skeletal muscle of man, dog, cat, rat, mouse, rabbit, guinea pig, cattle, sheep, pigeon, fowl and frog. Under the test conditions between 5 and 60 μmoles of substrate were split/g. fresh wt./hr. at 22°. 3. Like liver fructose diphosphatase, the muscle enzyme is inhibited by substrate concentrations above 0·1 mm, by AMP and by trace quantities of Zn2+, Fe2+ and Fe3+; it is `activated' by EDTA. Inhibitions by the above agents may account for the failure of previous authors to detect the enzyme. 4. Heart muscle of several vertebrate species and the smooth muscle of pigeon and fowl gizzard had no measurable activity. 5. The presence of fructose diphosphatase and the virtual absence of the enzyme systems converting pyruvate into phosphopyruvate means that lactate and pyruvate cannot be converted into glycogen in muscle, whereas the phosphorylated C3 compounds can. The reconversion into carbohydrate of lactate (which readily diffuses out of muscle) occurs in liver and kidney only. The reconversion of phosphorylated C3 intermediates (which cannot diffuse out of the tissue) can occur only within the muscle. 6. α-Glycerophosphate is probably the main intermediate requiring conversion into glycogen. The possible role of α-glycerophosphate formation in vertebrate muscle, already well established in insect muscle, is discussed. PMID:14346089

Marketing research on the angiotensin-converting enzyme inhibitors antihypertensive medicines.

PubMed

Boboia, Anamaria; Grigorescu, Marius Rareş; Turcu-Ştiolică, Adina

2017-01-01

The research aimed at investigating sales trends of angiotensin-converting enzyme inhibitors antihypertensive medicines, both in terms of quantity and value, in ten community pharmacies, for a period of three years. The research on the antihypertensive medicines consumption is important for highlighting the ever increasing impact of hypertension among the population. The methods used in this research were the following: marketing research, method of sampling, descriptive methods, retrospective analysis, method of comparison. The results showed that the drugs containing the active substances of the angiotensin converting enzyme inhibitors class had had significant increases in quantitative and value sales, bringing substantial revenues to pharmacies. From the quantitative perspective, the best-selling products were those containing Enalaprilum, while in terms of value, the best-selling medicines were those containing Perindoprilum. We evidenced that spectacular sales were also achieved for products that have Lisinoprilum, respectively Captoprilum, as active substances. The largest quantities were marketed for the Captopril Terapia® product and the highest earnings were recorded for the Prestarium® medicine. This paper approaches an interesting and topical issue, which can be helpful to professionals (pharmacists, doctors) and other categories, such as economists, statisticians, representatives of companies manufacturing medicines, as well as to hypertensive patients, as it could be used to warn population regarding the incidence of cardiovascular diseases, and, at the same time, trace sales trends in order to accomplish profitable business plans.

Injections of angiotensin-converting enzyme2 inhibitor MLN4760 into nucleus tractus solitarii reduce baroreceptor reflex sensitivity for heart rate control in rats

PubMed Central

Diz, Debra I.; Garcia-Espinosa, Maria A.; Gegick, Stephen; Tommasi, Ellen N.; Ferrario, Carlos M.; Tallant, E. Ann; Chappell, Mark C.; Gallagher, Patricia E.

2009-01-01

Injections of the angiotensin(1–7) [Ang(1–7)] antagonist [d-Ala7]-Ang(1–7) into the nucleus of the solitary tract (NTS) of Sprague–Dawley rats reduce baroreceptor reflex sensitivity (BRS) for control of heart rate by ~40%, whereas injections of the angiotensin II (Ang II) type 1 receptor antagonist candesartan increase BRS by 40% when reflex bradycardia is assessed. The enzyme angiotensin-converting enzyme 2 (ACE2) is known to convert Ang II to Ang(1–7). We report that ACE2 activity, as well as ACE and neprilysin activities, are present in plasma membrane fractions of the dorsomedial medulla of Sprague–Dawley rats. Moreover, we show that BRS for reflex bradycardia is attenuated (1.16±0.29 ms mmHg−1 before versus 0.33±0.11 ms mmHg−1 after; P < 0.05; n = 8) 30–60 min following injection of the selective ACE2 inhibitor MLN4760 (12 pmol in 120 nl) into the NTS. These findings support the concept that within the NTS, local synthesis of Ang(1–7) from Ang II is required for normal sensitivity for the baroreflex control of heart rate in response to increases in arterial pressure. PMID:18356558

Angiotensin-converting enzyme inhibitor (enalapril maleate) accelerates recovery of mouse skin from UVB-induced wrinkles

DOE Office of Scientific and Technical Information (OSTI.GOV)

Matsuura-Hachiya, Yuko; Arai, Koji Y.; Ozeki, Rieko

Highlights: •Angiotensin converting enzyme (ACE) increases in UVB-irradiated skin. •Administration of an ACE inhibitor improved UVB-induced skin wrinkle. •ACE inhibitor improved UVB-induced epidermal hypertrophy. •ACE inhibitor improved transepidermal water loss in the UVB-irradiated skin. -- Abstract: Angiotensin-converting enzyme (ACE) activity and angiotensin II signaling regulate cell proliferation, differentiation, and tissue remodeling, as well as blood pressure, while in skin, angiotensin II signaling is involved in wound healing, inflammation, and pathological scar formation. Therefore, we hypothesized that angiotensin II is also involved in photoaging of skin. In this study, we examined the effect of enalapril maleate, an ACE inhibitor, on recoverymore » of wrinkled skin of hairless mice exposed to long-term UVB irradiation. Immunohistochemical observation revealed that expression of ACE, angiotensin II, and angiotensin II type 1 (AT1) and type 2 (AT2) receptors in the skin was increased after UVB irradiation (3 times/week at increasing intensities for 8 weeks). Administration of enalapril maleate (5 times/week for 6 weeks, starting 1 week after 10-week irradiation) accelerated recovery from UVB-induced wrinkles, epidermal hyperplasia and epidermal barrier dysfunction, as compared with the vehicle control. Our results indicate that ACE and angiotensin II activity are involved in skin photoaging, and suggest that ACE inhibitor such as enalapril maleate may have potential for improvement of photoaged skin.« less

Review of Angiotensin-converting Enzyme Inhibitory Assay: Rapid Method in Drug Discovery of Herbal Plants

PubMed Central

Ahmad, Islamudin; Yanuar, Arry; Mulia, Kamarza; Mun’im, Abdul

2017-01-01

The renin-angiotensin-aldosterone system is a signaling pathway which responsible in the blood pressure regulation. Angiotensin-converting enzyme (ACE) is one of the key elements responsible for the hypertensive mechanism. It converts angiotensin-I to angiotensin-II. The discovery history of the ACE inhibitory activity assay method has been through a long stage for decades and development continues until today. The ACE inhibitory activity has become an effective screening method in the search for new antihypertensive agents from herbal plants. Some of in vitro assay methods were used to examine the activity of ACE inhibitors based on the substrate usage, such as; Cushman and Cheung Method using a substrate hippuryl-histidyl-leucine (HHL), Holmquist method using a substrate furanacryloyl-tripeptide, Elbl and Wagner method using a substrate benzoil-[l-14C] glicyl-L-histidine-L-leucine, Carmel and Yaron method using a substrate o-aminobenzoylglycyl-p-nitrophenylalanilproline, and Lam method using 3-hydroxybutyrylglycyl-glycyl-glycine as substrate. Several different methods to measure the results of enzymatic reactions or separating substrate with products, including spectrophotometric, fluorometric, high-performance liquid chromatography, electrophoresis, and radiochemistry. Application of the test method for screening the ACE inhibitors activity and investigation of active compounds from natural products can be done easily with this method, it is very helpful in research because the results obtained are simple, accurate, and rapid. PMID:28503045

Implications of the angiotensin converting enzyme gene insertion/deletion polymorphism in health and disease: a snapshot review

PubMed Central

Gard, Paul R

2010-01-01

This review considers the 250+ papers concerning the association of the angiotensin converting enzyme (ACE) gene insertion/deletion polymorphism (rs1799752) and various disease conditions published in 2009. The deletion allele occurs in approximately 55% of the population and is associated with increased activity of the ACE enzyme. It might be predicted that the D allele, therefore, might be associated with pathologies involving increased activity of the renin-angiotensin system. The D allele was seen to be associated with an increased risk of hypertension, pre-eclampsia, heart failure, cerebral infarct, diabetic nephropathy, encephalopathy, asthma, severe hypoglycaemia in diabetes, gastric cancer (in Caucasians) and poor prognosis following kidney transplant. On the positive side, the D allele appears to offer protection against schizophrenia and chronic periodontitis and confers greater up-per-body strength in old age. The I allele, meanwhile, offers improved endurance/athletic performance and aerobic capacity as determined by lung function tests, although it does increase the risk of oral squamous cell carcinoma and obstructive sleep apnoea in hypertensives. PMID:21537387

Optimization of Bromelain-Aided Production of Angiotensin I-Converting Enzyme Inhibitory Hydrolysates from Stone Fish Using Response Surface Methodology.

PubMed

Muhammad Auwal, Shehu; Zarei, Mohammad; Abdul-Hamid, Azizah; Saari, Nazamid

2017-03-31

The stone fish is an under-utilized sea cucumber with many nutritional and ethno-medicinal values. This study aimed to establish the conditions for its optimum hydrolysis with bromelain to generate angiotensin I-converting enzyme (ACE)-inhibitory hydrolysates. Response surface methodology (RSM) based on a central composite design was used to model and optimize the degree of hydrolysis (DH) and ACE-inhibitory activity. Process conditions including pH (4-7), temperature (40-70 °C), enzyme/substrate (E/S) ratio (0.5%-2%) and time (30-360 min) were used. A pH of 7.0, temperature of 40 °C, E/S ratio of 2% and time of 240 min were determined using a response surface model as the optimum levels to obtain the maximum ACE-inhibitory activity of 84.26% at 44.59% degree of hydrolysis. Hence, RSM can serve as an effective approach in the design of experiments to improve the antihypertensive effect of stone fish hydrolysates, which can thus be used as a value-added ingredient for various applications in the functional foods industries.

Characterization of angiotensin-I converting enzyme inhibiting peptide from Venerupis philippinarum with nano-liquid chromatography in combination with orbitrap mass spectrum detection and molecular docking

NASA Astrophysics Data System (ADS)

Shi, Lei; Wu, Tizhi; Sheng, Naijuan; Yang, Li; Wang, Qian; Liu, Rui; Wu, Hao

2017-06-01

The complexity and diversity of peptide mixture from protein hydrolysates make their characterization difficult. In this study, a method combining nano LC-MS/MS with molecular docking was applied to identifying and characterizing a peptide with angiotensin-I converting enzyme (ACE-I) inhibiting activity from Venerupis philippinarum hydrolysate. Firstly, ethanol supernatant of V. philippinarum hydrolysate was separated into active fractions with chromatographic methods such as ion-exchange chromatography and high performance liquid chromatography in combination. Then seven peptides from active fraction were identified according to the searching result of the MS/MS spectra against protein databases. Peptides were synthesized and subjected to ACE-I-inhibition assay. The peptide NTLTLIDTGIGMTK showed the highest potency with an IC50 of 5.75 μmol L-1. The molecular docking analysis showed that the ACE-I inhibiting peptide NTLTLIDTGIGMTK bond with residues Glu123, Glu403, Arg522, Glu376, Gln281 and Asn285 of ACE-I. Therefore, active peptides could be identified with the present method rather than the traditional purification and identification strategies. It may also be feasible to identify other food-derived peptides which target other enzymes and receptors with the method developed in this study.

Styrene Oxide Isomerase of Rhodococcus opacus 1CP, a Highly Stable and Considerably Active Enzyme

PubMed Central

Gröning, Janosch A. D.; Tischler, Dirk; Kaschabek, Stefan R.; Schlömann, Michael

2012-01-01

Styrene oxide isomerase (SOI) is involved in peripheral styrene catabolism of bacteria and converts styrene oxide to phenylacetaldehyde. Here, we report on the identification, enrichment, and biochemical characterization of a novel representative from the actinobacterium Rhodococcus opacus 1CP. The enzyme, which is strongly induced during growth on styrene, was shown to be membrane integrated, and a convenient procedure was developed to highly enrich the protein in active form from the wild-type host. A specific activity of about 370 U mg−1 represents the highest activity reported for this enzyme class so far. This, in combination with a wide pH and temperature tolerance, the independence from cofactors, and the ability to convert a spectrum of substituted styrene oxides, makes a biocatalytic application imaginable. First, semipreparative conversions were performed from which up to 760 μmol of the pure phenylacetaldehyde could be obtained from 130 U of enriched SOI. Product concentrations of up to 76 mM were achieved. However, due to the high chemical reactivity of the aldehyde function, SOI was shown to be the subject of an irreversible product inhibition. A half-life of 15 min was determined at a phenylacetaldehyde concentration of about 55 mM, indicating substantial limitations of applicability and the need to modify the process. PMID:22504818

Application of carbohydrate arrays coupled with mass spectrometry to detect activity of plant-polysaccharide degradative enzymes from the fungus Aspergillus niger

PubMed Central

van Munster, Jolanda M.; Thomas, Baptiste; Riese, Michel; Davis, Adrienne L.; Gray, Christopher J.; Archer, David B.; Flitsch, Sabine L.

2017-01-01

Renewables-based biotechnology depends on enzymes to degrade plant lignocellulose to simple sugars that are converted to fuels or high-value products. Identification and characterization of such lignocellulose degradative enzymes could be fast-tracked by availability of an enzyme activity measurement method that is fast, label-free, uses minimal resources and allows direct identification of generated products. We developed such a method by applying carbohydrate arrays coupled with MALDI-ToF mass spectrometry to identify reaction products of carbohydrate active enzymes (CAZymes) of the filamentous fungus Aspergillus niger. We describe the production and characterization of plant polysaccharide-derived oligosaccharides and their attachment to hydrophobic self-assembling monolayers on a gold target. We verify effectiveness of this array for detecting exo- and endo-acting glycoside hydrolase activity using commercial enzymes, and demonstrate how this platform is suitable for detection of enzyme activity in relevant biological samples, the culture filtrate of A. niger grown on wheat straw. In conclusion, this versatile method is broadly applicable in screening and characterisation of activity of CAZymes, such as fungal enzymes for plant lignocellulose degradation with relevance to biotechnological applications as biofuel production, the food and animal feed industry. PMID:28220903

DOE Office of Scientific and Technical Information (OSTI.GOV)

Alhenc-Gelas, F.; Weare, J.A.; Johnson, R.L. Jr.

CE (converting enzyme) was purified from human lung, and antisera were raised in rabbits. Antisera inhibited the activity of the purified enzyme from lung and kidney and the plasma CE of normal persons and sarcoid patients. With antisera at a titer of 1:100,000, a sensitive, direct RIA was developed. CE purified from lung or kidney and CE present in normal and in sarcoid plasma gave parallel logit-log displacement lines, suggesting immunological identity. The level of CE in normal human plasma was 400 +/- 131 ng/ml. In untreated sarcoid patients, the enzyme level and activity increased in parallel. There was amore » negative correlation between enzyme level and diffusing capacity of the lung for CO in sarcoid patients. Synthetic inhibitors such as captopril or MK 421 did not interfere with the RIA, permitting enzyme levels to be monitored in patients undergoing acute inhibitor therapy. During administration of MK 421, CE activity was negligible and plasma levels of CE did not change. In contrast, renin activity increased eightfold during the inhibitor therapy.« less

Angiotensin-converting enzyme inhibitory activity in Mexican Fresco cheese.

PubMed

Torres-Llanez, M J; González-Córdova, A F; Hernandez-Mendoza, A; Garcia, H S; Vallejo-Cordoba, B

2011-08-01

The objective of this study was to evaluate if Mexican Fresco cheese manufactured with specific lactic acid bacteria (LAB) presented angiotensin I-converting enzyme inhibitory (ACEI) activity. Water-soluble extracts (3 kDa) obtained from Mexican Fresco cheese prepared with specific LAB (Lactococcus, Lactobacillus, Enterococcus, and mixtures: Lactococcus-Lactobacillus and Lactococcus-Enterococcus) were evaluated for ACEI activity. Specific peptide fractions with high ACEI were analyzed using reverse phase-HPLC coupled to mass spectrometry for determination of amino acid sequence. Cheese containing Enterococcus faecium or a Lactococcus lactis ssp. lactis-Enterococcus faecium mixture showed the largest number of fractions with ACEI activity and the lowest half-maximal inhibitory concentration (IC(50); <10 μg/mL). Various ACEI peptides derived from β-casein [(f(193-205), f(193-207), and f(193-209)] and α(S1)-casein [f(1-15), f(1-22), f(14-23), and f(24-34)] were found. The Mexican Fresco cheese manufactured with specific LAB strains produced peptides with potential antihypertensive activity. Copyright © 2011 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.

Antioxidative and angiotensin-I-converting enzyme inhibitory potential of a Pacific Hake ( Merluccius productus ) fish protein hydrolysate subjected to simulated gastrointestinal digestion and Caco-2 cell permeation.

PubMed

Samaranayaka, Anusha G P; Kitts, David D; Li-Chan, Eunice C Y

2010-02-10

Pacific hake fish protein hydrolysate (FPH) with promising chemical assay based antioxidative capacity was studied for in vitro angiotensin-I-converting enzyme (ACE)-inhibitory potential, intestinal cell permeability characteristics, and intracellular antioxidative potential using the Caco-2 cell model system. FPH showed substrate-type inhibition of ACE with IC(50) of 161 microg of peptides/mL. HPLC analysis revealed that different peptides were responsible for antioxidative and ACE-inhibitory activity. FPH inhibited 2,2'-azobis(2-amidinopropane) dihydrochloride-induced oxidation in Caco-2 cells at noncytotoxic concentrations. In vitro simulated gastrointestinal digestion increased (P < 0.05) antioxidative capacity; ACE-inhibitory activity of FPH remained unchanged, although individual peptide fractions showed decreased or no activity after digestion. Some FPH peptides passed through Caco-2 cells: the permeates showed 2,2'-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) radical scavenging activity but no ACE-inhibitory activity. These results suggest the potential for application of Pacific hake FPH to reduce oxidative processes in vivo. Further studies are needed to assess prospective antihypertensive effects.

Angiotensin-I-converting enzyme and its relatives

PubMed Central

Riordan, James F

2003-01-01

Angiotensin-I-converting enzyme (ACE) is a monomeric, membrane-bound, zinc- and chloride-dependent peptidyl dipeptidase that catalyzes the conversion of the decapeptide angiotensin I to the octapeptide angiotensin II, by removing a carboxy-terminal dipeptide. ACE has long been known to be a key part of the renin angiotensin system that regulates blood pressure, and ACE inhibitors are important for the treatment of hypertension. There are two forms of the enzyme in humans, the ubiquitous somatic ACE and the sperm-specific germinal ACE, both encoded by the same gene through transcription from alternative promoters. Somatic ACE has two tandem active sites with distinct catalytic properties, whereas germinal ACE, the function of which is largely unknown, has just a single active site. Recently, an ACE homolog, ACE2, has been identified in humans that differs from ACE in being a carboxypeptidase that preferentially removes carboxy-terminal hydrophobic or basic amino acids; it appears to be important in cardiac function. ACE homologs (also known as members of the M2 gluzincin family) have been found in a wide variety of species, even in those that neither have a cardiovascular system nor synthesize angiotensin. X-ray structures of a truncated, deglycosylated form of germinal ACE and a related enzyme from Drosophila have been reported, and these show that the active site is deep within a central cavity. Structure-based drug design targeting the individual active sites of somatic ACE may lead to a new generation of ACE inhibitors, with fewer side-effects than currently available inhibitors. PMID:12914653

Perindopril

MedlinePlus

... in a class of medications called angiotensin-converting enzyme (ACE) inhibitors. It makes blood flow more smoothly ... if you are allergic to perindopril, angiotensin-converting enzyme (ACE) inhibitors such as benazepril (Lotensin, in Lotrel), ...

Screening of Zulu medicinal plants for angiotensin converting enzyme (ACE) inhibitors.

PubMed

Duncan, A C; Jäger, A K; van Staden, J

1999-12-15

Twenty plants used by traditional healers in South Africa for the treatment of high blood pressure were investigated for their anti-hypertensive properties, utilizing the angiotensin converting enzyme assay. A hit rate of 65% was achieved, with the highest inhibition (97%) obtained by Adenopodia spicata leaves. A further seven plants exhibited an inhibition greater than 70% and five more over 50%. The leaves of the plants showed the greatest levels of inhibition. There was little difference in the overall hit rate between ethanolic and aqueous extracts, although in most cases there was a marked difference in activity between aqueous and ethanolic extracts from the same species. Plants exhibiting inhibition levels greater than 50% were further tested for the presence of tannins in order to eliminate possible false positives. Active plants that did not contain tannins were Agapanthus africanus, Agave americana, Clausena anisata, Dietes iridioides, Mesembruanthemum spp., Stangeria eriopus and Tulbaghia violacea.

Release of IL-1beta via IL-1beta-converting enzyme in a skin dendritic cell line exposed to 2,4-dinitrofluorobenzene.

PubMed

Matos, Teresa J; Jaleco, Sara P; Gonçalo, Margarida; Duarte, Carlos B; Lopes, M Celeste

2005-08-14

We used a mouse fetal skin dendritic cell line (FSDC) to study the effect of the strong allergen 2,4-dinitrofluorobenzene (DNFB) on interleukin (IL)-1beta release and IL-1beta receptor immunoreactivity. Stimulation with DNFB (30 minutes) increased IL-1 release without changing the mRNA levels of the protein. Furthermore, DNFB increased transiently the interleukin-1beta-converting enzyme (ICE) activity, as measured with its fluorogenic substrate Z-Tyr-Val-Ala-Asp-AFC. The ICE inhibitor Z-YVAD-FMK prevented the release of IL-1beta evoked by DNFB. Incubation of the cells with DNFB (30 minutes) strongly increased IL-1beta receptor immunoreactivity. The rapid effect of DNFB on the release of mature IL-1beta, without inducing an increase of IL-1beta mRNA in FSDC, suggests a posttranslational modification of pro-IL-1beta by ICE activity.

Biocatalysis for the production of industrial products and functional foods from rice and other agricultural produce.

PubMed

Akoh, Casimir C; Chang, Shu-Wei; Lee, Guan-Chiun; Shaw, Jei-Fu

2008-11-26

Many industrial products and functional foods can be obtained from cheap and renewable raw agricultural materials. For example, starch can be converted to bioethanol as biofuel to reduce the current demand for petroleum or fossil fuel energy. On the other hand, starch can also be converted to useful functional ingredients, such as high fructose and high maltose syrups, wine, glucose, and trehalose. The conversion process involves fermentation by microorganisms and use of biocatalysts such as hydrolases of the amylase superfamily. Amylases catalyze the process of liquefaction and saccharification of starch. It is possible to perform complete hydrolysis of starch by using the fusion product of both linear and debranching thermostable enzymes. This will result in saving energy otherwise needed for cooling before the next enzyme can act on the substrate, if a sequential process is utilized. Recombinant enzyme technology, protein engineering, and enzyme immobilization are powerful tools available to enhance the activity of enzymes, lower the cost of enzyme through large scale production in a heterologous host, increase their thermostability, improve pH stability, enhance their productivity, and hence making it competitive with the chemical processes involved in starch hydrolysis and conversions. This review emphasizes the potential of using biocatalysis for the production of useful industrial products and functional foods from cheap agricultural produce and transgenic plants. Rice was selected as a typical example to illustrate many applications of biocatalysis in converting low-value agricultural produce to high-value commercial food and industrial products. The greatest advantages of using enzymes for food processing and for industrial production of biobased products are their environmental friendliness and consumer acceptance as being a natural process.

Production, optimisation and characterisation of angiotensin converting enzyme inhibitory peptides from sea cucumber (Stichopus japonicus) gonad.

PubMed

Zhong, Chan; Sun, Le-Chang; Yan, Long-Jie; Lin, Yi-Chen; Liu, Guang-Ming; Cao, Min-Jie

2018-01-24

In this study, production of bioactive peptides with angiotensin converting enzyme (ACE) inhibitory activity from sea cucumber (Stichopus japonicus) gonad using commercial protamex was optimised by response surface methodology (RSM). As a result, the optimal condition to achieve the highest ACE inhibitory activity in sea cucumber gonad hydrolysate (SCGH) was hydrolysis for 1.95 h and E/S of 0.75%. For further characterisation, three individual peptides (EIYR, LF and NAPHMR) were purified and identified. The peptide NAPHMR showed the highest ACE inhibitory activity with IC 50 of 260.22 ± 3.71 μM. NAPHMR was stable against simulated gastrointestinal digestion and revealed no significant cytotoxicity toward Caco-2 cells. Molecular docking study suggested that Arg, His and Asn residues in NAPHMR interact with the S2 pocket or Zn 2+ binding motifs of ACE via hydrogen or π-bonds, potentially contributing to ACE inhibitory effect. Sea cucumber gonad is thus a potential resource to produce ACE inhibitory peptides for preparation of functional foods.

Angiotensin converting enzyme (ACE) inhibitory, antihypertensive and antihyperlipidaemic activities of protein hydrolysates from Rhopilema esculentum.

PubMed

Liu, Xin; Zhang, Miansong; Zhang, Chao; Liu, Changheng

2012-10-15

Angiotensin-converting enzyme (ACE) inhibitory, antihypertensive and antihyperlipidaemic activities of protein hydrolysates (RPH) from the jellyfish Rhopilema esculentum were investigated. R. esculentum was hydrolysed sequentially with pepsin and papain, and then the hydrolysate was ultrafiltered with a 2000 Da cut-off membrane. It was found that RPH contained high levels of Gly, Glu, Pro, Asp and Ala, having potential ACE inhibitory activity in vitro with an IC(50) of 1.28 mg/ml. It was also found that systolic blood pressure was reduced markedly in spontaneously hypertensive rats after single and chronic oral administration of RPH, indicating that RPH had an antihypertensive effect. In addition, oral administration of RPH decreased total serum cholesterol and triglyceride, and increased high-density lipoprotein cholesterol in rats fed with high-fat diet. These results indicate that RPH may prove to be a promising functional food for the prevention and treatment of hypertension and hyperlipidaemia. Copyright © 2012 Elsevier Ltd. All rights reserved.

Aβ-degrading enzymes: potential for treatment of Alzheimer disease.

PubMed

Miners, James Scott; Barua, Neil; Kehoe, Patrick Gavin; Gill, Steven; Love, Seth

2011-11-01

There is increasing evidence that deficient clearance of β-amyloid (Aβ) contributes to its accumulation in late-onset Alzheimer disease (AD). Several Aβ-degrading enzymes, including neprilysin (NEP), insulin-degrading enzyme, and endothelin-converting enzyme reduce Aβ levels and protect against cognitive impairment in mouse models of AD. The activity of several Aβ-degrading enzymes rises with age and increases still further in AD, perhaps as a physiological response to minimize the buildup of Aβ. The age- and disease-related changes in expression of more recently recognized Aβ-degrading enzymes (e.g. NEP-2 and cathepsin B) remain to be investigated, and there is strong evidence that reduced NEP activity contributes to the development of cerebral amyloid angiopathy. Regardless of the role of Aβ-degrading enzymes in the development of AD, experimental data indicate that increasing the activity of these enzymes (NEP in particular) has therapeutic potential in AD, although targeting their delivery to the brain remains a major challenge. The most promising current approaches include the peripheral administration of agents that enhance the activity of Aβ-degrading enzymes and the direct intracerebral delivery of NEP by convection-enhanced delivery. In the longer term, genetic approaches to increasing the intracerebral expression of NEP or other Aβ-degrading enzymes may offer advantages.

[The changes of serum angiotensin converting enzyme activity in Type 2 diabetes and its vascular complications].

PubMed

Wu, H; Qu, S; Zhang, Y; Deng, J; Li, J; Zhou, J

2000-09-01

This investigation was made with reference to the changes of serum angiotensin converting enzyme (SACE) activity in type 2 diabetes and its vascular complications. SACE activity was studied in 127 type 2 diabetic patients and 90 healthy persons by using a spectrophotometric assay. The results showed SACE activity was obviously higher in diabetic patients (459.51 +/- 175.85 U) than in healthy persons (321.14 +/- 121.27 U); SACE activity was significantly higher in type 2 diabetic patients with diabetic nephropathy (548.27 +/- 166.60 U) than in patients without diabetic nephropathy (383.2 +/- 139.00 U), but there was no difference between patients with microalbuminuria and macroalbuminuria; no statistical difference was detected in SACE activity between diabetic patients with diabetic retinopathy (465.64 +/- 178.93 U) and without retinopathy (449.07 +/- 170.04 U); SACE activity was not associated with the course of diabetes, blood pressure, blood lipid and blood glucose. These data suggest that raised SACE activity might only play a role in the initiation of type 2 diabetes and diabetic nephropathy, but not relate to the progress of diabetic nephropathy, the onset of diabetic retinopathy and hypertension.

Identification and characterization of gamma-glutamylamine cyclotransferase, an enzyme responsible for gamma-glutamyl-epsilon-lysine catabolism.

PubMed

Oakley, Aaron J; Coggan, Marjorie; Board, Philip G

2010-03-26

Gamma-glutamylamine cyclotransferase (GGACT) is an enzyme that converts gamma-glutamylamines to free amines and 5-oxoproline. GGACT shows high activity toward gamma-glutamyl-epsilon-lysine, derived from the breakdown of fibrin and other proteins cross-linked by transglutaminases. The enzyme adopts the newly identified cyclotransferase fold, observed in gamma-glutamylcyclotransferase (GGCT), an enzyme with activity toward gamma-glutamyl-alpha-amino acids (Oakley, A. J., Yamada, T., Liu, D., Coggan, M., Clark, A. G., and Board, P. G. (2008) J. Biol. Chem. 283, 22031-22042). Despite the absence of significant sequence identity, several residues are conserved in the active sites of GGCT and GGACT, including a putative catalytic acid/base residue (GGACT Glu(82)). The structure of GGACT in complex with the reaction product 5-oxoproline provides evidence for a common catalytic mechanism in both enzymes. The proposed mechanism, combined with the three-dimensional structures, also explains the different substrate specificities of these enzymes. Despite significant sequence divergence, there are at least three subfamilies in prokaryotes and eukaryotes that have conserved the GGCT fold and GGCT enzymatic activity.

Identification and Characterization of γ-Glutamylamine Cyclotransferase, an Enzyme Responsible for γ-Glutamyl-ϵ-lysine Catabolism*

PubMed Central

Oakley, Aaron J.; Coggan, Marjorie; Board, Philip G.

2010-01-01

γ-Glutamylamine cyclotransferase (GGACT) is an enzyme that converts γ-glutamylamines to free amines and 5-oxoproline. GGACT shows high activity toward γ-glutamyl-ϵ-lysine, derived from the breakdown of fibrin and other proteins cross-linked by transglutaminases. The enzyme adopts the newly identified cyclotransferase fold, observed in γ-glutamylcyclotransferase (GGCT), an enzyme with activity toward γ-glutamyl-α-amino acids (Oakley, A. J., Yamada, T., Liu, D., Coggan, M., Clark, A. G., and Board, P. G. (2008) J. Biol. Chem. 283, 22031–22042). Despite the absence of significant sequence identity, several residues are conserved in the active sites of GGCT and GGACT, including a putative catalytic acid/base residue (GGACT Glu82). The structure of GGACT in complex with the reaction product 5-oxoproline provides evidence for a common catalytic mechanism in both enzymes. The proposed mechanism, combined with the three-dimensional structures, also explains the different substrate specificities of these enzymes. Despite significant sequence divergence, there are at least three subfamilies in prokaryotes and eukaryotes that have conserved the GGCT fold and GGCT enzymatic activity. PMID:20110353

Benazepril and Hydrochlorothiazide

MedlinePlus

... in a class of medications called angiotensin-converting enzyme (ACE) inhibitors. It works by decreasing certain chemicals ... benazepril (Lotensin); hydrochlorothiazide (HCTZ, Microzide, Oretic); angiotensin-converting enzyme (ACE) inhibitors such as captopril (Capoten), enalapril (Vasotec, ...

Lisinopril and Hydrochlorothiazide

MedlinePlus

... in a class of medications called angiotensin-converting enzyme (ACE) inhibitors. It works by decreasing certain chemicals ... to lisinopril; hydrochlorothiazide (HCTZ, Microzide, Oretic); angiotensin-converting enzyme (ACE) inhibitors such as benazepril (Lotensin), captopril (Capoten), ...

Enalapril and Hydrochlorothiazide

MedlinePlus

... in a class of medications called angiotensin-converting enzyme (ACE) inhibitors. It works by decreasing certain chemicals ... in Vaseretic); hydrochlorothiazide (HCTZ, Microzide, Oretic); angiotensin-converting enzyme (ACE) inhibitors such as benazepril (Lotensin, in Lotrel), ...

Alterations in Circulatory and Renal Angiotensin-Converting Enzyme and Angiotensin-Converting Enzyme 2 in Fetal Programmed Hypertension

PubMed Central

Shaltout, Hossam A.; Figueroa, Jorge P.; Rose, James C.; Diz, Debra I.; Chappell, Mark C.

2009-01-01

Antenatal betamethasone treatment is a widely accepted therapy to accelerate lung development and improve survival in preterm infants. However, there are reports that infants who receive antenatal glucocorticoids exhibit higher systolic blood pressure in their early adolescent years. We have developed an experimental model of programming whereby the offspring of pregnant sheep administered clinically relevant doses of betamethasone exhibit elevated blood pressure. We tested the hypothesis as to whether alterations in angiotensin-converting enzyme (ACE), ACE2, and neprilysin in serum, urine, and proximal tubules are associated with this increase in mean arterial pressure. Male sheep were administered betamethasone (2 doses of 0.17 mg/kg, 24 hours apart) or vehicle at the 80th day of gestation and delivered at term. Sheep were instrumented at adulthood (1.8 years) for direct conscious recording of mean arterial pressure. Serum and urine were collected and proximal tubules isolated from the renal cortex. Betamethasone-treated animals had elevated mean arterial pressure (97±3 versus 83±2 mm Hg; P<0.05) and a 25% increase in serum ACE activity (48.4±7.0 versus 36.0±2.7 fmol/mL per minute) but a 40% reduction in serum ACE2 activity (18.8±1.2 versus 31.4±4.4 fmol/mL per minute). In isolated proximal tubules, ACE2 activity and expression were 50% lower in the treated sheep with no significant change in ACE or neprilysin activities. We conclude that antenatal steroid treatment results in the chronic alteration of ACE and ACE2 in the circulatory and tubular compartments, which may contribute to the higher blood pressure in this model of fetal programming-induced hypertension. PMID:19047579

Effect of angiotensin-converting enzyme inhibitors on vascular endothelial function in hypertensive patients after intensive periodontal treatment.

PubMed

Rubio, María C; Lewin, Pablo G; De la Cruz, Griselda; Sarudiansky, Andrea N; Nieto, Mauricio; Costa, Osvaldo R; Nicolosi, Liliana N

2016-04-01

There is a relation between vascular endothelial function, atherosclerotic disease, and inflammation. Deterioration of endothelial function has been observed twenty-four hours after intensive periodontal treatment. This effect may be counteracted by the action of angiotensin-converting enzyme inhibitors, which improve endothelial function. The aim of the present study was to evaluate vascular endothelial function after intensive periodontal treatment, in hypertensive patients treated with angiotensinconverting enzyme inhibitors. A prospective, longitudinal, comparative study involving repeated measurements was conducted. Fifty-two consecutive patients with severe periodontal disease were divided into two groups, one comprising hypertensive patients treated with converting enzyme inhibitors and the other comprising patients with no clinical signs of pathology and not receiving angiotensin-converting enzyme inhibitors. Endothelial function was assessed by measuring postischemic dilation of the humeral artery (baseline echocardiography Doppler), and intensive periodontal treatment was performed 24h later. Endothelial function was re-assessed 24h and 15 days after periodontal treatment. Results were analyzed using the SPSS 20 statistical software package. Student's t test and MANOVA were calculated and linear regression analysis with 95% confidence intervals and α<0.05 was performed. Arterial dilation at 24 hours was lower compared to baseline in both groups; values corresponding to the groups receiving angiotensin-converting enzyme inhibitors were 11.89 ± 4.87 vs. 7.30 ± 2.90% (p<0.01) and those corresponding to the group not receiving ACE inhibitors were 12.72 ± 4.62 vs. 3.56 ± 2.39 (p<0.001). The differences between groups were statistically significant (p<0.001). The increase in endothelial dysfunction after intensive periodontal treatment was significantly lower in hypertensive patients treated with angiotensin-converting enzyme inhibitors. Endothelial function improved 15 days after periodontal treatment, reaching baseline values. These results support the protective effect of angiotensin converting enzyme inhibitors on the endothelial function after intensive periodontal treatment. Sociedad Argentina de Investigación Odontológica.

Angiotensin-converting enzyme genetic polymorphism: its impact on cardiac remodeling

PubMed Central

de Albuquerque, Felipe Neves; Brandão, Andréa Araujo; da Silva, Dayse Aparecida; Mourilhe-Rocha, Ricardo; Duque, Gustavo Salgado; Gondar, Alyne Freitas Pereira; Neves, Luiza Maceira de Almeida; Bittencourt, Marcelo Imbroinise; Pozzan, Roberto; de Albuquerque, Denilson Campos

2014-01-01

Background The role of angiotensin-converting enzyme genetic polymorphisms as a predictor of echocardiographic outcomes on heart failure is yet to be established. The local profile should be identified so that the impact of those genotypes on the Brazilian population could be identified. This is the first study on exclusively non-ischemic heart failure over a follow-up longer than 5 years. Objective To determine the distribution of angiotensin-converting enzyme genetic polymorphism variants and their relation with echocardiographic outcome of patients with non-ischemic heart failure. Methods Secondary analysis of the medical records of 111 patients and identification of the angiotensin-converting enzyme genetic polymorphism variants, classified as DD (Deletion/Deletion), DI (Deletion/Insertion) or II (Insertion/Insertion). Results The cohort means were as follows: follow-up, 64.9 months; age, 59.5 years; male sex, 60.4%; white skin color, 51.4%; use of beta-blockers, 98.2%; and use of angiotensin-converting-enzyme inhibitors or angiotensin receptor blocker, 89.2%. The angiotensin-converting enzyme genetic polymorphism distribution was as follows: DD, 51.4%; DI, 44.1%; and II, 4.5%. No difference regarding the clinical characteristics or treatment was observed between the groups. The final left ventricular systolic diameter was the only isolated echocardiographic variable that significantly differed between the angiotensin-converting enzyme genetic polymorphisms: 59.2 ± 1.8 for DD versus 52.3 ± 1.9 for DI versus 59.2 ± 5.2 for II (p = 0.029). Considering the evolutionary behavior, all echocardiographic variables (difference between the left ventricular ejection fraction at the last and first consultation; difference between the left ventricular systolic diameter at the last and first consultation; and difference between the left ventricular diastolic diameter at the last and first consultation) differed between the genotypes (p = 0.024; p = 0.002; and p = 0.021, respectively). Conclusion The distribution of the angiotensin-converting enzyme genetic polymorphisms differed from that of other studies with a very small number of II. The DD genotype was independently associated with worse echocardiographic outcome, while the DI genotype, with the best echocardiographic profile (increased left ventricular ejection fraction and decreased left ventricular diameters). PMID:24270863

Angiotensin-converting enzyme genetic polymorphism: its impact on cardiac remodeling.

PubMed

Albuquerque, Felipe Neves de; Brandão, Andréa Araujo; Silva, Dayse Aparecida da; Mourilhe-Rocha, Ricardo; Duque, Gustavo Salgado; Gondar, Alyne Freitas Pereira; Neves, Luiza Maceira de Almeida; Bittencourt, Marcelo Imbroinise; Pozzan, Roberto; Albuquerque, Denilson Campos de

2014-01-01

The role of angiotensin-converting enzyme genetic polymorphisms as a predictor of echocardiographic outcomes on heart failure is yet to be established. The local profile should be identified so that the impact of those genotypes on the Brazilian population could be identified. This is the first study on exclusively non-ischemic heart failure over a follow-up longer than 5 years. To determine the distribution of angiotensin-converting enzyme genetic polymorphism variants and their relation with echocardiographic outcome of patients with non-ischemic heart failure. Secondary analysis of the medical records of 111 patients and identification of the angiotensin-converting enzyme genetic polymorphism variants, classified as DD (Deletion/Deletion), DI (Deletion/Insertion) or II (Insertion/Insertion). The cohort means were as follows: follow-up, 64.9 months; age, 59.5 years; male sex, 60.4%; white skin color, 51.4%; use of beta-blockers, 98.2%; and use of angiotensin-converting-enzyme inhibitors or angiotensin receptor blocker, 89.2%. The angiotensin-converting enzyme genetic polymorphism distribution was as follows: DD, 51.4%; DI, 44.1%; and II, 4.5%. No difference regarding the clinical characteristics or treatment was observed between the groups. The final left ventricular systolic diameter was the only isolated echocardiographic variable that significantly differed between the angiotensin-converting enzyme genetic polymorphisms: 59.2 ± 1.8 for DD versus 52.3 ± 1.9 for DI versus 59.2 ± 5.2 for II (p = 0.029). Considering the evolutionary behavior, all echocardiographic variables (difference between the left ventricular ejection fraction at the last and first consultation; difference between the left ventricular systolic diameter at the last and first consultation; and difference between the left ventricular diastolic diameter at the last and first consultation) differed between the genotypes (p = 0.024; p = 0.002; and p = 0.021, respectively). The distribution of the angiotensin-converting enzyme genetic polymorphisms differed from that of other studies with a very small number of II. The DD genotype was independently associated with worse echocardiographic outcome, while the DI genotype, with the best echocardiographic profile (increased left ventricular ejection fraction and decreased left ventricular diameters).

Angiotensin-converting enzyme activity and cognitive impairment during hypoglycaemia in healthy humans.

PubMed

Pedersen-Bjergaard, Ulrik; Thomsen, Carsten E; Høgenhaven, Hans; Smed, Annelise; Kjaer, Troels W; Holst, Jens J; Dela, Flemming; Hilsted, Linda; Frandsen, Erik; Pramming, Stig; Thorsteinsson, Birger

2008-03-01

In type 1 diabetes increased risk of severe hypoglycaemia is associated with high angiotensin-converting enzyme (ACE) activity. We tested in healthy humans the hypothesis that this association is explained by the reduced ability of subjects with high ACE activity to maintain normal cognitive function during hypoglycaemia. Sixteen healthy volunteers selected by either particularly high or low serum ACE activity were subjected to hypoglycaemia (plasma glucose 2.7 mmol/L). Cognitive function was assessed by choice reaction tests. Despite a similar hypoglycaemic stimulus in the two groups, only the group with high ACE activity showed significant deterioration in cognitive performance during hypoglycaemia. In the high ACE group mean reaction time (MRT) in the most complex choice reaction task was prolonged and error rate (ER) was increased in contrast to the low ACE group. The total hypoglycaemic symptom response was greater in the high ACE group than in the low ACE group (p=0.031). There were no differences in responses of counterregulatory hormones or in concentrations of substrates between the groups. Healthy humans with high ACE activity are more susceptible to cognitive dysfunction and report higher symptom scores during mild hypoglycaemia than subjects with low ACE activity.

Ferredoxin-thioredoxin reductase: a catalytically active dithiol group links photoreduced ferredoxin to thioredoxin functional in photosynthetic enzyme regulation.

PubMed

Droux, M; Miginiac-Maslow, M; Jacquot, J P; Gadal, P; Crawford, N A; Kosower, N S; Buchanan, B B

1987-07-01

The mechanism by which the ferredoxin-thioredoxin system activates the target enzyme, NADP-malate dehydrogenase, was investigated by analyzing the sulfhydryl status of individual protein components with [14C]iodoacetate and monobromobimane. The data indicate that ferredoxin-thioredoxin reductase (FTR)--an iron-sulfur enzyme present in oxygenic photosynthetic organisms--is the first member of a thiol chain that links light to enzyme regulation. FTR possesses a catalytically active dithiol group localized on the 13 kDa (similar) subunit, that occurs in all species investigated and accepts reducing equivalents from photoreduced ferredoxin and transfers them stoichiometrically to the disulfide form of thioredoxin m. The reduced thioredoxin m, in turn, reduces NADP-malate dehydrogenase, thereby converting it from an inactive (S-S) to an active (SH) form. The means by which FTR is able to combine electrons (from photoreduced ferredoxin) with protons (from the medium) to reduce its active disulfide group remains to be determined.

Ferredoxin-thioredoxin reductase: a catalytically active dithiol group links photoreduced ferredoxin to thioredoxin functional in photosynthetic enzyme regulation

DOE Office of Scientific and Technical Information (OSTI.GOV)

Droux, M.; Miginiac-Maslow, M.; Jacquot, J.P.

The mechanism by which the ferredoxin-thioredoxin system activates the target enzyme, NADP-malate dehydrogenase, was investigated by analyzing the sulfhydryl status of individual protein components with (/sup 14/C)iodoacetate and monobromobimane. The data indicate that ferredoxin-thioredoxin reductase (FTR)--an iron-sulfur enzyme present in oxygenic photosynthetic organisms--is the first member of a thiol chain that links light to enzyme regulation. FTR possesses a catalytically active dithiol group localized on the 13 kDa (similar) subunit, that occurs in all species investigated and accepts reducing equivalents from photoreduced ferredoxin and transfers them stoichiometrically to the disulfide form of thioredoxin m. The reduced thioredoxin m, in turn,more » reduces NADP-malate dehydrogenase, thereby converting it from an inactive (S-S) to an active (SH) form. The means by which FTR is able to combine electrons (from photoreduced ferredoxin) with protons (from the medium) to reduce its active disulfide group remains to be determined.« less

Elevated serum angiotensin-converting enzyme (SACE) activity in acute pulmonary histoplasmosis.

PubMed

Davies, S F; Rohrbach, M S; Thelen, V; Kuritsky, J; Gruninger, R; Simpson, M L; DeRemee, R A

1984-03-01

Serum angiotensin-converting enzyme (SACE) levels were measured in 44 subjects six weeks after acute pulmonary histoplasmosis. All patients were infected in a common-source outbreak of histoplasmosis which occurred on one day. All patients had both strictly defined clinical and serologic evidence of infection. The SACE activity was elevated at six weeks compared to normal controls, and seven of the 44 had levels more than 2 SD above the normal mean. SACE levels were also measured at three and 24 weeks after acute infection in a smaller number of the same subjects. Serial observations demonstrated that all subjects (including those with normal and elevated SACE at six weeks) had a rise and fall in SACE activity following symptomatic acute pulmonary histoplasmosis. Our findings suggest that elevated SACE does not reliably separate sarcoidosis from histoplasmosis, although elevations in histoplasmosis are much less common and may occur only briefly following acute pulmonary histoplasmosis. More important, it seems that SACE activity rises acutely in all patients with symptomatic acute histoplasmosis and then falls gradually toward baseline over several months, coinciding temporally with the granulomatous response.

Preparation and characterization of novel bioactive peptides responsible for angiotensin I-converting enzyme inhibition from wheat germ.

PubMed

Matsui, T; Li, C H; Osajima, Y

1999-07-01

Reported is the preparation of wheat germ (WG) hydrolyzate with potent angiotensin I-converting enzyme (ACE) inhibitory activity, and the characterization of peptides responsible for ACE inhibition. Successful hydrolyzate with the most potent ACE inhibitory activity was obtained by 0.5 wt.%-8 h Bacillus licheniformis alkaline protease hydrolysis after 3.0 wt.%-3 h alpha-amylase treatment of defatted WG (IC50; 0.37 mg protein ml(-1)). The activity of WG hydrolyzate was markedly increased by ODS and subsequent AG50W purifications (IC50; 0.018 mg protein ml(-1)). As a result of isolations by high performance liquid chromatographies, 16 peptides with the IC50 value of less than 20 microM, composed of 2-7 amino acid residues were identified from the WG hydrolyzate. Judging from the high content (260 mg in 100 g of AG50W fraction) and powerful ACE inhibitory activity (IC50; 0.48 microM), Ile-Val-Tyr was identified as a main contributor to the ACE inhibition of the hydrolyzate.

The evolution of renin-angiotensin blockade: angiotensin-converting enzyme inhibitors as the starting point.

PubMed

Sica, Domenic A

2010-04-01

The renin-angiotensin system has been a target in the treatment of hypertension for close to three decades. Several medication classes that block specific aspects of this system have emerged as useful therapies, including angiotensin-converting enzyme inhibitors, angiotensin receptor blockers, and, most recently, direct renin inhibitors. There has been a natural history to the development of each of these three drug classes, starting with their use as antihypertensive agents; thereafter, in each case they have been employed as end-organ protective agents. To date, there has been scant evidence to favor angiotensin receptor blockers or direct renin inhibitors over angiotensin-converting enzyme inhibitors in treating hypertension or in affording end-organ protection; thus, angiotensin-converting enzyme inhibitors remain the standard of care when renin-angiotensin system blockade is warranted.

Localization and characterization of angiotensin II receptor binding and angiotensin converting enzyme in the human medulla oblongata.

PubMed

Allen, A M; Chai, S Y; Clevers, J; McKinley, M J; Paxinos, G; Mendelsohn, F A

1988-03-08

Angiotensin II receptor and angiotensin converting enzyme distributions in the human medulla oblongata were localised by quantitative in vitro autoradiography. Angiotensin II receptors were labelled with the antagonist analogue 125I-[Sar1, Ile8] AII while angiotensin converting enzyme was labelled with 125I-351A, a derivative of the specific converting enzyme inhibitor, lisinopril. Angiotensin II receptor binding and angiotensin converting enzyme are present in high concentrations in the nucleus of the solitary tract, the dorsal motor nucleus of vagus, the rostral and caudal ventrolateral reticular nucleus, and in a band connecting the dorsal and ventral regions. In the rostral and caudal ventrolateral reticular nucleus, angiotensin II receptors are distributed in a punctate pattern that registers with neuronal cell bodies. The distribution and density of these cell bodies closely resemble those of catecholamine-containing neurones mapped by others. In view of the known interactions of angiotensin II with both central and peripheral catecholamine-containing neurons of laboratory animals, the current anatomical findings suggest similar interactions between these neuroactive compounds in the human central nervous system. The presence of angiotensin II receptors and angiotensin converting enzyme in the nucleus of the solitary tract, dorsal motor nucleus of vagus, and rostral and caudal ventrolateral reticular nucleus demonstrates sites for central angiotensin II to exert its known actions on vasopressin release and autonomic functions including blood pressure control. These data also suggest a possible interaction between angiotensin II and central catecholeminergic systems.

Application of α-N-acetylgalactosaminidase and α-galactosidase in AB to O red blood cells conversion.

PubMed

Gao, Hongwei; Li, Subo; Tan, Yingxia; Ji, Shouping; Wang, Yingli; Bao, Guoqiang; Xu, Lijuan; Gong, Feng

2013-02-01

Enzymatical conversion of A or B RBCs into group O RBCs (ECORBCs) was achieved by using α-N-acetylgalactosaminidase and α-galactosidase, respectively. Now, we initiated AB to O-RBC conversion by using these two enzymes together. But α-N-acetylgalactosaminidase and α-galactosidase's preserving and their reaction buffer were quite different. The aim of this study is to confirm an available system for converting AB to O RBCs, especially to study the maximal permission amount of PCS which was brought to the system-accompanied enzyme addition. Enzyme activity was detected by using GalNAc-pNp or Gal-pNp as substrates. The efficiency of the conversion of A or B antigen was evaluated by routine method and measured by fluorescence-activated cell sorting analysis. The optimal buffer component and the doses of α-N-acetylgalactosaminidase and α-galactosidase were confirmed according to A and B antigen epitope removal efficiency. The activity of α-N-acetylgalactosaminidase and α-galactosidase was not decreased drastically when they were kept in PCS Buffer in 4°C. The optimal reaction buffer composed of glycine 250 mM and NaCl 3 mM, pH 6.8 and PCS less than 10%(v/v). For converting A(1)B to O RBCs completely, the doses of α-N-acetylgalactosaminidase and α-galactosidase were confirmed as 0.015 mg/ml packed RBCs(pRBCs) for A(1) antigen epitopes and 0.005 mg/ml pRBCs for B epitopes. Approximately 0.004 mg α-N-acetylgalactosaminidase and 0.005 mg α-galactosidase were required to convert 1 ml pRBCs. Our studies indicated that α-N-acetylgalactosaminidase and α-galactosidase were stable in PCS buffer and a modified protocol which was propitious to converting AB to O RBCs was provided.

Major amyloid-β-degrading enzymes, endothelin-converting enzyme-2 and neprilysin, are expressed by distinct populations of GABAergic interneurons in hippocampus and neocortex.

PubMed

Pacheco-Quinto, Javier; Eckman, Christopher B; Eckman, Elizabeth A

2016-12-01

Impaired clearance of amyloid-β peptide (Aβ) has been postulated to significantly contribute to the amyloid accumulation typical of Alzheimer's disease. Among the enzymes known to degrade Aβ in vivo are endothelin-converting enzyme (ECE)-1, ECE-2, and neprilysin (NEP), and evidence suggests that they regulate independent pools of Aβ that may be functionally significant. To better understand the differential regulation of Aβ concentration by its physiological degrading enzymes, we characterized the cell and region-specific expression pattern of ECE-1, ECE-2, and NEP by in situ hybridization and immunohistochemistry in brain areas relevant to Alzheimer's disease. In contrast to the broader distribution of ECE-1, ECE-2 and NEP were found enriched in GABAergic neurons. ECE-2 was majorly expressed by somatostatin-expressing interneurons and was active in isolated synaptosomes. NEP messenger RNA was found mainly in parvalbumin-expressing interneurons, with NEP protein localized to perisomatic parvalbuminergic synapses. The identification of somatostatinergic and parvalbuminergic synapses as hubs for Aβ degradation is consistent with the possibility that Aβ may have a physiological function related to the regulation of inhibitory signaling. Copyright © 2016 Elsevier Inc. All rights reserved.

Characterization of gossypol biosynthetic pathway

PubMed Central

Tian, Xiu; Ruan, Ju-Xin; Huang, Jin-Quan; Fang, Xin; Chen, Zhi-Wen; Hong, Hui; Wang, Ling-Jian; Mao, Ying-Bo; Lu, Shan; Zhang, Tian-Zhen; Chen, Xiao-Ya

2018-01-01

Gossypol and related sesquiterpene aldehydes in cotton function as defense compounds but are antinutritional in cottonseed products. By transcriptome comparison and coexpression analyses, we identified 146 candidates linked to gossypol biosynthesis. Analysis of metabolites accumulated in plants subjected to virus-induced gene silencing (VIGS) led to the identification of four enzymes and their supposed substrates. In vitro enzymatic assay and reconstitution in tobacco leaves elucidated a series of oxidative reactions of the gossypol biosynthesis pathway. The four functionally characterized enzymes, together with (+)-δ-cadinene synthase and the P450 involved in 7-hydroxy-(+)-δ-cadinene formation, convert farnesyl diphosphate (FPP) to hemigossypol, with two gaps left that each involves aromatization. Of six intermediates identified from the VIGS-treated leaves, 8-hydroxy-7-keto-δ-cadinene exerted a deleterious effect in dampening plant disease resistance if accumulated. Notably, CYP71BE79, the enzyme responsible for converting this phytotoxic intermediate, exhibited the highest catalytic activity among the five enzymes of the pathway assayed. In addition, despite their dispersed distribution in the cotton genome, all of the enzyme genes identified show a tight correlation of expression. Our data suggest that the enzymatic steps in the gossypol pathway are highly coordinated to ensure efficient substrate conversion. PMID:29784821

Angiotensin II-producing enzyme III from acidified serum of nephrectomized dogs.

PubMed

Haas, E; Lewis, L; Koshy, T J; Varde, A U; Renerts, L; Bagai, R C

1989-09-01

A highly active angiotensin-producing enzyme (enzyme III) was obtained from the serum of bilaterally nephrectomized dogs by acid treatment and ammonium sulfate fractionation. An inactive precursor (proenzyme III) was converted to enzyme III during prolonged storage (or by treatment with acid or with cathepsin G or by incubation at 38 degrees C as described in the following paper). Enzyme III reacted maximally at pH 7.7 and it produced up to 400 ng of angiotensin II/mL serum/h (i.e., amounts 4000 times higher than that generated by the endogenous renin present in serum after bilateral nephrectomy). Enzyme III produced angiotensin II at identical rates when either dog angiotensinogen or angiotensin I was used as substrate, but the rate was 710 times higher with synthetic tetradecapeptide renin substrate. Enzyme III is not identical to renin, cathepsin G, tonin, enzyme I, enzyme II, the calcium-dependent angiotensin I-converting enzyme, or the calcium-independent carboxy peptidase, which acts by sequential cleavage of angiotensin I. Enzyme III was inhibited by alpha-1-antitrypsin, diisopropyl fluorophosphate, and lima bean trypsin inhibitor (hence it is a serine proteinase). It was not inhibited by Captopril, Teprotide, or Enalapril. It had been reported previously that cathepsin G released from neutrophil granulocytes, by producing high local concentrations of angiotensin II, may provide a mobile means for modulating blood flow in tissue microvasculature during the inflammatory response. The present study offers a new, additional pathway, by enzyme III, for a similar rapid formation of angiotensin II from serum protein substrate or angiotensin I.

/sup 67/Ga citrate scanning and serum angiotensin converting enzyme levels in sarcoidosis

DOE Office of Scientific and Technical Information (OSTI.GOV)

Gupta, R.G.; Bekerman, C.; Sicilian, L.

1982-09-01

/sup 67/Ga citrate scans and serum angiotensin converting enzyme (ACE) levels were obtained in 54 patients with sarcoidosis and analyzed in relation to clinical manifestations. /sup 67/Ga scans were abnormal in 97% of patients with clinically active disease (n . 30) and in 71% of patients with inactive disease (n . 24). Serum ACE levels were abnormally high (2 standard deviations above the control mean) in 73% of patients with clinically active disease and in 54% of patients with inactive disease. Serum ACE levels correlated significantly with /sup 67/Ga uptake score (r..436; p less than .005). The frequency of abnormalmore » /sup 67/Ga scans and elevated serum ACE levels suggests that inflammatory activity with little or no clinical expression is common in sarcoidosis. Abnormal /sup 67/Ga scans were highly sensitive (97%) but had poor specificity (29%) to clinical disease activity. The accuracy of negative prediction of clinical activity by normal scans (87%) was better than the accuracy of positive prediction of clinical activity by abnormal scans (63%). /sup 67/Ga scans can be used to support the clinical indentification of inactive sacoidosis.« less

Absence of diamine oxidase activity from rabbit and rat lungs.

PubMed Central

Rao, S B; Rao, K S; Mehendale, H M

1986-01-01

To study the presence of diamine oxidase (DAO) activity in any tissue with putrescine as the substrate, it is necessary to use inhibitors to block all pathways that could further metabolize gamma-aminobutyraldehyde, which is the product of enzyme reaction. It is also necessary to inhibit any enzyme that may convert putrescine into higher polyamines. By this approach it was observed that lung tissue of both rat and rabbit exhibited no DAO activity. DAO activity was observed in the rat and rabbit intestine, the former showing 3 times as much activity as the latter. The other potential pathways of putrescine metabolism are of no consequence in the rat and rabbit intestine and lungs. PMID:3087348

Expression and regulation of estrogen-converting enzymes in ectopic human endometrial tissue.

PubMed

Fechner, Sabine; Husen, Bettina; Thole, Hubert; Schmidt, Markus; Gashaw, Isabella; Kimmig, Rainer; Winterhager, Elke; Grümmer, Ruth

2007-10-01

To investigate the regulation of estrogen-converting enzymes in human ectopic endometrial tissue. Animal study. Academic medical center. Sixty female nude mice with implanted human endometrial tissue. Twenty-two premenopausal women undergoing endometrial biopsy or hysterectomy. Human endometrial tissue was implanted into the peritoneal cavity of nude mice, and the effect of therapeutic drugs on transcription of steroid receptors and estrogen-converting enzymes was analyzed. Transcript levels of steroid hormone receptors, 17beta-hydroxysteroid dehydrogenase type 1 and 2, aromatase, and steroid sulfatase as well as proliferation rate were analyzed in the human ectopic endometrial tissue. Steroid receptors and estrogen-converting enzymes were expressed in the ectopic human endometrial fragments. Application of medroxyprogesterone acetate, dydrogesterone, danazol, and the aromatase inhibitor finrozole significantly inhibited aromatase transcription. In addition, danazol caused a significant decrease in transcription of steroid sulfatase, and finrozole, of 17beta-hydroxysteroid dehydrogenase type 1 in parallel to a decrease in proliferation rate in the ectopic human endometrial tissue. Pharmacological regulation of transcription of estrogen-converting enzymes in human endometrium cultured in nude mice may help to develop new therapeutic concepts based on local regulation of estrogen metabolism in endometriosis.

Topographic effects on denitrification in drained agricultural fields

USDA-ARS?s Scientific Manuscript database

Denitrification is affected by soil moisture, while soil moisture can be affected by topography. Therefore, denitrification can be spatially correlated to topographic gradients. Three prior converted fields on the Delmarva Peninsula were sampled spatially for denitrification enzyme activity. The up...

Immobilized enzymes to convert N-sulfo, N-acetyl heparosan to a critical intermediate in the production of bioengineered heparin.

PubMed

Xiong, Jian; Bhaskar, Ujjwal; Li, Guoyun; Fu, Li; Li, Lingyun; Zhang, Fuming; Dordick, Jonathan S; Linhardt, Robert J

2013-09-10

Heparin is a critically important anticoagulant drug that is prepared from pig intestine. In 2007-2008, there was a crisis in the heparin market when the raw material was adulterated with the toxic polysaccharide, oversulfated chondroitin sulfate, which was associated with 100 deaths in the U.S. alone. As the result of this crisis, our laboratory and others have been actively pursuing alternative sources for this critical drug, including synthetic heparins and bioengineered heparin. In assessing the bioengineering processing costs it has become clear that the use of both enzyme-catalyzed cofactor recycling and enzyme immobilization will be needed for commercialization. In the current study, we examine the use of immobilization of C₅-epimerase and 2-O-sulfotransferase involved in the first enzymatic step in the bioengineered heparin process, as well as arylsulfotransferase-IV involved in cofactor recycling in all three enzymatic steps. We report the successful immobilization of all three enzymes and their use in converting N-sulfo, N-acetyl heparosan into N-sulfo, N-acetyl 2-O-sulfo heparin. Copyright © 2013 Elsevier B.V. All rights reserved.

Immobilization of xanthine oxidase on a polyaniline silicone support.

PubMed

Nadruz, W; Marques, E T; Azevedo, W M; Lima-Filho, J L; Carvalho, L B

1996-03-01

A polyaniline silicone support to immobilize xanthine oxidase is proposed as a reactor coil to monitor the action of xanthine oxidase on hypoxanthine, xanthine and 6-mercaptopurine. A purified xanthine oxidase immobilized on this support lost 80% of the initial activity after 12 min of use. Co-immobilization of superoxide dismutase and catalase increased the stability of immobilized xanthine oxidase so that the derivative maintained 79% of its initial activity after 4.6 h of continuous use in which 1.5 mumol purine bases were converted by the immobilized enzyme system. There is no evidence of either polyaniline or protein leaching from the coil during 3 h of continuous use. When solutions (10 ml) of hypoxanthine, xanthine and 6-mercaptopurine were circulated individually through the xanthine oxidase-superoxide dismutase-catalase-polyaniline coil (1 mm internal diameter and 3 m in length, 3 ml internal volume) activities of 8.12, 11.17 and 1.09 nmol min-1 coil-1, respectively, were obtained. The advantages of the reactor configuration and the redox properties of the polymer, particularly with respect to immobilized oxidoreductases, make this methodology attractive for similar enzyme systems. This immobilized enzyme system using polyaniline-silicone as support converted 6-mercaptopurine to 6-thiouric acid with equal efficiency as resins based on polyacrylamide and polyamide 11.

Inhibitory properties of bambara groundnut protein hydrolysate and peptide fractions against angiotensin-converting enzymes, renin and free radicals.

PubMed

Arise, Abimbola K; Alashi, Adeola M; Nwachukwu, Ifeanyi D; Malomo, Sunday A; Aluko, Rotimi E; Amonsou, Eric O

2017-07-01

An increased rate of high blood pressure has led to critical human hypertensive conditions in most nations. In the present study, bambara protein hydrolysates (BPHs) obtained using three different proteases (alcalase, trypsin and pepsin) and their peptide fractions (molecular weight: 10, 5, 3 and 1 kDa) were investigated for antihypertensive and antioxidant activities. Alcalase hydrolysate contained the highest amount of low molecular weight (LMW) peptides compared to pepsin and trypsin hydrolysates. LMW peptides fractions (<1 kDa) exhibited the highest inhibitory activity against angiotensin-converting enzyme (ACE) for all the enzymes hydrolysates. For renin inhibition, alcalase hydrolysate showed the highest inhibition at 59% compared to other hydrolysates and their corresponding membrane fractions. The antioxidant power of bambara protein hydrolysates and peptide fractions was evaluated through the inhibition of linoleic acid peroxidation and ABTS scavenging activity. Among the hydrolysates, alcalase exhibited the highest inhibition of linoleic acid oxidation. Furthermore, all BPHs were able to scavenge ABTS •+ to a three-fold greater extent compared to the isolate. BPH and LMW peptide fractions could potentially serve as useful ingredients in the formulation of functional foods and nutraceuticals against high blood pressure and oxidative stress. © 2016 Society of Chemical Industry. © 2016 Society of Chemical Industry.

Marketing research on the angiotensin-converting enzyme inhibitors antihypertensive medicines

PubMed Central

BOBOIA, ANAMARIA; GRIGORESCU, MARIUS RAREŞ; TURCU - ŞTIOLICĂ, ADINA

2017-01-01

Background and aims The research aimed at investigating sales trends of angiotensin-converting enzyme inhibitors antihypertensive medicines, both in terms of quantity and value, in ten community pharmacies, for a period of three years. The research on the antihypertensive medicines consumption is important for highlighting the ever increasing impact of hypertension among the population. Methods The methods used in this research were the following: marketing research, method of sampling, descriptive methods, retrospective analysis, method of comparison. Results The results showed that the drugs containing the active substances of the angiotensin converting enzyme inhibitors class had had significant increases in quantitative and value sales, bringing substantial revenues to pharmacies. From the quantitative perspective, the best-selling products were those containing Enalaprilum, while in terms of value, the best-selling medicines were those containing Perindoprilum. We evidenced that spectacular sales were also achieved for products that have Lisinoprilum, respectively Captoprilum, as active substances. The largest quantities were marketed for the Captopril Terapia® product and the highest earnings were recorded for the Prestarium® medicine. Conclusion This paper approaches an interesting and topical issue, which can be helpful to professionals (pharmacists, doctors) and other categories, such as economists, statisticians, representatives of companies manufacturing medicines, as well as to hypertensive patients, as it could be used to warn population regarding the incidence of cardiovascular diseases, and, at the same time, trace sales trends in order to accomplish profitable business plans. PMID:28246502

Molecular imprinting of enzymes with water-insoluble ligands for nonaqueous biocatalysis.

PubMed

Rich, Joseph O; Mozhaev, Vadim V; Dordick, Jonathan S; Clark, Douglas S; Khmelnitsky, Yuri L

2002-05-15

Attaining higher levels of catalytic activity of enzymes in organic solvents is one of the major challenges in nonaqueous enzymology. One of the most successful strategies for enhancing enzyme activity in organic solvents involves tuning the enzyme active site by molecular imprinting with substrates or their analogues. Unfortunately, numerous imprinters of potential importance are poorly soluble in water, which significantly limits the utility of this method. In the present study, we have developed strategies that overcome this limitation of the molecular-imprinting technique and that thus expand its applicability beyond water-soluble ligands. The solubility problem can be addressed either by converting the ligands into a water-soluble form or by adding relatively high concentrations of organic cosolvents, such as tert-butyl alcohol and 1,4-dioxane, to increase their solubility in the lyophilization medium. We have succeeded in applying both of these strategies to produce imprinted thermolysin, subtilisin, and lipase TL possessing up to 26-fold higher catalytic activity in the acylation of paclitaxel and 17beta-estradiol compared to nonimprinted enzymes. Furthermore, we have demonstrated for the first time that molecular imprinting and salt activation, applied in combination, produce a strong additive activation effect (up to 110-fold), suggesting different mechanisms of action involved in these enzyme activation techniques.

Genetically Engineered Materials for Biofuels Production

NASA Astrophysics Data System (ADS)

Raab, Michael

2012-02-01

Agrivida, Inc., is an agricultural biotechnology company developing industrial crop feedstocks for the fuel and chemical industries. Agrivida's crops have improved processing traits that enable efficient, low cost conversion of the crops' cellulosic components into fermentable sugars. Currently, pretreatment and enzymatic conversion of the major cell wall components, cellulose and hemicellulose, into fermentable sugars is the most expensive processing step that prevents widespread adoption of biomass in biofuels processes. To lower production costs we are consolidating pretreatment and enzyme production within the crop. In this strategy, transgenic plants express engineered cell wall degrading enzymes in an inactive form, which can be reactivated after harvest. We have engineered protein elements that disrupt enzyme activity during normal plant growth. Upon exposure to specific processing conditions, the engineered enzymes are converted into their active forms. This mechanism significantly lowers pretreatment costs and enzyme loadings (>75% reduction) below those currently available to the industry.

Focus on increased serum angiotensin-converting enzyme level: From granulomatous diseases to genetic mutations.

PubMed

Lopez-Sublet, Marilucy; di Lanzacco, Lorenzo Caratti; Jan Danser, A H; Lambert, Michel; Elourimi, Ghassan; Persu, Alexandre

2018-06-18

Angiotensin I-converting enzyme (ACE) is a well-known zinc-metallopeptidase that converts angiotensin I to the potent vasoconstrictor angiotensin II and degrades bradykinin, a powerful vasodilator, and as such plays a key role in the regulation of vascular tone and cardiac function. Increased circulating ACE (cACE) activity has been reported in multiple diseases, including but not limited to granulomatous disorders. Since 2001, genetic mutations leading to cACE elevation have also been described. This review takes advantage of the identification of a novel ACE mutation (25-IVS25 + 1G > A) in two Belgian pedigrees to summarize current knowledge about the differential diagnosis of cACE elevation, based on literature review and the experience of our centre. Furthermore, we propose a practical approach for the evaluation and management of patients with elevated cACE and discuss in which cases search for genetic mutations should be considered. Copyright © 2018. Published by Elsevier Inc.

State-of-the-art: Immunosuppression and biologic therapy.

PubMed

Sandborn, William J

2010-01-01

Azathioprine and 6-mercaptopurine are orally administered immunosuppressive drugs which are effective for the treatment of Crohn's disease and ulcerative colitis. Azathioprine is rapidly converted to 6-mercaptopurine after administration. 6-Mercaptopurine is then either converted to the putative active metabolites, the 6-thioguinine nucleotides, or inactivated by the enzyme xanthine oxidase to 6-thiouric acid or alternatively inactivated to 6-methylmercaptopurine by the enzyme thiopurine methyltransferase. Thiopurine methyltransferase activity is genetically determined, with one in 300 patients having low or absent enzyme activity, one in 10 patients having intermediate enzyme activity, and 9 in 10 patients having normal enzyme activity. Patients with intermediate or low thiopurine methyltransferase activity are at risk for early leukopenia. Higher erythrocyte 6-thioguinine nucleotide concentrations are associated with a greater likelihood of clinical response. Azathioprine is modestly effective for Crohn's disease and ulcerative colitis. Toxicity associated with azathioprine includes infection and lymphoma. Anti-TNF therapy with infliximab, adalimumab, and certolizumab pegol is effective for induction and maintenance treatment of Crohn's disease, and infliximab is effective for ulcerative colitis. Toxicity associated with anti-TNF therapy includes infection and lymphoma. Combination therapy with infliximab and azathioprine is more effective for inducing and maintaining steroid-free remission and mucosal healing then monotherapy with either drug alone. Strategies to reduce immunogenicity of anti-TNF agents include combination therapy with azathioprine and administration of a loading dose followed by systematic maintenance dosing. Higher serum trough concentrations of infliximab occur more frequently in patients receiving combination therapy with azathioprine and are associated with better clinical outcomes. Combination therapy is associated with an increased relative risk of opportunistic infection, but is not associated with an increased absolute risk of serious infection. Clinical practice should change such that combination therapy with an anti-TNF agent and azathioprine replace azathioprine in patients failing first line therapy with mesalamine and/or steroids. Copyright © 2010 S. Karger AG, Basel.

Ligand-binding specificity and promiscuity of the main lignocellulolytic enzyme families as revealed by active-site architecture analysis.

PubMed

Tian, Li; Liu, Shijia; Wang, Shuai; Wang, Lushan

2016-03-24

Biomass can be converted into sugars by a series of lignocellulolytic enzymes, which belong to the glycoside hydrolase (GH) families summarized in CAZy databases. Here, using a structural bioinformatics method, we analyzed the active site architecture of the main lignocellulolytic enzyme families. The aromatic amino acids Trp/Tyr and polar amino acids Glu/Asp/Asn/Gln/Arg occurred at higher frequencies in the active site architecture than in the whole enzyme structure. And the number of potential subsites was significantly different among different families. In the cellulase and xylanase families, the conserved amino acids in the active site architecture were mostly found at the -2 to +1 subsites, while in β-glucosidase they were mainly concentrated at the -1 subsite. Families with more conserved binding amino acid residues displayed strong selectivity for their ligands, while those with fewer conserved binding amino acid residues often exhibited promiscuity when recognizing ligands. Enzymes with different activities also tended to bind different hydroxyl oxygen atoms on the ligand. These results may help us to better understand the common and unique structural bases of enzyme-ligand recognition from different families and provide a theoretical basis for the functional evolution and rational design of major lignocellulolytic enzymes.

Structure and Mechanism of MbtI, the Salicylate Synthase from Mycobacterium tuberculosis

DOE Office of Scientific and Technical Information (OSTI.GOV)

Zwahlen,J.; Kolappan, S.; Zhou, R.

2007-01-01

MbtI (rv2386c) from Mycobacterium tuberculosis catalyzes the initial transformation in mycobactin biosynthesis by converting chorismate to salicylate. We report here the structure of MbtI at 2.5 {angstrom} resolution and demonstrate that isochorismate is a kinetically competent intermediate in the synthesis of salicylate from chorismate. At pH values below 7.5 isochorismate is the dominant product while above this pH value the enzyme converts chorismate to salicylate without the accumulation of isochorismate in solution. The salicylate and isochorismate synthase activities of MbtI are Mg{sup 2+}-dependent, and in the absence of Mg{sup 2+} MbtI has a promiscuous chorismate mutase activity similar to thatmore » of the isochorismate pyruvate lyase, PchB, from Pseudomonas aeruginosa. MbtI is part of a larger family of chorismate-binding enzymes descended from a common ancestor (the MST family), that includes the isochorismate synthases and anthranilate synthases. The lack of active site residues unique to pyruvate eliminating members of this family, combined with the observed chorismate mutase activity, suggests that MbtI may exploit a sigmatropic pyruvate elimination mechanism similar to that proposed for PchB. Using a combination of structural, kinetic, and sequence based studies we propose a mechanism for MbtI applicable to all members of the MST enzyme family.« less

Is substance P released from slices of the rat spinal cord inactivated by peptidase(s) distinct from both 'enkephalinase' and 'angiotensin-converting enzyme'?

PubMed

Mauborgne, A; Bourgoin, S; Benoliel, J J; Hamon, M; Cesselin, F

1991-02-25

Studies on the effects of peptidase inhibitors on substance P-like immunoreactive material (SPLI) released by K(+)-induced depolarization from slices of the rat spinal cord showed that bacitracin was the most potent agent to protect SPLI from degradation. Captopril and thiorphan which inhibit, respectively, angiotensin I converting enzyme and endopeptidase-24.11 also protected SPLI from degradation. However other inhibitors of these two enzymes, kelatorphan for endopeptidase-24.11 and enalaprilat for angiotensin I converting enzyme were essentially inactive, indicating that both enzymes are probably not involved in the degradation of endogenous substance P. Instead, the non-additive protecting effect of bacitracin, captopril and thiorphan might be due to the blockade of some 'bacitracin-sensitive enzyme' playing a key role in the catabolism of SP within the rat spinal cord.

Optimization of Bromelain-Aided Production of Angiotensin I-Converting Enzyme Inhibitory Hydrolysates from Stone Fish Using Response Surface Methodology

PubMed Central

Auwal, Shehu Muhammad; Zarei, Mohammad; Abdul-Hamid, Azizah; Saari, Nazamid

2017-01-01

The stone fish is an under-utilized sea cucumber with many nutritional and ethno-medicinal values. This study aimed to establish the conditions for its optimum hydrolysis with bromelain to generate angiotensin I-converting enzyme (ACE)-inhibitory hydrolysates. Response surface methodology (RSM) based on a central composite design was used to model and optimize the degree of hydrolysis (DH) and ACE-inhibitory activity. Process conditions including pH (4–7), temperature (40–70 °C), enzyme/substrate (E/S) ratio (0.5%–2%) and time (30–360 min) were used. A pH of 7.0, temperature of 40 °C, E/S ratio of 2% and time of 240 min were determined using a response surface model as the optimum levels to obtain the maximum ACE-inhibitory activity of 84.26% at 44.59% degree of hydrolysis. Hence, RSM can serve as an effective approach in the design of experiments to improve the antihypertensive effect of stone fish hydrolysates, which can thus be used as a value-added ingredient for various applications in the functional foods industries. PMID:28362352

DOE Office of Scientific and Technical Information (OSTI.GOV)

Nally, J.V. Jr.; Clarke, H.S. Jr.; Grecos, G.P.

In an effort to improve on the noninvasive detection of renal artery stenosis, we investigated the effect of angiotensin converting enzyme inhibition on computer-assisted /sup 99m/Tc-diethylenetriaminepentaacetic acid (DTPA) renal flow studies in a canine model of two-kidney, one clip hypertension and compared these findings with clearances of inulin and p-aminohippuric acid in the stenotic and contralateral kidney before and after converting enzyme inhibition. The /sup 99m/Tc-DTPA renal flow study with the converting enzyme inhibitor captopril (1.5 mg/kg bolus with 1.5 mg/min infusion) showed an increased sensitivity in the detection of unilateral renal artery stenosis over the use of the /supmore » 99m/Tc-DTPA study alone. Captopril induced striking alterations that were most evident in the 15-minute /sup 99m/Tc-DTPA renal flow study, in which all nine curves exhibited severely blunted uptake and excretion of the radionuclide. These changes were reversed during a recovery study without converting enzyme inhibition and were not seen when blood pressure was lowered with nitroprusside to a level similar to that observed during converting enzyme inhibition. The changes shown by the /sup 99m/Tc-DTPA study during converting enzyme inhibition correlated with a decrease in the glomerular filtration rate of the stenotic kidney. Captopril infusion significantly decreased the glomerular filtration rate of the stenotic kidney (16.0 +/- 3.1 vs 11.0 +/- 2.5 mg/min, p less than 0.03) but not of the contralateral kidney (32.4 +/- 2.6 vs 28.4 +/- 2.8 mg/min).« less

Angiotensin I converting enzyme inhibitory activity and antihypertensive effect in spontaneously hypertensive rats of cobia (Rachycentron canadum) head papain hydrolysate.

PubMed

Yang, Ping; Jiang, Yuchuan; Hong, Pengzhi; Cao, Wenhong

2013-06-01

Cobia head protein hydrolysate (CHPH) with angiotensin I converting enzyme (ACE) inhibitory activity was prepared with papain. The 3 kDa ultrafiltration filtrate CHPH-IV of the hydrolysate exerted a potent ACE inhibitory activity with IC50 being 0.24 mg/mL. The fractions with molecular weight located between 1749 Da and 173 Da represented up 66.96% of CHPH-IV, and those between 494 Da and 173 Da represented up 31.37% of CHPH-IV. It was found that the ACE inhibitory activity of CHPH-IV was intensified from IC50 0.24 mg/mL to 0.17 mg/mL after incubation with gastrointestinal proteases. The CHPH-IV significantly decreased the systolic blood pressure in a dose-dependent manner after oral administration to spontaneously hypertensive rats (SHR) at dose of 150 mg/kg, 600 mg/kg and 1200 mg/kg body weight. These results suggested that CHPH-IV from cobia head protein hydrolysate by papain could serve as a source of peptides with antihypertensive activity in functional food industry.

Angiotensin I-converting enzyme-inhibitory activity of the Norwegian autochthonous cheeses Gamalost and Norvegia after in vitro human gastrointestinal digestion.

PubMed

Qureshi, T M; Vegarud, G E; Abrahamsen, R K; Skeie, S

2013-02-01

The angiotensin I-converting enzyme (ACE) inhibitory activity of Gamalost cheese, its pH 4.6-soluble fraction, and Norvegia cheese was monitored before and after digestion with human gastric and duodenal juices. Both Gamalost and Norvegia cheeses showed an increased ACE-inhibitory activity during gastrointestinal digestion. However, only Norvegia showed pronounced increased activity after duodenal digestion. More peptides were detected in digested Gamalost compared with digested Norvegia. Most of the peptides in Gamalost were derived from β-casein (CN), some originated from α(s1)-CN, and only a very few originated from α(s2)-CN and κ-CN. In general, the number of peptides increased during gastrointestinal digestion, whereas some peptides were further degraded and disappeared; however, surprisingly, a few peptides remained stable. The aromatic amino acids, such as Tyr, Phe, and Trp; the positively charged amino acids (Arg and Lys); and Leu increased after simulated gastrointestinal digestion of Gamalost and Norvegia. After in vitro gastrointestinal digestion, both Gamalost and Norvegia showed high ACE-inhibitory activity, which may contribute in lowering of mild hypertension. Copyright © 2013 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.

Structural requirements of choline derivatives for 'conversion' of pneumococcal amidase. A new single-step procedure for purification of this autolysin.

PubMed

Sanz, J M; Lopez, R; Garcia, J L

1988-05-23

Tertiary amines appear to be the minimal structure needed to convert in vitro the inactive form (E-form) of pneumococcal amidase to the catalytic active form (C-form). Diethylethanolamine was one of the compounds that converted the E-form, a finding that has been used successfully to develop an affinity chromatography system in DEAE-cellulose for the rapid and efficient purification of lytic enzymes of pneumococcus and its bacteriophages.

Short communication: Assessing antihypertensive activity in native and model Queso Fresco cheeses.

PubMed

Paul, M; Van Hekken, D L

2011-05-01

Hispanic-style cheeses are one of the fastest growing varieties in the United States, making up approximately 2% of the total cheese production in this country. Queso Fresco is one of most popular Hispanic-style cheeses. Protein extracts from several varieties of Mexican Queso Fresco and model Queso Fresco were analyzed for potential antihypertensive activity. Many Quesos Frescos obtained from Mexico are made from raw milk and therefore the native microflora is included in the cheese-making process. Model Queso Fresco samples were made from pasteurized milk and did not utilize starter cultures. Water-soluble protein extracts from 6 Mexican Quesos Frescos and 12 model cheeses were obtained and assayed for their ability to inhibit angiotensin-converting enzyme, implying potential as foods that can help to lower blood pressure. All model cheeses displayed antihypertensive activity, but mainly after 8 wk of aging when they were no longer consumable, whereas the Mexican samples did display some angiotensin-converting enzyme inhibitory action after minimal aging. Copyright © 2011 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.

Bioavailability of angiotensin I-converting enzyme (ACE) inhibitory peptides derived from Virgibacillus halodenitrificans SK1-3-7 proteinases hydrolyzed tilapia muscle proteins.

PubMed

Toopcham, Tidarat; Mes, Jurriaan J; Wichers, Harry J; Roytrakul, Sittiruk; Yongsawatdigul, Jirawat

2017-04-01

The angiotensin I-converting enzyme (ACE) inhibitory activity of protein hydrolysates from tilapia muscle fractions, namely mince (M), washed mince (WM), and sarcoplasmic protein (SP), were investigated. Each fraction was hydrolyzed by Virgibacillus halodenitrificans SK1-3-7 proteinases for up to 24h. After 8h of hydrolysis, the M hydrolysate (48% degree of hydrolysis (DH)) showed the highest ACE inhibitory activity, with an IC 50 value of 0.54mg/ml, while the SP hydrolysate exhibited the lowest DH and ACE inhibition. In vitro gastrointestinal digestion reduced the ACE inhibitory activity of the M hydrolysate but enhanced its transport across Caco-2 cell monolayers. The transported peptides were found to contain 3-4 amino acid residues showing strong ACE inhibition. The novel ACE inhibitory peptide with the highest inhibition was found to be MCS, with an IC 50 value of 0.29μM. Therefore, tilapia mince hydrolyzed by V. halodenitrificans proteinases contained ACE inhibitory peptides that are potentially bioavailable. Copyright © 2016 Elsevier Ltd. All rights reserved.

Assay for Angiotensin-Converting Enzyme.

ERIC Educational Resources Information Center

Russo, Salvatore F.

1983-01-01

Describes a three-hour experiment designed to introduce students to chemistry of the angiotensis-converting enzyme, illustrate design of a quenched fluorescence substrate, and examine considerations necessary in designing a clinical assay. Includes background information on the biochemistry of hypertension, reagents/materials needed, procedures…

EFFECTS OF INCUBATION TEMPERATURE AND ESTROGEN EXPOSURE ON AROMATASE ACTIVITY IN THE BRAIN AND GONADS OF EMBRYONIC ALLIGATORS

EPA Science Inventory

During embryogenesis, incubation temperature and the hormonal environment influence gonadal differentiation of some reptiles, including all crocodilians. Current evidence suggests that aromatase, the enzyme that converts androgens to estrogens, has a role in sexual differentiatio...

Discovery and structural characterisation of new fold type IV-transaminases exemplify the diversity of this enzyme fold

PubMed Central

Pavkov-Keller, Tea; Strohmeier, Gernot A.; Diepold, Matthias; Peeters, Wilco; Smeets, Natascha; Schürmann, Martin; Gruber, Karl; Schwab, Helmut; Steiner, Kerstin

2016-01-01

Transaminases are useful biocatalysts for the production of amino acids and chiral amines as intermediates for a broad range of drugs and fine chemicals. Here, we describe the discovery and characterisation of new transaminases from microorganisms which were enriched in selective media containing (R)-amines as sole nitrogen source. While most of the candidate proteins were clearly assigned to known subgroups of the fold IV family of PLP-dependent enzymes by sequence analysis and characterisation of their substrate specificity, some of them did not fit to any of these groups. The structure of one of these enzymes from Curtobacterium pusillum, which can convert d-amino acids and various (R)-amines with high enantioselectivity, was solved at a resolution of 2.4 Å. It shows significant differences especially in the active site compared to other transaminases of the fold IV family and thus indicates the existence of a new subgroup within this family. Although the discovered transaminases were not able to convert ketones in a reasonable time frame, overall, the enrichment-based approach was successful, as we identified two amine transaminases, which convert (R)-amines with high enantioselectivity, and can be used for a kinetic resolution of 1-phenylethylamine and analogues to obtain the (S)-amines with e.e.s >99%. PMID:27905516

Harnessing the natural inhibitory domain to control TNFα Converting Enzyme (TACE) activity in vivo.

PubMed

Wong, Eitan; Cohen, Tal; Romi, Erez; Levin, Maxim; Peleg, Yoav; Arad, Uri; Yaron, Avraham; Milla, Marcos E; Sagi, Irit

2016-12-16

Dysregulated activity of A Disintegrin And Metalloproteinase 17 (ADAM17)/TNFα Converting Enzyme (TACE) is associated with inflammatory disorders and cancer progression by releasing regulatory membrane-tethered proteins like TNFα, IL6R and EGFR ligands. Although specific inhibition of TACE is thought to be a viable strategy for inflammatory disorders and for malignancies treatment, the generation of effective inhibitors in vivo has been proven to be challenging. Here we report on the development of a protein inhibitor that leverages the endogenous modulator of TACE. We have generated a stable form of the auto-inhibitory TACE prodomain (TPD), which specifically inhibits in vitro and cell-surface TACE, but not the related ADAM10, and effectively modulated TNFα secretion in cells. TPD significantly attenuated TACE-mediated disease models of sepsis, rheumatoid arthritis (RA) and inflammatory bowel disease (IBD), and reduced TNFα in synovial fluids from RA patients. Our results demonstrate that intervening with endogenous ADAM sheddase modulatory mechanisms holds potential as a general strategy for the design of ADAM inhibitors.

Release of IL-1β via IL-1β-Converting Enzyme in a Skin Dendritic Cell Line Exposed to 2,4-Dinitrofluorobenzene

PubMed Central

Matos, Teresa J.; Jaleco, Sara P.; Gonçalo, Margarida; Duarte, Carlos B.; Lopes, M. Celeste

2005-01-01

We used a mouse fetal skin dendritic cell line (FSDC) to study the effect of the strong allergen 2,4-dinitrofluorobenzene (DNFB) on interleukin (IL)-1β release and IL-1β receptor immunoreactivity. Stimulation with DNFB (30 minutes) increased IL-1β release without changing the mRNA levels of the protein. Furthermore, DNFB increased transiently the interleukin-1β-converting enzyme (ICE) activity, as measured with its fluorogenic substrate Z-Tyr-Val-Ala-Asp-AFC. The ICE inhibitor Z-YVAD-FMK prevented the release of IL-1β evoked by DNFB. Incubation of the cells with DNFB (30 minutes) strongly increased IL-1β receptor immunoreactivity. The rapid effect of DNFB on the release of mature IL-1β, without inducing an increase of IL-1β mRNA in FSDC, suggests a posttranslational modification of pro-IL-1β by ICE activity. PMID:16106098

The relationship between angiotensin-converting enzyme (ACE) insertion (I) / deletion (D) polymorphism, serum ACE activity and bone mineral density (BMD) in older Chinese.

PubMed

Zhang, Ya-Feng; Wang, Hong; Cheng, Qiong; Qin, Ling; Tang, Nelson Ls; Leung, Ping-Chong; Kwok, Timothy Cy

2017-01-01

In this study, we set out to investigate the relationship between angiotensin-converting enzyme ( ACE) I/D polymorphism, serum ACE activity and bone mineral density (BMD) in older Chinese. A standardized, structured, face-to-face interview was performed to collect demographic information. BMD was measured using dual-energy X-ray absorptiometry (DXA). I/D genotypes of ACE were determined by polymerase chain reaction (PCR) amplification. Serum ACE activity was determined photometrically by a commercially available kinetic kit. Multiple linear regression analysis was used to examine the relationship between ACE I/D polymorphism, serum ACE activity and BMD. A total of 1567 males and 1760 females were selected for analyzing the relationship between ACE I/D polymorphism and BMD. There was no significant difference in spine BMD, total hip BMD and femur neck BMD among different ACE I/D genotypes both in males and females. A total of 1699 males and 1739 females were selected for analyzing the relationship between serum ACE activity and BMD. There was also no significant difference in spine BMD, total hip BMD and femur neck BMD among different serum ACE activity groups both in males and females. There was no relationship between ACE I/D polymorphism, serum ACE activity and BMD in older Chinese.

Demonstration of extrapulmonary activity of angiotensin converting enzyme in intact tissue preparations.

PubMed Central

Lembeck, F.; Griesbacher, T.; Eckhardt, M.

1990-01-01

1. The activity of angiotensin converting enzyme (ACE) has been studied on functional parameters of intact isolated preparations of extrapulmonary tissues. The conversion of angiotensin I (A I) to angiotensin II (A II) and the cleavage of bradykinin (BK) were used as indicators of ACE activity. Captopril was employed as a specific inhibitor of ACE. 2. Captopril augmented the BK-induced contractions of the rat isolated uterus, the BK- and substance P-induced contractions of the guinea-pig ileum, and the BK-induced venoconstriction in the isolated perfused ear of the rabbit. Degradation of BK by ACE was calculated to be 52% in the rat uterus and 75% in the rabbit perfused ear. 3. Captopril inhibited the A I-induced contractions of the rat isolated colon, the A I-induced vasoconstriction in the isolated perfused ear of the rabbit and the rise in blood pressure induced by i.a. injections of A I in pithed rats. Conversion of A I to A II was calculated to be 13% in the rat colon and 26% in the rabbit perfused ear. 4. From estimations of the A II activity (bioassay on the rat colon) in the effluent of the perfused ear of the rabbit after injections of A I into the arterial inflow cannula it was calculated that approximately one tenth of A I was converted to A II during a single passage through the ear (less than 15 s).(ABSTRACT TRUNCATED AT 250 WORDS) PMID:2164861

Angiotensin-(1-7) augments endothelium-dependent relaxations of porcine coronary arteries to bradykinin by inhibiting angiotensin-converting enzyme 1.

PubMed

Raffai, Gábor; Khang, Gilson; Vanhoutte, Paul M

2014-05-01

Angiotensin-converting enzyme 2 (ACE2) converts angiotensin II to angiotensin-(1-7) that activates Mas receptors, inhibits ACE1, and modulates bradykinin receptor sensitivity. This in vitro study compared the direct and indirect effects of angiotensin-(1-7), the ACE1 inhibitor captopril, and diminazene aceturate (DIZE) an alleged ACE2 activator in rings of porcine coronary arteries, by measuring changes of isometric tension. Angiotensin-(1-7), captopril, and DIZE did not cause significant changes in tension before or after desensitization of bradykinin receptors in preparations contracted with U46619. Bradykinin caused concentration-dependent and endothelium-dependent relaxations that were not affected by DIZE but were potentiated to a similar extent by angiotensin-(1-7) and captopril, given alone or in combination. Bradykinin responses potentiated by angiotensin-(1-7) and captopril were not affected by the BK1 antagonist SSR240612 and remained augmented in the presence of either N-nitro-L-arginine methyl ester hydrochloride plus indomethacin or TRAM-34 plus UCL-1684. ACE2 was identified in the coronary endothelium by immunofluorescence, but its basal activity was not influenced by DIZE. These results suggest that in coronary arteries, angiotensin-(1-7) and captopril both improves NO bioavailability and enhances endothelium-dependent hyperpolarization to bradykinin solely by ACE1 inhibition. Endothelial ACE2 activity cannot be increased by DIZE to produce local adequate amounts of angiotensin-(1-7) to influence vascular tone.

Targeting of antibody-conjugated plasminogen activators to the pulmonary vasculature.

PubMed

Muzykantov, V R; Barnathan, E S; Atochina, E N; Kuo, A; Danilov, S M; Fisher, A B

1996-11-01

Thrombolytic therapy has not been widely used for pulmonary embolism due to less than optimal results with conventional plasminogen activators. We propose a new approach to deliver plasminogen activators to the luminal surface of the pulmonary vasculature to potentially improve dissolution of pulmonary thromboemboli. Our previous studies have documented that a monoclonal antibody (mAb) to angiotensin-converting enzyme (anti-angiotensin-converting enzyme mAb 9B9) accumulates in the lungs of various animal species after systemic administration. We coupled 125I-labeled biotinylated plasminogen activators (single-chain urokinase plasminogen activator, tissue-type plasminogen activator and streptokinase) to biotinylated mAb 9B9, using streptavidin as a cross-linker. The fibrinolytic activity of plasminogen activators was not changed significantly by either biotinylation or by coupling to streptavidin. Antibody-conjugated plasminogen activators bind to the antigen immobilized in plastic wells and provide lysis of fibrin clots formed in these wells. Therefore, antibody-conjugated plasminogen activators bound to their target antigen retain their capacity to activate plasminogen. One hour after i.v. injection of mAb 9B9-conjugated radiolabeled biotinylated single-chain urokinase plasminogen activator, biotinylated tissue-type plasminogen activator or biotinylated-streptokinase in rats, the level of radiolabel was 7.4 +/- 0.8, 5.9 +/- 0.4 and 3.6 +/- 0.4% of injected dose/g (ID/g) of lung tissue vs. 0.5 +/- 0.01, 0.3 +/- 0.01 and 0.6 +/- 0.3% ID/g after injection of the same activators conjugated with control mouse IgG (P < .01 in all cases). Injection of mAb 9B9-conjugated radiolabeled plasminogen activator led to its rapid pulmonary uptake with a peak value 6.2 +/- 1.2% ID/g attained 3 hr after injection. One day later, 2.2 +/- 0.5% of the injected radioactivity was found per gram of lung tissue, although the blood level was 0.13 +/- 0.03% ID/g (lung/blood ratio 16.7 +/- 0.3). Therefore, conjugation of plasminogen activators with anti-angiotensin-converting enzyme mAb 9B9 provides their specific targeting to and prolonged association with the pulmonary vasculature. These results provide a basis for study of the local pulmonary fibrinolysis by mAb 9B9-conjugated plasminogen activators.

Sacubitril/Valsartan: The Newest Addition to the Toolbox for Guideline-Directed Medical Therapy of Heart Failure.

PubMed

Rodgers, Jo E

2017-06-01

Sacubitril/valsartan combines a neprilysin inhibitor with an angiotensin receptor blocker. As an inhibitor of neprilysin, an enzyme that degrades biologically active natriuretic peptides, this first-in-class therapy increases levels of circulating natriuretic peptides, resulting in natriuretic, diuretic, and vasodilatory effects. In patients with chronic New York Heart Association class II-IV heart failure with reduced ejection fraction, the PARADIGM-HF trial demonstrated that sacubitril/valsartan significantly reduced the primary endpoint of cardiovascular mortality and heart failure hospitalization, compared with enalapril. The rate of all-cause mortality was also significantly reduced. Subsequently, the American College of Cardiology/American Heart Association/Heart Failure Society of America recently updated guideline recommendations for Stage C patients with heart failure with reduced ejection fraction to recommend angiotensin-converting enzyme inhibitors, angiotensin receptor blockers, or sacubitril/valsartan in conjunction with other evidence-based therapies to reduce morbidity and mortality. Several analyses have suggested the cost-effectiveness of this new therapy. To ensure tolerability, initiating the lower dosage form of sacubitril/valsartan is warranted in patients with severe renal impairment, moderate hepatic impairment, and low blood pressure, and close monitoring is warranted in such patients. A 36-hour washout period is recommended when switching patients from an angiotensin-converting enzyme inhibitor to sacubitril/valsartan. Similarly, sacubitril/valsartan is contraindicated in patients receiving concomitant angiotensin-converting enzyme inhibitor or angiotensin receptor blocker and those with a history of angioedema. Copyright © 2017 Elsevier Inc. All rights reserved.

[Association between visual improvement after photocoagulation and the use of angiotensin converting enzyme inhibitors in diabetic macular oedema].

PubMed

Somilleda-Ventura, Selma Alin; García-Rubio, Yatzul Zuhaila; Razo Blanco-Hernández, Dulce Milagros; Lima-Gómez, Virgilio

2016-01-01

Angiotensin converting enzyme inhibitors are effective in delaying the progression of diabetic retinopathy. It is unknown if their use is associated with a better visual outcome in patients with diabetic macular oedema. A non-experimental, comparative, longitudinal and retrospective study was performed on patients with diabetic macular oedema treated by focal photocoagulation, and with systemic arterial hypertension treated with angiotensin converting enzyme inhibitors (Group 1), and without hypertension (Group 2). The dependent variable was the proportion with visual improvement, operatively defined as the gain of one or more lines of vision three weeks after photocoagulation. The independent variable was the use of angiotensin converting enzyme inhibitors. The proportion of eyes with visual improvement after treatment was compared between groups using the Chi squared (χ(2)) test. A total of 33 eyes (51.6%) were assigned to group 1, and 31 (48.2%), to group 2. The mean of visual acuity improved after three weeks, compared with baseline (p=0.002). The proportion of eyes with visual improvement did not differ between patients treated with angiotensin converting enzyme inhibitors (45.5%) and those that did not use them (51.6%, p=0.4). There was no statistical difference in the proportion of eyes with visual improvement between patients treated with angiotensin converting enzyme inhibitors and in those where they were not used. There is no support for the inhibition of angiotensin II in addition to photocoagulation for improving the outcome in patients with diabetic macular oedema. Copyright © 2015 Academia Mexicana de Cirugía A.C. Published by Masson Doyma México S.A. All rights reserved.

Morphine biosynthesis in opium poppy involves two cell types: sieve elements and laticifers.

PubMed

Onoyovwe, Akpevwe; Hagel, Jillian M; Chen, Xue; Khan, Morgan F; Schriemer, David C; Facchini, Peter J

2013-10-01

Immunofluorescence labeling and shotgun proteomics were used to establish the cell type-specific localization of morphine biosynthesis in opium poppy (Papaver somniferum). Polyclonal antibodies for each of six enzymes involved in converting (R)-reticuline to morphine detected corresponding antigens in sieve elements of the phloem, as described previously for all upstream enzymes transforming (S)-norcoclaurine to (S)-reticuline. Validated shotgun proteomics performed on whole-stem and latex total protein extracts generated 2031 and 830 distinct protein families, respectively. Proteins corresponding to nine morphine biosynthetic enzymes were represented in the whole stem, whereas only four of the final five pathway enzymes were detected in the latex. Salutaridine synthase was detected in the whole stem, but not in the latex subproteome. The final three enzymes converting thebaine to morphine were among the most abundant active latex proteins despite a limited occurrence in laticifers suggested by immunofluorescence labeling. Multiple charge isoforms of two key O-demethylases in the latex were revealed by two-dimensional immunoblot analysis. Salutaridine biosynthesis appears to occur only in sieve elements, whereas conversion of thebaine to morphine is predominant in adjacent laticifers, which contain morphine-rich latex. Complementary use of immunofluorescence labeling and shotgun proteomics has substantially resolved the cellular localization of morphine biosynthesis in opium poppy.

Fine-Mapping Angiotensin-Converting Enzyme Gene: Separate QTLs Identified for Hypertension and for ACE Activity

PubMed Central

Chung, Chia-Min; Wang, Ruey-Yun; Fann, Cathy S. J.; Chen, Jaw-Wen; Jong, Yuh-Shiun; Jou, Yuh-Shan; Yang, Hsin-Chou; Kang, Chih-Sen; Chen, Chien-Chung; Chang, Huan-Cheng; Pan, Wen-Harn

2013-01-01

Angiotensin-converting enzyme (ACE) has been implicated in multiple biological system, particularly cardiovascular diseases. However, findings associating ACE insertion/deletion polymorphism with hypertension or other related traits are inconsistent. Therefore, in a two-stage approach, we aimed to fine-map ACE in order to narrow-down the function-specific locations. We genotyped 31 single nucleotide polymorphisms (SNPs) of ACE from 1168 individuals from 305 young-onset (age ≤40) hypertension pedigrees, and found four linkage disequilibrium (LD) blocks. A tag-SNP, rs1800764 on LD block 2, upstream of and near the ACE promoter, was significantly associated with young-onset hypertension (p = 0.04). Tag-SNPs on all LD blocks were significantly associated with ACE activity (p-value: 10–16 to <10–33). The two regions most associated with ACE activity were found between exon13 and intron18 and between intron 20 and 3′UTR, as revealed by measured haplotype analysis. These two major QTLs of ACE activity and the moderate effect variant upstream of ACE promoter for young-onset hypertension were replicated by another independent association study with 842 subjects. PMID:23469169

Gender difference of serum angiotensin-converting enzyme (ACE) activity in DD genotype of ACE insertion/deletion polymorphism in elderly Chinese.

PubMed

Zhang, Ya-Feng; Cheng, Qiong; Tang, Nelson L S; Chu, Tanya T W; Tomlinson, Brian; Liu, Fan; Kwok, Timothy C Y

2014-12-01

In this study we investigated the gender difference of serum angiotensin-converting enzyme (ACE) activity in a population of Hong Kong-dwelling elderly Chinese. A total of 1767 (843 male, 924 female) Hong Kong-dwelling elderly Chinese were recruited. ACE I/D genotypes were identified by polymerase chain reaction amplification and serum ACE activity was determined using a commercially available kinetic kit. ACE I/D genotype distribution was compared by chi-square test, the correlation between ACE I/D polymorphism and serum ACE activity was analysed by ANOVA test and gender difference of serum ACE activity of different genotypes was compared by independent sample t-test. No statistically significant difference of genotype distribution between male and female subjects was found. Serum ACE activity was significantly correlated with ACE genotype. Overall, there was no gender difference of serum ACE activity; however, when sub-grouping the subjects by ACE I/D genotype, male subjects with DD genotype had higher serum ACE activity than female subjects with DD genotype. No significant gender difference of genotype distribution was found in elderly Chinese. Serum ACE activity was significantly correlated with ACE I/D polymorphism in elderly Chinese. Male subjects with DD genotype had higher serum ACE activity than female subjects with DD genotype. © The Author(s) 2013.

Substrate tunnels in enzymes: structure-function relationships and computational methodology.

PubMed

Kingsley, Laura J; Lill, Markus A

2015-04-01

In enzymes, the active site is the location where incoming substrates are chemically converted to products. In some enzymes, this site is deeply buried within the core of the protein, and, in order to access the active site, substrates must pass through the body of the protein via a tunnel. In many systems, these tunnels act as filters and have been found to influence both substrate specificity and catalytic mechanism. Identifying and understanding how these tunnels exert such control has been of growing interest over the past several years because of implications in fields such as protein engineering and drug design. This growing interest has spurred the development of several computational methods to identify and analyze tunnels and how ligands migrate through these tunnels. The goal of this review is to outline how tunnels influence substrate specificity and catalytic efficiency in enzymes with buried active sites and to provide a brief summary of the computational tools used to identify and evaluate these tunnels. © 2015 Wiley Periodicals, Inc.

Identification and partial characterization of a latent ATP, Mg-dependent protein phosphatase in rabbit skeletal muscle cytosol.

PubMed

Vandenheede, J R; Staquet, S; Merlevede, W

1989-05-04

Fractionation of rabbit skeletal muscle cytosol on Aminohexyl-Sepharose has resulted in the identification of a latent ATP, Mg-dependent protein phosphatase whose catalytic subunit is in the active conformation, but is inhibited by the presence of more than one modulator unit. The partially purified enzyme is converted to an inactive, kinase FA-dependent form upon incubation at 30 degrees C unless modulator-specific polyclonal antibodies are added to the preparation. The immunoglobulins also relieve the inhibition which is responsible for the low basal phosphatase activity of the enzyme, and they counteract all of the heat-stable inhibitor activity present in the preparation. Addition of free catalytic subunit abolishes the inhibition of the latent enzyme in a dose-dependent way, but cannot prevent the inactivation process. The inactivated phosphatase and the original latent enzyme exhibit the same apparent Mr in sucrose density-gradient centrifugation (70,000) and in gel filtration (110,000).

Epilactose production by 2 cellobiose 2-epimerases in natural milk.

PubMed

Krewinkel, Manuel; Gosch, Maria; Rentschler, Eva; Fischer, Lutz

2014-01-01

It was reported recently that cellobiose 2-epimerases (CE) from various aerobic microorganisms convert lactose to epilactose in defined buffer systems. In this study, we showed that CE from 2 mesophilic microorganisms, Flavobacterium johnsoniae and Pedobacter heparinus, were capable of converting lactose to prebiotic epilactose not only in buffer but also in a complex milk system. First, the 2 enzymes were separately cloned, recombinantly expressed in Escherichia coli, and purified by column chromatography. The production of F. johnsoniae CE was carried out in a stirred-tank reactor, indicating that future upscaling is possible. The bioconversions of milk lactose were carried out at an industrially relevant low temperature of 8°C to avoid undesired microbial contaminations or chemical side reactions. Both enzymes were reasonably active at this low temperature, because of their origin from mesophilic organisms. The enzymes showed different operational stabilities over a 24-h time-course. A conversion yield of about 30 to 33% epilactose was achieved with both enzymes. No side products were detected other than epilactose. Therefore, CE may introduce an added value for particular dairy products by in situ production of the prebiotic sugar epilactose. Copyright © 2014 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.

Flavonoids-Rich Orthosiphon stamineus Extract as New Candidate for Angiotensin I-Converting Enzyme Inhibition: A Molecular Docking Study.

PubMed

Shafaei, Armaghan; Sultan Khan, Md Shamsuddin; F A Aisha, Abdalrahim; Abdul Majid, Amin Malik Shah; Hamdan, Mohammad Razak; Mordi, Mohd Nizam; Ismail, Zhari

2016-11-09

This study aims to evaluate the in vitro angiotensin-converting enzyme (ACE) inhibition activity of different extracts of Orthosiphon stamineus (OS) leaves and their main flavonoids, namely rosmarinic acid (RA), sinensetin (SIN), eupatorin (EUP) and 3'-hydroxy-5,6,7,4'-tetramethoxyflavone (TMF). Furthermore, to identify possible mechanisms of action based on structure-activity relationships and molecular docking. The in vitro ACE inhibition activity relied on determining hippuric acid (HA) formation from ACE-specific substrate (hippuryl-histidyl-leucine (HHL)) by the action of ACE enzyme. A High Performance Liquid Chromatography method combined with UV detection was developed and validated for measurement the concentration of produced HA. The chelation ability of OS extract and its reference compounds was evaluated by tetramethylmurexide reagent. Furthermore, molecular docking study was performed by LeadIT-FlexX : BioSolveIT's LeadIT program. OS ethanolic extract (OS-E) exhibited highest inhibition and lowest IC 50 value (45.77 ± 1.17 µg/mL) against ACE compared to the other extracts. Among the tested reference compounds, EUP with IC 50 15.35 ± 4.49 µg/mL had highest inhibition against ACE and binding ability with Zn (II) (56.03% ± 1.26%) compared to RA, TMF and SIN. Molecular docking studies also confirmed that flavonoids inhibit ACE via interaction with the zinc ion and this interaction is stabilized by other interactions with amino acids in the active site. In this study, we have demonstrated that changes in flavonoids active core affect their capacity to inhibit ACE. Moreover, we showed that ACE inhibition activity of flavonoids compounds is directly related to their ability to bind with zinc ion in the active site of ACE enzyme. It was also revealed that OS extract contained high amount of flavonoids other than RA, TMF, SIN and EUP. As such, application of OS extract is useful as inhibitors of ACE.

Purification and characterization of angiotensin I converting enzyme inhibition peptides from sandworm Sipunculus nudus

NASA Astrophysics Data System (ADS)

Sun, Xueping; Wang, Man; Liu, Buming; Sun, Zhenliang

2017-10-01

Three angiotensin I converting enzyme (ACE) inhibition peptides were isolated from sandworm Sipunculus nudus protein hydrolysate prepared using protamex. Consecutive purification methods, including size exclusion chromatography and reverse-phase high performance liquid chromatography (RP-HPLC), were used to isolate the ACE inhibition peptides. The amino acid sequences of the peptides were identified as Ile-Asn-Asp, Val-Glu-Pro-Gly and Leu-Ala-Asp-Glu-Phe. The IC50 values of the purified peptides for ACE inhibition activity were 34.72 μmol L-1, 20.55 μmol L-1 and 22.77 μmol L-1, respectively. These results suggested that S. nudus proteins contain specific peptides that can be released by enzymatic hydrolysis. This study may provide an experimental basis for further systematic research, rational development and clinical utilization of sandworm resources.

The carboxypeptidase angiotensin converting enzyme (ACE) shapes the MHC class I peptide repertoire

PubMed Central

Shen, Xiao Z.; Billet, Sandrine; Lin, Chentao; Okwan-Duodu, Derick; Chen, Xu; Lukacher, Aron E.; Bernstein, Kenneth E.

2011-01-01

The surface presentation of peptides by major histocompatibility complex (MHC) class I molecules is critical to CD8+ T cell mediated adaptive immune responses. Aminopeptidases are implicated in the editing of peptides for MHC class I loading, but C-terminal editing is thought due to proteasome cleavage. By comparing genetically deficient, wild-type and over-expressing mice, we now identify the dipeptidase angiotensin-converting enzyme (ACE) as playing a physiologic role in peptide processing for MHC class I. ACE edits the C-termini of proteasome-produced class I peptides. The lack of ACE exposes novel antigens but also abrogates some self-antigens. ACE has major effects on surface MHC class I expression in a haplotype-dependent manner. We propose a revised model of MHC class I peptide processing by introducing carboxypeptidase activity. PMID:21964607

Novel angiotensin I-converting enzyme inhibitory peptides isolated from Alcalase hydrolysate of mung bean protein.

PubMed

Li, Guan-Hong; Wan, Ju-Zhen; Le, Guo-Wei; Shi, Yong-Hui

2006-08-01

Mung bean protein isolates were hydrolyzed for 2 h by Alcalase. The generated hydrolysate showed angiotensin I-converting enzyme (ACE) inhibitory activity with the IC(50) value of 0.64 mg protein/ml. Three kinds of novel ACE inhibitory peptides were isolated from the hydrolysate by Sephadex G-15 and reverse-phase high performance liquid chromatography (RP-HPLC). These peptides were identified by amino acid composition analysis and matrix assisted-laser desorption/ionization time-of-flight tandem mass spectrometry (MALDI-TOF MS/MS), as Lys-Asp-Tyr-Arg-Leu, Val-Thr-Pro-Ala-Leu-Arg and Lys-Leu-Pro-Ala-Gly-Thr-Leu-Phe with the IC(50) values of 26.5 microM, 82.4 microM and 13.4 microM, respectively. Copyright (c) 2006 European Peptide Society and John Wiley & Sons, Ltd.

Preparation and activity of bubbling-immobilized cellobiase within chitosan-alginate composite.

PubMed

Wang, Fang; Su, Rong-Xin; Qi, Wei; Zhang, Ming-Jia; He, Zhi-Min

2010-01-01

Cellobiase can hydrolyze cellobiose into glucose; it plays a key role in the process of cellulose hydrolysis by reducing the product inhibition. To reuse the enzyme and improve the economic value of cellulosic ethanol, cellobiase was immobilized using sodium alginate and chitosan as carriers by the bubbling method. The immobilization conditions were optimized as follows: enzyme loading of 100 U cellobiase/g carrier, 30 min immobilization, 3.5 wt% sodium alginate, 0.25 wt% chitosan, and 2 wt% calcium chloride. Compared to free enzyme, the immobilized cellobiase had a decreased apparent K(m) and the maximum activity at a lower pH, indicating its higher acidic and thermal stability. The immobilized cellobiase was further tested in the hydrolysis of cellobiose and various cellulosic substrates (microcrystalline cellulose, filter paper, and ammonia-pretreated corn cobs). Together with cellulases, the immobilized cellobiase converted the cellulosic substrates into glucose with the rate and extent similar to the free enzyme.

Mining novel starch-converting Glycoside Hydrolase 70 enzymes from the Nestlé Culture Collection genome database: The Lactobacillus reuteri NCC 2613 GtfB.

PubMed

Gangoiti, Joana; van Leeuwen, Sander S; Meng, Xiangfeng; Duboux, Stéphane; Vafiadi, Christina; Pijning, Tjaard; Dijkhuizen, Lubbert

2017-08-30

The Glycoside hydrolase (GH) family 70 originally was established for glucansucrases of lactic acid bacteria (LAB) converting sucrose into α-glucan polymers. In recent years we have identified 3 subfamilies of GH70 enzymes (designated GtfB, GtfC and GtfD) as 4,6-α-glucanotransferases, cleaving (α1 → 4)-linkages in maltodextrins/starch and synthesizing new (α1 → 6)-linkages. In this work, 106 putative GtfBs were identified in the Nestlé Culture Collection genome database with ~2700 genomes, and the L. reuteri NCC 2613 one was selected for further characterization based on variations in its conserved motifs. Using amylose the L. reuteri NCC 2613 GtfB synthesizes a low-molecular-mass reuteran-like polymer consisting of linear (α1 → 4) sequences interspersed with (α1 → 6) linkages, and (α1 → 4,6) branching points. This product specificity is novel within the GtfB subfamily, mostly comprising 4,6-α-glucanotransferases synthesizing consecutive (α1 → 6)-linkages. Instead, its activity resembles that of the GtfD 4,6-α-glucanotransferases identified in non-LAB strains. This study demonstrates the potential of large-scale genome sequence data for the discovery of enzymes of interest for the food industry. The L. reuteri NCC 2613 GtfB is a valuable addition to the starch-converting GH70 enzyme toolbox. It represents a new evolutionary intermediate between families GH13 and GH70, and provides further insights into the structure-function relationships of the GtfB subfamily enzymes.

Relative lipophilicities and structural-pharmacological considerations of various angiotensin-converting enzyme (ACE) inhibitors.

PubMed

Ranadive, S A; Chen, A X; Serajuddin, A T

1992-11-01

Lipophilicities of seven structurally diverse angiotensin-converting enzyme (ACE) inhibitors, viz., captopril, zofenoprilat, enalaprilat, ramiprilat, lisinopril, fosinoprilat, and ceronapril (SQ29852), were compared by determining their octanol-water distribution coefficients (D) under physiological pH conditions. The distribution co-efficients of zofenopril, enalapril, ramipril and fosinopril, which are the prodrug forms of zofenoprilat, enalaprilat, ramiprilat, and fosinoprilat, respectively, were also determined. Attempts were made to correlate lipophilicities with the reported data for oral absorption, protein binding, ACE inhibitory activity, propensity for biliary excretion, and penetration across the blood-brain barrier for these therapeutic entities. Better absorption of prodrugs compared to their respective active forms is in agreement with their greater lipophilicities. Captopril, lisinopril, and ceronapril are orally well absorbed despite their low lipophilicities, suggesting involvement of other factors such as a carrier-mediated transport process. Of all the compounds studied, the two most lipophilic ACE inhibitors, fosinoprilat and zofenoprilat, exhibit a rank-order correlation with respect to biliary excretion. This may explain the dual routes of elimination (renal and hepatic) observed with fosinoprilat in humans. The more lipophilic compounds also exhibit higher protein binding. Both the lipophilicity and a carrier-mediated process may be involved in penetration of some of these drugs into brain. For structurally similar compounds, in vitro ACE inhibitory activity increased with the increase in lipophilicity. However, no clear correlation between lipophilicity and ACE inhibitory activity emerged when different types of inhibitors are compared, possibly because their interactions with enzymes are primarily ionic in nature.

Identification of interleukin-8 converting enzyme as cathepsin L.

PubMed

Ohashi, Kensaku; Naruto, Masanobu; Nakaki, Toshio; Sano, Emiko

2003-06-26

IL-8 is produced by various cells, and the NH(2)-terminal amino acid sequence of IL-8 displays heterogeneity among cell types. The mature form of IL-8 has 72 amino acids (72IL-8), while a precursor form (77IL-8) of IL-8 has five additional amino acids to the 72IL-8 NH(2)-terminal. However, it has been unclear how IL-8 is processed to yield the mature form. In this study, converting enzyme was purified as a single 31-kDa band on silver-stained polyacrylamide gel from 160 l of cultured fibroblast supernatant by sequential chromatography. NH(2)-terminal amino acid sequence analysis revealed a sequence, EAPRSVDWRE, which was identified as a partial sequence of cathepsin L. Polyclonal antibodies raised against cathepsin L recognized the purified converting enzyme on Western blot. Moreover, human hepatic cathepsin L cleaved 77IL-8 between Arg(5) and Ser(6), which is the same cleavage site as the putative converting enzyme, resulting in 72IL-8 formation. These data indicate that the converting enzyme of the partially purified fraction of the human fibroblast culture supernatant was cathepsin L. Furthermore, 72IL-8 was sevenfold more potent than 77IL-8 in a neutrophil chemotaxis assay. These results show that cathepsin L is secreted from human fibroblasts in response to external stimuli and plays an important role in IL-8 processing in inflammatory sites.

Enzymatic Conversion of CO2 to Bicarbonate in Functionalized Mesoporous Silica

DOE Office of Scientific and Technical Information (OSTI.GOV)

Yu, Yuehua; Chen, Baowei; Qi, Wen N.

2012-05-01

We report here that carbonic anhydrase (CA), the fastest enzyme that can covert carbon dioxide to bicarbonate, can be spontaneously entrapped in functionalized mesoporous silica (FMS) with super-high loading density (up to 0.5 mg of protein/mg of FMS) due to the dominant electrostatic interaction. The binding of CA to HOOC-FMS can result in the protein’s conformational change comparing to the enzyme free in solution, but can be overcome with increased protein loading density. The higher the protein loading density, the less conformational change, hence the higher enzymatic activity and the higher enzyme immobilization efficiency. The electrostatically bound CA can bemore » released by changing pH. The released enzyme still displayed the native conformational structure and the same high enzymatic activity as that prior to the enzyme entrapment. This work opens up a new approach converting carbon dioxide to biocarbonate in a biomimetic nanoconfiguration that can be integrated with the other part of biosynthesis process for the assimilation of carbon dioxide.« less

THE EFFECT OF ATRAZINE ON DIMETHYL SULFUR IN MARINE PHYTOPLANKTON

EPA Science Inventory

It is anticipated that under stress, the cellular DMSP concentration should drop, as sulfur is transferred from the DMSP pool to DMS. Sulfur in the DMS pool will be transferred to the DMSO pool as radicals are scavenged. Enzyme activities such as DMSP lyase, which converts D...

Influence of iodinated contrast media on the activities of histamine inactivating enzymes diamine oxidase and histamine N-methyltransferase in vitro.

PubMed

Kuefner, M A; Feurle, J; Petersen, J; Uder, M; Schwelberger, H G

2014-01-01

Iodinated contrast media can cause pseudoallergic reactions associated with histamine release in significant numbers of patients. To clarify whether these adverse reactions may be aggravated by a compromised histamine catabolism we asked if radiographic contrast agents in vitro inhibit the histamine inactivating enzymes diamine oxidase (DAO) and histamine N-methyltransferase (HMT). Nine iodinated contrast agents were tested in vitro. Following pre-incubation of purified porcine kidney DAO and recombinant human HMT with 0.1-10mM of the respective contrast medium (H2O and specific inhibitors of DAO and HMT as controls) enzyme activities were determined by using radiometric micro assays. None of the contrast media irrespective of their structure showed significant inhibition of the activities of DAO and HMT. Pre-incubation of the enzymes with specific inhibitors led to complete inhibition of the respective enzymatic activity. The iodinated contrast media tested in vitro did not exhibit inhibition of histamine converting enzymes at physiologically relevant concentrations. However due to the in vitro character of this study these results do not directly reflect the in vivo situation. Copyright © 2012 SEICAP. Published by Elsevier Espana. All rights reserved.

The granulocyte-angiotensin system. Angiotensin I-converting activity of cathepsin G.

PubMed

Klickstein, L B; Kaempfer, C E; Wintroub, B U

1982-12-25

Cathepsin G, an Mr = 26,000-29,000 cationic human neutrophil lysosomal serine protease, releases angiotensin II from angiotensinogen and was, therefore, examined for angiotensin I-converting activity. Cathepsin G-dependent angiotensin I conversion was detected by a high performance liquid chromatography assay which permitted independent quantitation of angiotensin I and angiotensin II and detection of angiotensin degradation products. 1.8-5.0 X 10(-9) M cathepsin G converted angiotensin I (3.3 X 10(-4) M) to angiotensin II without further degradation of angiotensin II. The pH optimum for cathepsin G-catalyzed angiotensin I conversion was pH 7.0-7.5, and the Km and Kcat were 2.2 X 10(-4) M and 3.4 s-1, respectively. In contrast to dipeptidyl hydrolase-converting enzyme, cathepsin G did not inactivate bradykinin, did not cleave hippuryl-His-Leu, and was not inhibited by 10(-4) M Captopril or SQ 20881. Purified human neutrophils stimulated with 2.5 X 10(-6) M-10(-10) M fMet-Leu-Phe released angiotensin-converting activity with a Km of 3.3 X 10(-4) M. That the angiotensin-converting activity released from neutrophils was attributable to cathepsin G was indicated by similar susceptibility to inhibitors and adsorption by goat antibody to cathepsin G. The granulocyte-angiotensin system provides a mechanism for the local generation of angiotensin II at sites of neutrophil accumulation and may be of significance in regulation of blood flow in tissue microvasculature.

Insights into the Hypertensive Effects of Tityus serrulatus Scorpion Venom: Purification of an Angiotensin-Converting Enzyme-Like Peptidase.

PubMed

Cajado-Carvalho, Daniela; Kuniyoshi, Alexandre Kazuo; Duzzi, Bruno; Iwai, Leo Kei; Oliveira, Úrsula Castro de; Junqueira de Azevedo, Inácio de Loiola Meirelles; Kodama, Roberto Tadashi; Portaro, Fernanda Vieira

2016-11-24

The number of cases of envenomation by scorpions has grown significantly in Brazil since 2007, with the most severe cases being caused by the Tityus serrulatus scorpion. Although envenomed patients mostly suffer neurotoxic manifestations, other symptoms, such as hypertension, cannot be exclusively attributed to neurotoxins. Omics analyses have detected plentiful amounts of metalloproteases in T. serrulatus venom. However, the roles played by these enzymes in envenomation are still unclear. Endeavoring to investigate the functions of scorpion venom proteases, we describe here for the first time an Angiotensin I-Converting Enzyme-like peptidase (ACE-like) purified from T. serrulatus venom. The crude venom cleaved natural and fluorescent substrates and these activities were inhibited by captopril. Regarding the serum neutralization, the scorpion antivenom was more effective at blocking the ACE-like activity than arachnid antivenom, although neither completely inhibited the venom cleavage action, even at higher doses. ACE-like was purified from the venom after three chromatographic steps and its identity was confirmed by mass spectrometric and transcriptomic analyses. Bioinformatics analysis showed homology between the ACE-like transcript sequences from Tityus spp. and human testis ACE. These findings advance our understanding of T. serrulatus venom components and may improve treatment of envenomation victims, as ACE-like may contribute to envenomation symptoms, especially the resulting hypertension.

Angiotensin-converting enzyme in Spodoptera littoralis: molecular characterization, expression and activity profile during development.

PubMed

Lemeire, Els; Vanholme, Bartel; Van Leeuwen, Thomas; Van Camp, John; Smagghe, Guy

2008-02-01

The characterization of the full-length angiotensin-converting enzyme (ACE) cDNA sequence of the lepidopteran Spodoptera littoralis is reported in this study. The predicted open reading frame encodes a 647 amino acids long protein (SlACE) and shows 63.6% identity with the Bombyx mori ACE sequence. A 3D-model, consisting of 26 alpha-helices and three beta-sheets, was predicted for the sequence. SlACE expression was studied in the embryonic, larval and pupal stages of S. littoralis and in different tissues of the last larval stage by reverse-transcribed PCR. This revealed that the gene is expressed throughout the life cycle and especially in brain, gut and fat body tissue of the last stage. These results are in agreement with a role of ACE in the metabolism of neuropeptides and gut hormones. In addition, ACE activity has been studied in more detail during development, making use of a fluorescent assay. High ACE peptidase activity coincides with every transition state, from embryo to larva, from larva to larva and from larva to pupa. A peak value in activity occurs during the early pupal stage. These results indicate the importance of SlACE during metamorphosis and reveal the high correlation of ACE activity with the insect's development, which is regulated by growth and developmental hormones.

Aronia melanocarpa Elliot reduces the activity of angiotensin i-converting enzyme-in vitro and ex vivo studies.

PubMed

Sikora, Joanna; Broncel, Marlena; Mikiciuk-Olasik, Elżbieta

2014-01-01

The aim of the study was to analyze the effects of two-month supplementation with chokeberry preparation on the activity of angiotensin I-converting enzyme (ACE) in patients with metabolic syndrome (MS). During the in vitro stage of the study, we determined the concentration of chokeberry extract, which inhibited the activity of ACE by 50% (IC50). The participants (n = 70) were divided into three groups: I-patients with MS who received chokeberry extract supplements, II-healthy controls, and III-patients with MS treated with ACE inhibitors. After one and two months of the experiment, a decrease in ACE activity corresponded to 25% and 30%, respectively. We documented significant positive correlations between the ACE activity and the systolic (r = 0.459, P = 0.048) and diastolic blood pressure, (r = 0.603, P = 0.005) and CRP. The IC50 of chokeberry extract and captopril amounted to 155.4 ± 12.1 μg/mL and 0.52 ± 0.18 μg/mL, respectively. Our in vitro study revealed that chokeberry extract is a relatively weak ACE inhibitor. However, the results of clinical observations suggest that the favorable hypotensive action of chokeberry polyphenols may be an outcome of both ACE inhibition and other pleotropic effects, for example, antioxidative effect.

Increasing brain angiotensin converting enzyme 2 activity decreases anxiety-like behavior in male mice by activating central Mas receptors

PubMed Central

Wang, Lei; de Kloet, Annette D.; Pati, Dipanwita; Hiller, Helmut; Smith, Justin A.; Pioquinto, David J.; Ludin, Jacob A.; Oh, S. Paul; Katovich, Michael J.; Frazier, Charles J.; Raizada, Mohan K.; Krause, Eric G.

2016-01-01

Over-activation of brain renin-angiotensin system (RAS) has been implicated in the etiology of anxiety disorders. Angiotensin converting enzyme (ACE2) inhibits RAS activity by converting angiotensin II, the effector peptide of RAS, to angiotensin-(1-7), which activates Mas receptors (MasR). Whether increasing brain ACE2 activity reduces anxiety by stimulating central MasR is unknown. To test the hypothesis that increasing brain ACE2 activity reduces anxiety-like behavior via central MasR stimulation, we generated male mice overexpressing ACE2 (ACE2 KI mice) and wild type littermate controls (WT). ACE2 KI mice explored the open arms of the elevated plus maze (EPM) significantly more than WT, suggesting increasing ACE2 activity is anxiolytic. Central delivery of diminazene aceturate, an ACE2 activator, to C57BL/6 mice also reduced anxiety-like behavior in the EPM, but centrally administering ACE2 KI mice A-779, a MasR antagonist, abolished their anxiolytic phenotype, suggesting that ACE2 reduces anxiety-like behavior by activating central MasR. To identify the brain circuits mediating these effects, we measured Fos, a marker of neuronal activation, subsequent to EPM exposure and found that ACE2 KI mice had decreased Fos in the bed nucleus of stria terminalis but had increased Fos in the basolateral amygdala (BLA). Within the BLA, we determined that ~62% of GABAergic neurons contained MasR mRNA and expression of MasR mRNA was upregulated by ACE2 overexpression, suggesting that ACE2 may influence GABA neurotransmission within the BLA via MasR activation. Indeed, ACE2 overexpression was associated with increased frequency of spontaneous inhibitory postsynaptic currents (indicative of presynaptic release of GABA) onto BLA pyramidal neurons and central infusion of A-779 eliminated this effect. Collectively, these results suggest that ACE2 may reduce anxiety-like behavior by activating central MasR that facilitate GABA release onto pyramidal neurons within the BLA. PMID:26767952

[Biochemical basis of the single theory of aging. Part II. The cell aerobic status, the hypoxia resistance and proliferation].

PubMed

Kirova, Iu I; Borodulin, V B

2009-01-01

Cells of an organism have different parameters of morphology, metabolism, isoenzyme composition, proliferation and respiration. These differences are derivatives of the cell aerobic status. The primary oxygen acceptors are the "macroscopic" cells (neurons, cardiocytes). In these obligatory aerobic cells oxygen is converted into metabolic water directly by the cytochrome oxidase activity. The secondary oxygen acceptors are the "microscopic" cells (other single-nucleus cells). In these facultative aerobic cells oxygen is converted into hydrogen peroxide. The intracellular labile peroxide pool of oxygen is formed by the oxidase, cytochrome P450, superoxide dismutase, and the mitochondrial cyan-resistance oxidase. The mitochondrial isoenzymes of catalase, glutation peroxidase, and thioredoxin reductase convert hydrogen peroxide into molecular oxygen and form high local oxygen concentration as the major factor for the cytochrome oxidase activity. The hypoxia resistance is increased by the growth of the functional activity of the peroxide-generative and peroxide-mobilizative enzyme systems.

Survey of enzyme activity responsible for phenolic off-flavour production by Dekkera and Brettanomyces yeast.

PubMed

Harris, Victoria; Ford, Christopher M; Jiranek, Vladimir; Grbin, Paul R

2009-01-01

Volatile phenols are produced by Dekkera yeasts and are of organoleptic importance in alcoholic beverages. The key compound in this respect is 4-ethylphenol, responsible for the medicinal and phenolic aromas in spoiled wines. The microbial synthesis of volatile phenols is thought to occur in two steps, beginning with naturally occurring hydroxycinnamic acids (HCAs). The enzyme phenolic acid decarboxylase (PAD) converts HCAs to vinyl derivatives, which are the substrates of a second enzyme, postulated to be a vinylphenol reductase (VPR), whose activity results in the formation of ethylphenols. Here, both steps of the pathway are investigated, using cell extracts from a number of Dekkera and Brettanomyces species. Dekkera species catabolise ferulic, caffeic and p-coumaric acids and possess inducible enzymes with similar pH and temperature optima. Brettanomyces does not decarboxylate HCAs but does metabolise vinylphenols. Dekkera species form ethylphenols but the VPR enzyme appears to be highly unstable in cell extracts. A partial protein sequence for PAD was determined from Dekkera anomala and may indicate the presence of a novel enzyme in this genus.

Enzyme-triggered compound release using functionalized antimicrobial peptide derivatives† †Electronic supplementary information (ESI) available. See DOI: 10.1039/c6sc04435b Click here for additional data file.

PubMed Central

Kashibe, Masayoshi; Matsumoto, Kengo; Hori, Yuichiro

2017-01-01

Controlled release is one of the key technologies for medical innovation, and many stimulus-responsive nanocarriers have been developed to utilize this technology. Enzyme activity is one of the most useful stimuli, because many enzymes are specifically activated in diseased tissues. However, controlled release stimulated by enzyme activity has not been frequently reported. One of the reasons for this is the lack of versatility of carriers. Most of the reported stimulus-responsive systems involve a sophisticated design and a complicated process for the synthesis of stimulus-responsive nanocarrier components. The purpose of this study was to develop versatile controlled release systems triggered by various stimuli, including enzyme activity, without modifying the nanocarrier components. We developed two controlled release systems, both of which comprised a liposome as the nanocarrier and a membrane-damaging peptide, temporin L (TL), and its derivatives as the release-controllers. One system utilized branched peptides for proteases, and the other utilized phosphopeptides for phosphatases. In our systems, the target enzymes converted the non-membrane-damaging TL derivatives into membrane-damaging peptides and released the liposome inclusion. We demonstrated the use of our antimicrobial peptide-based controlled release systems for different enzymes and showed the promise of this technology as a novel theranostic tool. PMID:28451373

Lactic acid bacteria: inhibition of angiotensin converting enzyme in vitro and in vivo.

PubMed

Fuglsang, Anders; Rattray, Fergal P; Nilsson, Dan; Nyborg, Niels C B

2003-01-01

A total of 26 strains of wild-type lactic acid bacteria, mainly belonging to Lactococcus lactis and Lactobacillus helveticus, were assayed in vitro for their ability to produce a milk fermentate with inhibitory activity towards angiotensin converting enzyme (ACE). It was clear that the test strains in this study, in general, produce inhibitory substances in varying amounts. Using a spectrophotometric assay based on amino group derivatization with ortho-phthaldialdehyde as a measure of relative peptide content, it was shown that there is a significant correlation between peptide formation and ACE inhibition, indicating that peptide measurement constitutes a convenient selection method. The effect of active fermentates on in vivo ACE activity was demonstrated in normotensive rats. The pressor effect of angiotensin I (0.3 microg/kg) upon intravenous injection was significantly lower when rats were pre-fed with milks fermented using two strains of Lactobacillus helveticus. An increased response to bradykinin (10 microg/kg, intravenously injected) was observed using one of these fermented milks. It is concluded that Lactobacillus helveticus produces substances which in vivo can give rise to an inhibition of ACE. The inhibition in vivo was low compared to what can be achieved with classical ACE inhibitors. The clinical relevance of this finding is discussed. This work is the first in which an effect of fermented milk on ACE in vivo has been demonstrated, measured as decreased ability to convert angiotensin I to angiotensin II.

Enzymes involved in branched-chain amino acid metabolism in humans.

PubMed

Adeva-Andany, María M; López-Maside, Laura; Donapetry-García, Cristóbal; Fernández-Fernández, Carlos; Sixto-Leal, Cristina

2017-06-01

Branched-chain amino acids (leucine, isoleucine and valine) are structurally related to branched-chain fatty acids. Leucine is 2-amino-4-methyl-pentanoic acid, isoleucine is 2-amino-3-methyl-pentanoic acid, and valine is 2-amino-3-methyl-butanoic acid. Similar to fatty acid oxidation, leucine and isoleucine produce acetyl-coA. Additionally, leucine generates acetoacetate and isoleucine yields propionyl-coA. Valine oxidation produces propionyl-coA, which is converted into methylmalonyl-coA and succinyl-coA. Branched-chain aminotransferase catalyzes the first reaction in the catabolic pathway of branched-chain amino acids, a reversible transamination that converts branched-chain amino acids into branched-chain ketoacids. Simultaneously, glutamate is converted in 2-ketoglutarate. The branched-chain ketoacid dehydrogenase complex catalyzes the irreversible oxidative decarboxylation of branched-chain ketoacids to produce branched-chain acyl-coA intermediates, which then follow separate catabolic pathways. Human tissue distribution and function of most of the enzymes involved in branched-chain amino acid catabolism is unknown. Congenital deficiencies of the enzymes involved in branched-chain amino acid metabolism are generally rare disorders. Some of them are associated with reduced pyruvate dehydrogenase complex activity and respiratory chain dysfunction that may contribute to their clinical phenotype. The biochemical phenotype is characterized by accumulation of the substrate to the deficient enzyme and its carnitine and/or glycine derivatives. It was established at the beginning of the twentieth century that the plasma level of the branched-chain amino acids is increased in conditions associated with insulin resistance such as obesity and diabetes mellitus. However, the potential clinical relevance of this elevation is uncertain.

Application of liquid chromatography-electrospray ionization mass spectrometry for study of steroid-converting enzymes.

PubMed

Miksík, Ivan; Mikulíková, Katerina; Pácha, Jirí; Kucka, Marek; Deyl, Zdenek

2004-02-05

A high-performance liquid chromatography-atmospheric pressure ionization-electrospray ionization mass spectrometry (HPLC-API-ESI-MS) method was developed for the analysis of steroids in a study of steroid-converting enzymes. Separations ware done on a Zorbax Eclipse XDB-C18 column (eluted with a linear methanol-water-acetic acid gradient) and identification of the steroids involved was done by API-ESI-MS using positive ion mode and extracted ion analysis. The applicability of the present method for studying steroid metabolism was proven in assaying two steroid-converting enzymes (20beta-hydroxysteroid dehydrogenase and 11beta-hydroxysteroid dehydrogenase) in various biological samples (rat and chicken intestine, chicken oviduct).

Characterization of Two Late-Stage Enzymes Involved in Fosfomycin Biosynthesis in Pseudomonads.

PubMed

Olivares, Philip; Ulrich, Emily C; Chekan, Jonathan R; van der Donk, Wilfred A; Nair, Satish K

2017-02-17

The broad-spectrum phosphonate antibiotic fosfomycin is currently in use for clinical treatment of infections caused by both Gram-positive and Gram-negative uropathogens. The antibiotic is biosynthesized by various streptomycetes, as well as by pseudomonads. Notably, the biosynthetic strategies used by the two genera share only two steps: the first step in which primary metabolite phosphoenolpyruvate (PEP) is converted to phosphonopyruvate (PnPy) and the terminal step in which 2-hydroxypropylphosphonate (2-HPP) is converted to fosfomycin. Otherwise, distinct enzymatic paths are employed. Here, we biochemically confirm the last two steps in the fosfomycin biosynthetic pathway of Pseudomonas syringae PB-5123, showing that Psf3 performs the reduction of 2-oxopropylphosphonate (2-OPP) to (S)-2-HPP, followed by the Psf4-catalyzed epoxidation of (S)-2-HPP to fosfomycin. Psf4 can also accept (R)-2-HPP as a substrate but instead performs an oxidation to make 2-OPP. We show that the combined activities of Psf3 and Psf4 can be used to convert racemic 2-HPP to fosfomycin in an enantioconvergent process. X-ray structures of each enzyme with bound substrates provide insights into the stereospecificity of each conversion. These studies shed light on the reaction mechanisms of the two terminal enzymes in a distinct pathway employed by pseudomonads for the production of a potent antimicrobial agent.

Molecular diversity of tuliposide A-converting enzyme in the tulip.

PubMed

Nomura, Taiji; Tsuchigami, Aya; Ogita, Shinjiro; Kato, Yasuo

2013-01-01

Tuliposide A-converting enzyme (TCEA) catalyzes the conversion of 6-tuliposide A to its lactonized aglycon, tulipalin A, in the tulip (Tulipa gesneriana). The TgTCEA gene, isolated previously from petals, was transcribed in all tulip tissues but not in the bulbs despite the presence of TCEA activity, which allowed prediction of the presence of a TgTCEA isozyme gene preferentially expressed in the bulbs. Here, the TgTCEA-b gene, the TgTCEA homolog, was identified in bulbs. TgTCEA-b polypeptides showed approximately 77% identity to the petal TgTCEA. Functional characterization of the recombinant enzyme verified that TgTCEA-b encoded the TCEA. Moreover, the TgTCEA-b was found to be localized to plastids, as found for the petal TgTCEA. Transcript analysis revealed that TgTCEA-b was functionally transcribed in the bulb scales, unlike the TgTCEA gene, whose transcripts were absent there. In contrast, TgTCEA-b transcripts were in the minority in other tissues where TgTCEA transcripts were dominant, indicating a tissue preference for the transcription of those isozyme genes.

Role of tumour necrosis factor alpha converting enzyme (TACE/ADAM17) and associated proteins in coronary artery disease and cardiac events.

PubMed

Chemaly, Melody; McGilligan, Victoria; Gibson, Mark; Clauss, Matthias; Watterson, Steven; Alexander, H Denis; Bjourson, Anthony John; Peace, Aaron

2017-12-01

Tumour necrosis factor alpha converting enzyme (TACE/ADAM17) is a member of the A disintegrin and metalloproteinase (ADAM) family of ectodomain shedding proteinases. It regulates many inflammatory processes by cleaving several transmembrane proteins, including tumour necrosis factor alpha (TNFα) and its receptors tumour necrosis factor alpha receptor 1 and tumour necrosis factor alpha receptor 2. There is evidence that TACE is involved in several inflammatory diseases, such as ischaemia, heart failure, arthritis, atherosclerosis, diabetes and cancer as well as neurological and immune diseases. This review summarizes the latest discoveries regarding the mechanism of action and regulation of TACE. It also focuses on the role of TACE in atherosclerosis and coronary artery disease (CAD), highlighting clinical studies that have investigated its expression and protein activity. The multitude of substrates cleaved by TACE make this enzyme an attractive target for therapy and a candidate for biomarker research and development in CAD. Crown Copyright © 2017. Published by Elsevier Masson SAS. All rights reserved.

Novel angiotensin I-converting enzyme inhibitory peptides produced in fermented milk by specific wild Lactococcus lactis strains.

PubMed

Rodríguez-Figueroa, J C; González-Córdova, A F; Torres-Llanez, M J; Garcia, H S; Vallejo-Cordoba, B

2012-10-01

The ability of specific wild Lactococcus lactis strains to hydrolyze milk proteins to release angiotensin I-converting enzyme (ACE) inhibitory peptides was evaluated. The peptide profiles were obtained from the <3 kDa water-soluble extract and subsequently fractionated by reversed-phase HPLC. The fractions with the lowest half-maximal inhibitory concentration estimated values (peptide concentration necessary to inhibit ACE activity by 50%) were Lc. lactis NRRL B-50571 fraction (F)1 (0.034 ± 0.002 μg/mL; mean ± SD) and Lc. lactis NRRL B-50572B F 0005 (0.041 ± 0.003 μg/mL; mean ± SD). All peptide fractions were analyzed by reversed-phase HPLC tandem mass spectrometry. Twenty-one novel peptide sequences associated with ACE inhibitory (ACEI) activity were identified. Several novel ACEI peptides presented peptides encrypted with proven hypotensive activity. In conclusion, specific wild Lc. lactis strains were able to hydrolyze milk proteins to generate potent ACEI peptides. However, further studies are necessary to find out the relationship between Lc. lactis strain proteolytic systems and their ability to biogenerate hypotensive peptides. Copyright © 2012 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.

Presence of angiotensin converting enzyme isoforms in larval lepidoptera (Spodoptera littoralis).

PubMed

Lemeire, E; Van Camp, J; Smagghe, G

2007-01-01

In this research the presence of angiotensin converting enzyme (ACE) in larvae of the lepidopteran Spodoptera littoralis was evaluated. Making use of the substrate Abz-FRK-(Dnp)P-OH and the specific inhibitor captopril at 10 microM, ACE activity was determined in a fluorescence assay for intact larvae, hemolymph, head, midgut and dorsal tissue. In dorsal tissue and hemolymph, ACE activity was highest. These data are consistent with a possible role for ACE in contractions of the dorsal vessel and metabolism of circulating peptide hormones in the hemolymph. After the presence of ACE was confirmed, a sequential procedure of anion exchange and size exclusion chromatography was applied to purify ACE from whole wandering larvae (last stage). With this procedure, three different ACE pools were collected that cleaved the fluorogenic substrate Abz-FRK-(Dnp)P-OH. Activity could be inhibited by a final concentration of 2.5 microM captopril. In addition, two out of three samples eluted at different salt concentration and thus ACE 1, 2 and 3 represent at least two different ACE isoforms. These data reveal that ACE is present in S. littoralis and that at least two out of three isolated ACE forms are truly isoforms.

21 CFR 862.1090 - Angiotensin converting enzyme (A.C.E.) test system.

Code of Federal Regulations, 2010 CFR

2010-04-01

... 21 Food and Drugs 8 2010-04-01 2010-04-01 false Angiotensin converting enzyme (A.C.E.) test system. 862.1090 Section 862.1090 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES CLINICAL CHEMISTRY AND CLINICAL TOXICOLOGY DEVICES Clinical Chemistry...

21 CFR 862.1090 - Angiotensin converting enzyme (A.C.E.) test system.

Code of Federal Regulations, 2011 CFR

2011-04-01

... 21 Food and Drugs 8 2011-04-01 2011-04-01 false Angiotensin converting enzyme (A.C.E.) test system. 862.1090 Section 862.1090 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES CLINICAL CHEMISTRY AND CLINICAL TOXICOLOGY DEVICES Clinical Chemistry...

21 CFR 862.1090 - Angiotensin converting enzyme (A.C.E.) test system.

Code of Federal Regulations, 2012 CFR

2012-04-01

... 21 Food and Drugs 8 2012-04-01 2012-04-01 false Angiotensin converting enzyme (A.C.E.) test system. 862.1090 Section 862.1090 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES CLINICAL CHEMISTRY AND CLINICAL TOXICOLOGY DEVICES Clinical Chemistry...

21 CFR 862.1090 - Angiotensin converting enzyme (A.C.E.) test system.

Code of Federal Regulations, 2013 CFR

2013-04-01

... 21 Food and Drugs 8 2013-04-01 2013-04-01 false Angiotensin converting enzyme (A.C.E.) test system. 862.1090 Section 862.1090 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES CLINICAL CHEMISTRY AND CLINICAL TOXICOLOGY DEVICES Clinical Chemistry...

Biological production of organic compounds

DOEpatents

Yu, Jianping; Wang, Bo; Paddock, Troy; Carrieri, Damian; Maness, Pin-Ching; Seibert, Michael

2018-03-13

Methods of producing ethylene oxide and ethylene glycol are disclosed herein. Ethylene produced by cyanobacteria engineered to express ethylene-forming enzymes may be converted to ethylene oxide by bacteria engineered to express a monooxygenase enzyme. Ethylene oxide may be converted to ethylene glycol by exposure to an acidic solution. The methods may be performed in a bioreactor.

Serum protease activity in chronic kidney disease patients: The GANI_MED renal cohort

PubMed Central

Wolke, Carmen; Teumer, Alexander; Endlich, Karlhans; Endlich, Nicole; Rettig, Rainer; Stracke, Sylvia; Fiene, Beate; Aymanns, Simone; Felix, Stephan B; Hannemann, Anke

2016-01-01

Serum or plasma proteases have been associated with various diseases including cancer, inflammation, or reno-cardiovascular diseases. We aimed to investigate whether the enzymatic activities of serum proteases are associated with the estimated glomerular filtration rate (eGFR) in patients with different stages of chronic kidney disease (CKD). Our study population comprised 268 participants of the “Greifswald Approach to Individualized Medicine” (GANI_MED) cohort. Enzymatic activity of aminopeptidase A, aminopeptidase B, alanyl (membrane) aminopeptidase, insulin-regulated aminopeptidase, puromycin-sensitive aminopeptidase, leucine aminopeptidase 3, prolyl-endopeptidase (PEP), dipeptidyl peptidase 4 (DPP4), angiotensin I-converting enzyme, and angiotensin I-converting enzyme 2 (ACE2) proteases was measured in serum. Linear regression of the respective protease was performed on kidney function adjusted for age and sex. Kidney function was modeled either by the continuous Modification of Diet in Renal Disease (MDRD)-based eGFR or dichotomized by eGFR < 15 mL/min/1.73 m2 or <45 mL/min/1.73 m2, respectively. Results with a false discovery rate below 0.05 were deemed statistically significant. Among the 10 proteases investigated, only the activities of ACE2 and DPP4 were correlated with eGFR. Patients with lowest eGFR exhibited highest DPP4 and ACE2 activities. DPP4 and PEP were correlated with age, but all other serum protease activities showed no associations with age or sex. Our data indicate that ACE2 and DPP4 enzymatic activity are associated with the eGFR in patients with CKD. This finding distinguishes ACE2 and DPP4 from other serum peptidases analyzed and clearly indicates that further analyses are warranted to identify the precise role of these serum ectopeptidases in the pathogenesis of CKD and to fully elucidate underlying molecular mechanisms. Impact statement • Renal and cardiac diseases are very common and often occur concomitantly, resulting in increased morbidity and mortality. Understanding of molecular mechanisms linking both diseases is limited, available fragmentary data point to a role of the renin–angiotensin system (RAS) and, in particular, Ras-related peptidases. • Here, a comprehensive analysis of serum peptidase activities in patients with different stages of chronic kidney disease (CKD) is presented, with special emphasis given to RAS peptidases • The serum activities of the peptidases angiotensin I-converting enzyme 2 and dipeptidyl peptidase 4 were identified as closely associated with kidney function, specifically with the estimated glomerular filtration rate. The findings are discussed in the context of available data suggesting protective roles for both enzymes in reno-cardiac diseases. • The data add to our understanding of pathomechanisms underlying development and progression of CKD and indicate that both enzymes might represent potential pharmacological targets for the preservation of renal function. PMID:28038565

Serum protease activity in chronic kidney disease patients: The GANI_MED renal cohort.

PubMed

Wolke, Carmen; Teumer, Alexander; Endlich, Karlhans; Endlich, Nicole; Rettig, Rainer; Stracke, Sylvia; Fiene, Beate; Aymanns, Simone; Felix, Stephan B; Hannemann, Anke; Lendeckel, Uwe

2017-03-01

Serum or plasma proteases have been associated with various diseases including cancer, inflammation, or reno-cardiovascular diseases. We aimed to investigate whether the enzymatic activities of serum proteases are associated with the estimated glomerular filtration rate (eGFR) in patients with different stages of chronic kidney disease (CKD). Our study population comprised 268 participants of the "Greifswald Approach to Individualized Medicine" (GANI_MED) cohort. Enzymatic activity of aminopeptidase A, aminopeptidase B, alanyl (membrane) aminopeptidase, insulin-regulated aminopeptidase, puromycin-sensitive aminopeptidase, leucine aminopeptidase 3, prolyl-endopeptidase (PEP), dipeptidyl peptidase 4 (DPP4), angiotensin I-converting enzyme, and angiotensin I-converting enzyme 2 (ACE2) proteases was measured in serum. Linear regression of the respective protease was performed on kidney function adjusted for age and sex. Kidney function was modeled either by the continuous Modification of Diet in Renal Disease (MDRD)-based eGFR or dichotomized by eGFR < 15 mL/min/1.73 m 2 or <45 mL/min/1.73 m 2 , respectively. Results with a false discovery rate below 0.05 were deemed statistically significant. Among the 10 proteases investigated, only the activities of ACE2 and DPP4 were correlated with eGFR. Patients with lowest eGFR exhibited highest DPP4 and ACE2 activities. DPP4 and PEP were correlated with age, but all other serum protease activities showed no associations with age or sex. Our data indicate that ACE2 and DPP4 enzymatic activity are associated with the eGFR in patients with CKD. This finding distinguishes ACE2 and DPP4 from other serum peptidases analyzed and clearly indicates that further analyses are warranted to identify the precise role of these serum ectopeptidases in the pathogenesis of CKD and to fully elucidate underlying molecular mechanisms. Impact statement • Renal and cardiac diseases are very common and often occur concomitantly, resulting in increased morbidity and mortality. Understanding of molecular mechanisms linking both diseases is limited, available fragmentary data point to a role of the renin-angiotensin system (RAS) and, in particular, Ras-related peptidases. • Here, a comprehensive analysis of serum peptidase activities in patients with different stages of chronic kidney disease (CKD) is presented, with special emphasis given to RAS peptidases • The serum activities of the peptidases angiotensin I-converting enzyme 2 and dipeptidyl peptidase 4 were identified as closely associated with kidney function, specifically with the estimated glomerular filtration rate. The findings are discussed in the context of available data suggesting protective roles for both enzymes in reno-cardiac diseases. • The data add to our understanding of pathomechanisms underlying development and progression of CKD and indicate that both enzymes might represent potential pharmacological targets for the preservation of renal function.

Forkhead Box Transcription Factors of the FOXA Class Are Required for Basal Transcription of Angiotensin-Converting Enzyme 2

PubMed Central

Pedersen, Kim Brint; Chodavarapu, Harshita

2017-01-01

Angiotensin-converting enzyme 2 (ACE2) has protective effects on a wide range of morbidities associated with elevated angiotensin-II signaling. Most tissues, including pancreatic islets, express ACE2 mainly from the proximal promoter region. We previously found that hepatocyte nuclear factors 1α and 1β stimulate ACE2 expression from three highly conserved hepatocyte nuclear factor 1 binding motifs in the proximal promoter region. We hypothesized that other highly conserved motifs would also affect ACE2 expression. By systematic mutation of conserved elements, we identified five regions affecting ACE2 expression, of which two regions bound transcriptional activators. One of these is a functional FOXA binding motif. We further identified the main protein binding the FOXA motif in 832/13 insulinoma cells as well as in mouse pancreatic islets as FOXA2. PMID:29082356

Reduction of mean arterial pressure and proteinuria by the effect of ACEIs (Lisinopril) in Kurdish hypertensive patients in Hawler City.

PubMed

Muslih, A I

2012-06-30

The angiotensin converting enzyme inhibitors (ACEIs) are a group of pharmaceuticals that are used primarily in treatment of hypertension and congestive heart failure, in some cases as the drugs of first choice. The renin-angiotensin system is activated in response to hypotension, decreased sodium concentration in the distal tubule, decreased blood volume and in renal sympathetic nerve stimulation. This study examines the effects of angiotensin converting enzyme inhibitor (Lisinopril) on blood pressure (BP) 131 ± 2.4 and proteinuria 0.198 ± 0.005 in Kurd hypertensive patients, mean arterial blood pressure and proteinuria excretion were measured weekly along the period of 12 weeks. Lisinopril significantly reduced mean arterial blood pressure, and attenuated proteinuria level in patients subjected to this study in lisinopril 10mg dose dependent manner (p<0.05, n=24). In conclusion, lisinopril is of beneficial of renoprotection and in lowering BP.

Characterizing the distribution of steroid sulfatase during embryonic development: when and where might metabolites of maternal steroids be reactivated?

PubMed

Paitz, Ryan T; Duffield, Kristin R; Bowden, Rachel M

2017-12-15

All vertebrate embryos are exposed to maternally derived steroids during development. In placental vertebrates, metabolism of maternal steroids by the placenta modulates embryonic exposure, but how exposure is regulated in oviparous vertebrates is less clear. Recent work in oviparous vertebrates has demonstrated that steroids are not static molecules, as they can be converted to more polar steroid sulfates by sulfotransferase enzymes. Importantly, these steroid sulfates can be converted back to the parent compound by the enzyme steroid sulfatase (STS). We investigated when and where STS was present during embryonic development in the red-eared slider turtle, Trachemys scripta We report that STS is present during all stages of development and in all tissues we examined. We conclude that STS activity may be particularly important for regulating maternal steroid exposure in oviparous vertebrates. © 2017. Published by The Company of Biologists Ltd.

An Iterative, Bimodular Nonribosomal Peptide Synthetase that Converts Anthranilate and Tryptophan into Tetracyclic Asperlicins

PubMed Central

Gao, Xue; Jiang, Wei; Jiménez-Osés, Gonzalo; Choi, Moon Seok; Houk, Kendall N.; Tang, Yi; Walsh, Christopher T.

2013-01-01

The bimodular 276 kDa nonribosomal peptide synthetase AspA from Aspergillus alliaceus, heterologously expressed in Saccharomyces cerevisiae, converts tryptophan and two molecules of the aromatic β-amino acid anthranilate (Ant) into a pair of tetracyclic peptidyl alkaloids asperlicin C and D in a ratio of 10:1. The first module of AspA activates and processes two molecules of Ant iteratively to generate a tethered Ant-Ant-Trp-S-enzyme intermediate on module two. Release is postulated to involve tandem cyclizations, in which the first step is the macrocyclization of the linear tripeptidyl-S-enzyme, by the terminal condensation (CT) domain to generate the regioisomeric tetracyclic asperlicin scaffolds. Computational analysis of the transannular cyclization of the 11-membered macrocyclic intermediate shows that asperlicin C is the kinetically favored product due to the high stability of a conformation resembling the transition state for cyclization, while asperlicin D is thermodynamically more stable. PMID:23890005

[Prospective observational study of angiotensin converting enzyme inhibitors-induced hyperkalemia in hospitalized patients with chronic renal failure].

PubMed

Ben Mahmoud, Lobna; Ghozzi, Hanene; Kammoun, Khawla; Hakim, Ahmed; Kharrat, Mahmoud; Ben Hmida, Mohamed; Jarraya, Faical; Sahnoun, Zouheir; Zeghal, Khaled; Hachicha, Jamil

2013-04-01

To study the incidence and risk factors of angiotensin converting enzyme inhibitors-induced hyperkalemia in hospitalized patients with hypertension and preexisting chronic renal failure. Two-months prospective observational study was used including all hospitalized patients older than 18 years with a history of hypertension, non-dialyzed chronic renal failure and who had angiotensin converting enzyme prescription at the time of the admission. Hyperkalemia greater than or equal to 5 mmol/L was detected in these patients. The studied variables were demographic, clinical, biological and therapeutic. Eight patients, among 27 included, had a hyperkalemia (2963%). They were 73±15 years old. Factors that predispose to hyperkalemia were present in all patients. Hyperkalemia was associated in six cases with decompensation of renal function. The age was associated with hyperkalaemia in patients treated with angiotensin converting enzyme inhibitors (RC=1.21; IC95 1,11-1,46; P=0,021). Diabetes is a possible risk factor (OR=59 021 et, 95 0.93 to 2410, P=0.053). Compared with patients who did not develop hyperkalemia, the occurrence of hyperkalemia in patients included was associated with a longer duration of hospitalization (OR=130, 95 112 to 160, P=0. 022). The prescription of angiotensin converting enzyme inhibitors in the elderly with chronic renal failure and diabetes requires careful monitoring of serum potassium. Copyright © 2012 Association Société de néphrologie. Published by Elsevier SAS. All rights reserved.

Genetic and physiological characterization of the purine salvage pathway in the archaebacterium Methanobacterium thermoautotrophicum Marburg.

PubMed Central

Worrell, V E; Nagle, D P

1990-01-01

The enzymes involved in the purine interconversion pathway of wild-type and purine analog-resistant strains of Methanobacterium thermoautotrophicum Marburg were assayed by radiometric and spectrophotometric methods. Wild-type cells incorporated labeled adenine, guanine, and hypoxanthine, whereas mutant strains varied in their ability to incorporate these bases. Adenine, guanine, hypoxanthine, and xanthine were activated by phosphoribosyltransferase activities present in wild-type cell extracts. Some mutant strains simultaneously lost the ability to convert both guanine and hypoxanthine to the respective nucleotide, suggesting that the same enzyme activates both bases. Adenosine, guanosine, and inosine phosphorylase activities were detected for the conversion of base to nucleoside. Adenine deaminase activity was detected at low levels. Guanine deaminase activity was not detected. Nucleoside kinase activities for the conversion of adenosine, guanosine, and inosine to the respective nucleotides were detected by a new assay. The nucleotide-interconverting enzymes AMP deaminase, succinyl-AMP synthetase, succinyl-AMP lyase, IMP dehydrogenase, and GMP synthetase were present in extracts; GMP reductase was not detected. The results indicate that this autotrophic methanogen has a complex system for the utilization of exogenous purines. PMID:2345148

Characterization of PgPepO, a bacterial homologue of endothelin-converting enzyme-1.

PubMed

Carson, Julie A; Ansai, Toshihiro; Awano, Shuji; Yu, Weixian; Takehara, Tadamichi; Turner, Anthony J

2002-08-01

PgPepO is a homologue of endothelin-converting enzyme-1 (ECE-1), with which it shares 31% identity. PgPepO was isolated from the periodontal pathogen Porphyromonas gingivalis. Recent studies have suggested a link between periodontal and cardiovascular disease, and several groups have suggested that bacterial and viral infections may contribute to the latter. P. gingivalis possesses the ability to invade, and multiply within, aortic endothelial cells and has been localized to atherosclerotic plaques. PgPepO was expressed and purified to homogeneity and we have begun detailed functional analysis, in terms of substrate preference and inhibitor specificity, in order to provide active-site comparisons with other members of the neprilysin (NEP)/ECE family. PgPepO possesses similar substrate specificity to ECE-1 and has been shown to cleave big endothelin-1 (big ET-1), big ET-2 and big ET-3, converting the substrates into their respective mature endothelin peptides. Substance P, angiotensin I, angiotensin II and neurotensin are all cleaved at multiple sites by PgPepO and the kinetics of these reactions have been compared. The potent vasoconstrictor urotensin II is not hydrolysed by PgPepO. Cleavage of bradykinin by PgPepO occurs at the Pro(7)-Phe(8) bond and is inhibited by the NEP and ECE-1 inhibitor phosphoramidon in a pH-dependent fashion (IC(50) =10 microM at pH 7.0) but not by thiorphan, an NEP-specific inhibitor. PgPepO activity is completely inhibited by EDTA. Characterization of this enzyme is important in elucidating possible links between periodontal pathogens and cardiovascular disorders such as atherosclerosis, and provides an opportunity to gain structural information on a bacterial protein with striking similarity to human ECE-1.

Heterologous Expression of a Bioactive β-Hexosyltransferase, an Enzyme Producer of Prebiotics, from Sporobolomyces singularis

PubMed Central

Dagher, Suzanne F.; Azcarate-Peril, M. Andrea

2013-01-01

Galacto-oligosaccharides (GOS) are indigestible dietary fibers that are able to reach the lower gastrointestinal tract to be selectively fermented by health-promoting bacteria. In this report, we describe the heterologous expression of an optimized synthetically produced version of the β-hexosyltransferase gene (Bht) from Sporobolomyces singularis. The Bht gene encodes a glycosyl hydrolase (EC 3.2.1.21) that acts as galactosyltransferase, able to catalyze a one-step conversion of lactose to GOS. Expression of the enzyme in Escherichia coli yielded an inactive insoluble protein, while the methylotrophic yeast Pichia pastoris GS115 produced a bioactive β-hexosyltransferase (rBHT). The enzyme exhibited faster kinetics at pHs between 3.5 and 6 and at temperatures between 40 and 50°C. Enzyme stability improved at temperatures lower than 40°C, and glucose was found to be a competitive inhibitor of enzymatic activity. P. pastoris secreted a fraction of the bioactive rBHT into the fermentation broth, while the majority of the enzyme remained associated with the outer membrane. Both the secreted and the membrane-associated forms were able to efficiently convert lactose to GOS. Additionally, resting cells with membrane-bound enzyme converted 90% of the initial lactose into GOS at 68% yield (g/g) (the maximum theoretical is 75%) with no secondary residual (glucose or galactose) products. This is the first report of a bioactive BHT from S. singularis that has been heterologously expressed. PMID:23241974

Impaired swim bladder inflation in early-life stage fathead minnows exposed to a deiodinase inhibitor, iopanoic acid (article)

EPA Science Inventory

The present study investigated whether inhibition of deiodinase, the enzyme which converts thyroxine (T4) to the more biologically-active form, 3,5,3'-triiodothyronine (T3), would impact inflation of the posterior and/or anterior chamber of the swim bladder, processes previously ...

Impaired swim bladder inflation in early-life stage fathead minnows exposed to a deiodinase inhibitor, iopanoic acid (presentation)

EPA Science Inventory

The present study investigated whether inhibition of deiodinase, the enzyme which converts thyroxine (T4) to the more biologically-active form, 3,5,3'-triiodothyronine (T3), would impact inflation of the posterior and/or anterior chamber of the swim bladder, processes previously ...

Impaired swim bladder inflation in early-life stage fathead minnows exposed to a deiodinase inhibitor, iopanoic acid

EPA Science Inventory

The thyroid axis plays a critical role in teleost fish development. The present study investigated whether inhibition of deiodinase, the enzyme which converts thyroxine (T4), to the more biologically-active form, 3,5,3'-triiodothyronine (T3), would impact inflation of the posteri...

Angiotensin-converting enzyme inhibitors in patients with coronary artery disease and absence of heart failure or left ventricular systolic dysfunction: an overview of long-term randomized controlled trials.

PubMed

Danchin, Nicolas; Cucherat, Michel; Thuillez, Christian; Durand, Eric; Kadri, Zena; Steg, Philippe G

2006-04-10

Results of randomized trials of angiotensin-converting enzyme inhibitors in patients with coronary artery disease (CAD) and preserved left ventricular function are conflicting. We undertook this study to determine whether long-term prescription of angiotensin-converting enzyme inhibitors decreases major cardiovascular events and mortality in patients who have CAD and no evidence of left ventricular systolic dysfunction. We searched MEDLINE, EMBASE, and IPA databases, the Cochrane Controlled Trials Register (1990-2004), and reports from scientific meetings (2003-2004), and we reviewed secondary sources. Search terms included angiotensin-converting enzyme inhibitors, coronary artery disease, randomi(s)zed controlled trials, clinical trials, and myocardial infarction. Eligible studies included randomized controlled trials in patients who had CAD and no heart failure or left ventricular dysfunction, with follow-up omicronf 2 years or longer. Of 1146 publications screened, 7 met our selection criteria and included a total of 33 960 patients followed up for a mean of 4.4 years. Five trials included only patients with documented CAD. One trial included patients with documented CAD (80%) or patients who had diabetes mellitus and 1 or more additional risk factors, and another trial included patients who had CAD, a history of transient ischemic attack, or intermittent claudication. Treatment with angiotensin-converting enzyme inhibitors decreased overall mortality (odds ratio, 0.86; 95% confidence interval, 0.79-0.93), cardiovascular mortality (odds ratio, 0.81; 95% confidence interval, 0.73-0.90), myocardial infarction (odds ratio, 0.82; 95% confidence interval, 0.75-0.89), and stroke (odds ratio, 0.77; 95% confidence interval, 0.66-0.88). Other end points, including resuscitation after cardiac arrest, myocardial revascularization, and hospitalization because of heart failure, were also reduced. Angiotensin-converting enzyme inhibitors reduce total mortality and major cardiovascular end points in patients who have CAD and no left ventricular systolic dysfunction or heart failure.

N- vs. C-Domain Selectivity of Catalytic Inactivation of Human Angiotensin Converting Enzyme by Lisinopril-Coupled Transition Metal Chelates

PubMed Central

Hocharoen, Lalintip; Joyner, Jeff C.; Cowan, J. A.

2014-01-01

The N- and C-terminal domains of human somatic Angiotensin I Converting Enzyme (sACE-1) demonstrate distinct physiological functions, with resulting interest in the development of domain-selective inhibitors for specific therapeutic applications. Herein, the activity of lisinopril-coupled transition metal chelates were tested for both reversible binding and irreversible catalytic inactivation of sACE-1. C/N domain binding selectivity ratios ranged from 1 to 350, while rates of irreversible catalytic inactivation of the N- and C-domains were found to be significantly greater for the N-domain, suggesting a more optimal orientation of the M-chelate-lisinopril complexes within the active site of the N-domain of sACE-1. Finally, the combined effect of binding selectivity and inactivation selectivity was assessed for each catalyst (double-filter selectivity factors), and several catalysts were found to cause domain-selective catalytic inactivation. The results of this study demonstrate the ability to optimize the target selectivity of catalytic metallopeptides through both binding and orientation factors (double-filter effect). PMID:24228790

N- versus C-domain selectivity of catalytic inactivation of human angiotensin converting enzyme by lisinopril-coupled transition metal chelates.

PubMed

Hocharoen, Lalintip; Joyner, Jeff C; Cowan, J A

2013-12-27

The N- and C-terminal domains of human somatic angiotensin I converting enzyme (sACE-1) demonstrate distinct physiological functions, with resulting interest in the development of domain-selective inhibitors for specific therapeutic applications. Herein, the activity of lisinopril-coupled transition metal chelates was tested for both reversible binding and irreversible catalytic inactivation of each domain of sACE-1. C/N domain binding selectivity ratios ranged from 1 to 350, while rates of irreversible catalytic inactivation of the N- and C-domains were found to be significantly greater for the N-domain, suggesting a more optimal orientation of M-chelate-lisinopril complexes within the active site of the N-domain of sACE-1. Finally, the combined effect of binding selectivity and inactivation selectivity was assessed for each catalyst (double-filter selectivity factors), and several catalysts were found to cause domain-selective catalytic inactivation. The results of this study demonstrate the ability to optimize the target selectivity of catalytic metallopeptides through both binding and catalytic factors (double-filter effect).

Brain angiotensin-(1-7)/Mas axis: A new target to reduce the cardiovascular risk to emotional stress.

PubMed

Fontes, Marco Antônio Peliky; Martins Lima, Augusto; Santos, Robson Augusto Souza dos

2016-04-01

Emotional stress is now considered a risk factor for several diseases including cardiac arrhythmias and hypertension. It is well known that the activation of neuroendocrine and autonomic mechanisms features the response to emotional stress. However, its link to cardiovascular diseases and the regulatory mechanisms involved remain to be further comprehended. The renin-angiotensin system (RAS) plays an important role in homeostasis on all body systems. Specifically in the brain, the RAS regulates a number of physiological aspects. Recent data indicate that the activation of angiotensin-converting enzyme/angiotensin II/AT1 receptor axis facilitates the emotional stress responses. On the other hand, growing evidence indicates that its counterregulatory axis, the angiotensin-converting enzyme 2 (ACE2)/(Ang)iotensin-(1-7)/Mas axis, reduces anxiety and attenuates the physiological responses to emotional stress. The present review focuses on angiotensin-(1-7)/Mas axis as a promising target to attenuate the physiological response to emotional stress reducing the risk of cardiovascular diseases. Copyright © 2015 Elsevier Ltd. All rights reserved.

Potential Therapeutic Applications of Mucuna pruriens Peptide Fractions Purified by High-Performance Liquid Chromatography as Angiotensin-Converting Enzyme Inhibitors, Antioxidants, Antithrombotic and Hypocholesterolemic Agents.

PubMed

Herrera-Chalé, Francisco; Ruiz-Ruiz, Jorge Carlos; Betancur-Ancona, David; Segura-Campos, Maira Rubi

2016-02-01

A Mucuna pruriens protein concentrate was hydrolyzed with a digestive (pepsin-pancreatin) enzymatic system. The soluble portion of the hydrolysate was fractionated by ultrafiltration and the ultrafiltered peptide fraction (PF) with lower molecular weight was purified by reversed-phase high-performance liquid chromatography. The PF obtained were evaluated by testing the biological activity in vitro. Fractions showed that the ability to inhibit the angiotensin-converting enzyme had IC50 values that ranged from 2.7 to 6.2 μg/mL. Trolox equivalent antioxidant capacity values ranged from 132.20 to 507.43 mM/mg. The inhibition of human platelet aggregation ranged from 1.59% to 11.11%, and the inhibition of cholesterol micellar solubility ranged from 0.24% to 0.47%. Hydrophobicity, size, and amino acid sequence could be factors in determining the biological activity of peptides contained in fractions. This is the first report that M. pruriens peptides act as antihypertensives, antioxidants, and inhibitors for human platelet aggregation and cholesterol micellar solubility in vitro.

Graphitized Porous Carbon for Rapid Screening of Angiotensin-Converting Enzyme Inhibitory Peptide GAMVVH from Silkworm Pupa Protein and Molecular Insight into Inhibition Mechanism.

PubMed

Tao, Mengliang; Sun, Huaju; Liu, Long; Luo, Xuan; Lin, Guoyou; Li, Renbo; Zhao, Zhenxia; Zhao, Zhongxing

2017-10-04

A novel hydrophobic hexapeptide with high angiotensin-converting enzyme (ACE) inhibitory activity was screened from silkworm pupa protein (SPP) hydrolysate via graphitized porous carbon and reverse-phase high-performance liquid chromatography methods. Graphitized porous carbon derived from dopamine, possessing high surface area and high graphitic carbon, was used to rapidly screen and enrich hydrophobic peptides from SPP hydrolysate. The ACE inhibition pattern and mechanism of the purified peptide were also systematically studied by the classic Lineweaver-Burk model and by molecular docking/dynamic simulation. The novel hydrophobic hexapeptide was identified as Gly-Ala-Met-Val-Val-His (GAMVVH, IC 50 = 19.39 ± 0.21 μM) with good thermal/antidigestive stabilities. Lineweaver-Burk plots revealed that GAMVVH behaved as a competitive ACE inhibitor. It formed hydrogen bonds with S1 and S2 pockets of ACE and established competitive coordination with Zn(II) of ACE. The synergy of hydrogen bonds with active pockets and Zn(II) coordination efficiently changed the three-dimensional structure of ACE and thus inhibited bioactivity of ACE.

Antihypertensive Effects, Molecular Docking Study, and Isothermal Titration Calorimetry Assay of Angiotensin I-Converting Enzyme Inhibitory Peptides from Chlorella vulgaris.

PubMed

Xie, Jingli; Chen, Xujun; Wu, Junjie; Zhang, Yanyan; Zhou, Yan; Zhang, Lujia; Tang, Ya-Jie; Wei, Dongzhi

2018-02-14

The aim of this work is to explore angiotensin I-converting enzyme (ACE) inhibitory peptides from Chlorella vulgaris (C. vulgaris) and discover the inhibitory mechanism of the peptides. After C. vulgaris proteins were gastrointestinal digested in silico, several ACE inhibitory peptides with C-terminal tryptophan were screened. Among them, two novel noncompetitive ACE inhibitors, Thr-Thr-Trp (TTW) and Val-His-Trp (VHW), exhibited the highest inhibitory activity indicated by IC 50 values 0.61 ± 0.12 and 0.91 ± 0.31 μM, respectively. Both the peptides were demonstrated stable against gastrointestinal digestion and ACE hydrolysis. The peptides were administrated to spontaneously hypertensive rats (SHRs) in the dose 5 mg/kg body weight, and VHW could decrease 50 mmHg systolic blood pressure of SHRs (p < 0.05). Molecular docking displayed that both TTW and VHW formed six hydrogen bonds with active site pockets of ACE. Besides, isothermal titration calorimetry assay discovered that VHW could form more stable complex with ACE than TTW. Therefore, VHW was an excellent ACE inhibitor.

Limonoate dehydrogenase from Arthrobacter globiformis: the native enzyme and its N-terminal sequence.

PubMed

Suhayda, C G; Omura, M; Hasegawa, S

1995-09-01

Bitter limonoids in citrus juice lower the quality and value of commercial juices. Limonoate dehydrogenase converts the precursor of bitter limonin, limonoate A-ring lactone, to nonbitter 17-dehydrolimonoate A-ring lactone. This enzyme was isolated from Arthrobacter globiformis cells by a combination of ammonium sulfate fractionation, Cibacron Blue affinity chromatography and DEAE ion exchange HPLC. Using this protocol a 428-fold purification of the enzyme was obtained. Gel filtration HPLC indicated a M(r) of 118,000 for the native enzyme. SDS-PAGE indicated an individual subunit M(r) of 31,000. N-Terminal sequencing of the protein provided a sequence of the first 16 amino acid residues. Since LDH activity in citrus is very low, cloning the gene for this bacterial enzyme into citrus trees should enhance the natural debittering mechanism in citrus fruit.

Rapid conversion of angiotensin I to angiotensin II by neutrophil and mast cell proteinases.

PubMed

Reilly, C F; Tewksbury, D A; Schechter, N M; Travis, J

1982-08-10

Human neutrophil cathepsin G and human skin mast cell chymase rapidly convert angiotensin I to angiotensin II with only minor cleavage elsewhere in the molecule. The rate of cleavage is consistent with a potential role for either or both of these enzymes in an alternate pathway for angiotensin II synthesis. Since neither enzyme in inhibited by captopril, an angiotensin converting enzyme inactivator, it is possible that leukocyte and mast cell enzymes may play a significant role in the development of abnormally high local concentrations of angiotensin II, associated with various inflammatory processes.

Whole-Cell Biocatalysis for Producing Ginsenoside Rd from Rb1 Using Lactobacillus rhamnosus GG.

PubMed

Ku, Seockmo; You, Hyun Ju; Park, Myeong Soo; Ji, Geun Eog

2016-07-28

Ginsenosides are the major active ingredients in ginseng used for human therapeutic plant medicines. One of the most well-known probiotic bacteria among the various strains on the functional food market is Lactobacillus rhamnosus GG. Biocatalytic methods using probiotic enzymes for producing deglycosylated ginsenosides such as Rd have a growing significance in the functional food industry. The addition of 2% cellobiose (w/v) to glucose-free de Man-Rogosa-Sharpe broths notably induced β-glucosidase production from L. rhamnosus GG. Enzyme production and activity were optimized at a pH, temperature, and cellobiose concentration of 6.0, 40°C, and 2% (w/v), respectively. Under these controlled conditions, β-glucosidase production in L. rhamnosus GG was enhanced by 25-fold. Additionally, whole-cell homogenates showed the highest β-glucosidase activity when compared with disrupted cell suspensions; the cell disruption step significantly decreased the β-glucosidase activity. Based on the optimized enzyme conditions, whole-cell L. rhamnosus GG was successfully used to convert ginsenoside Rb1 into Rd.

Mineralisation of 14C-labelled synthetic lignin and ligninolytic enzyme activities of litter-decomposing basidiomycetous fungi.

PubMed

Steffen, K T; Hofrichter, M; Hatakka, A

2000-12-01

Within a screening program, 27 soil litter-decomposing basidiomycetes were tested for ligninolytic enzyme activities using agar-media containing 2,2'-azinobis(3-ethylbenzthiazoline-6-sulphonate), a humic acid or Mn2+ ions as indicator substrates. Most active species were found within the family Strophariaceae (Agrocybe praecox, Stropharia coronilla, S. rugosoannulata) and used for mineralisation experiments with a 14C-ring-labelled synthetic lignin (14C-DHP). The fungi mineralised around 25% of the lignin to 14CO2 within 12 weeks of incubation in a straw environment; about 20% of the lignin was converted to water-soluble fragments. Mn-peroxidase was found to be the predominant ligninolytic enzyme of all three fungi in liquid culture and its production was strongly enhanced in the presence of Mn2+ ions. The results of this study demonstrate that certain ubiquitous litter-decomposing basidiomycetes possess ligninolytic activities similar to the wood-decaying white-rot fungi, the most efficient lignin degraders in nature.

Angiotensin I-converting enzyme inhibitors potentiate bradykinin's inotropic effects independently of blocking its inactivation.

PubMed

Minshall, R D; Erdös, E G; Vogel, S M

1997-08-04

The positive inotropic effects of bradykinin (BK) and 2 analogs resistant to angiotensin I-converting enzyme (ACE) were potentiated on isolated guinea pig atrial preparations by enalaprilat. The stable BK analogs, dextran-BK and [Hyp3-Tyr(Me)8]-BK, were as active as BK. Pretreatment for 5 min with enalaprilat augmented the maximal positive inotropic effect of [Hyp3-Tyr(Me)8]-BK 2.8-fold, from 19% to 53% and that of BK from 28% to 42% over baseline; inotropic responses to dextran-BK (1 microM) were similarly increased. The activity of atrial ACE, a zinc-requiring enzyme, was completely inhibited by 8-hydroxyquinoline-5-sulfonic acid (QSA, 10 mM), which raised the maximal inotropic effect of BK to 39% above baseline. This value rose to 67% when in addition to QSA, 1 microM enalaprilat was added; enalaprilat thus, potentiated the effects of BK independently of enzyme inhibition. The positive inotropic effects to BK and its analogs decline with time in the presence of these agonists. After 10 min of exposure, the response to 1 microM [Hyp3-Tyr(Me)8]-BK decreased to about half, and after 20 min, to 0. Enalaprilat, when present in the tissue bath, prevented the decline in inotropy; even after tachyphylaxis occurred, it reversed this decrease in activity when added. The effects of 1 microM [Hyp3-Tyr(Me)8]-BK, in the absence or presence of enalaprilat, were abolished by the BK B2 receptor antagonist icatibant (0.75 microM). The results indicate that ACE inhibitors, by potentiating the BK effects and blocking BK B2-receptor desensitization, may contribute to the beneficial cardiac effects of BK independently of blocking its inactivation.

Renin-Angiotensin Inhibitors Decrease Recurrence after Transurethral Resection of Bladder Tumor in Patients with Nonmuscle Invasive Bladder Cancer.

PubMed

Blute, Michael L; Rushmer, Timothy J; Shi, Fangfang; Fuller, Benjamin J; Abel, E Jason; Jarrard, David F; Downs, Tracy M

2015-11-01

Prior reports suggest that renin-angiotensin system inhibition may decrease nonmuscle invasive bladder cancer recurrence. We evaluated whether angiotensin converting enzyme inhibitor or angiotensin receptor blocker treatment at initial surgery was associated with decreased recurrence or progression in patients with nonmuscle invasive bladder cancer. Using an institutional bladder cancer database we identified 340 patients with data available on initial transurethral resection of bladder tumor. Progression was defined as an increase to stage T2. Cox proportional hazards models were used to evaluate associations with recurrence-free and progression-free survival. Median patient age was 69.6 years. During a median followup of 3 years (IQR 1.3-6.1) 200 patients (59%) had recurrence and 14 (4.1%) had stage progression. Of those patients 143 were receiving angiotensin converting enzyme inhibitor/angiotensin receptor blockers at the time of the first transurethral resection. On univariate analysis factors associated with improved recurrence-free survival included carcinoma in situ (p = 0.040), bacillus Calmette-Guérin therapy (p = 0.003) and angiotensin converting enzyme inhibitor/angiotensin receptor blocker therapy (p = 0.009). Multivariate analysis demonstrated that patients treated with bacillus Calmette-Guérin therapy (HR 0.68, 95% CI 0.47-0.87, p = 0.002) or angiotensin converting enzyme inhibitor/angiotensin receptor blocker therapy (HR 0.61, 95% CI 0.45-0.84, p = 0.005) were less likely to experience tumor recurrence. The 5-year recurrence-free survival rate was 45.6% for patients treated with angiotensin converting enzyme inhibitor/angiotensin receptor blockers and 28.1% in those not treated with angiotensin converting enzyme inhibitor/angiotensin receptor blockers (p = 0.009). Subgroup analysis was performed to evaluate nonmuscle invasive bladder cancer pathology (Ta, T1 and carcinoma in situ) in 85 patients on bacillus Calmette-Guérin therapy alone and in 52 in whom it was combined with angiotensin converting enzyme inhibitor/angiotensin receptor blocker. Multivariate analysis revealed that patients treated with bacillus Calmette-Guérin alone (HR 2.19, 95% CI 1.01-4.77, p = 0.04) showed worse recurrence-free survival compared to patients treated with bacillus Calmette-Guérin and angiotensin converting enzyme inhibitor/angiotensin receptor blocker (stage Ta HR 0.45, 95% CI 0.21-0.98, p = 0.04). Pharmacological inhibition of the renin-angiotensin system is associated with improved outcomes in patients with bladder cancer. Renin-angiotensin system inhibitor administration in nonmuscle invasive bladder cancer cases should be studied in a prospective randomized trial. Copyright © 2015 American Urological Association Education and Research, Inc. Published by Elsevier Inc. All rights reserved.

Combined enzyme mediated fermentation of cellulous and xylose to ethanol by Schizosaccharoyces pombe, cellulase, .beta.-glucosidase, and xylose isomerase

DOEpatents

Lastick, Stanley M.; Mohagheghi, Ali; Tucker, Melvin P.; Grohmann, Karel

1994-01-01

A process for producing ethanol from mixed sugar streams from pretreated biomass comprising xylose and cellulose using enzymes to convert these substrates to fermentable sugars; selecting and isolating a yeast Schizosaccharomyces pombe ATCC No. 2476, having the ability to ferment these sugars as they are being formed to produce ethanol; loading the substrates with the fermentation mix composed of yeast, enzymes and substrates; fermenting the loaded substrates and enzymes under anaerobic conditions at a pH range of between about 5.0 to about 6.0 and at a temperature range of between about 35.degree. C. to about 40.degree. C. until the fermentation is completed, the xylose being isomerized to xylulose, the cellulose being converted to glucose, and these sugars being concurrently converted to ethanol by yeast through means of the anaerobic fermentation; and recovering the ethanol.

Combined enzyme mediated fermentation of cellulose and xylose to ethanol by Schizosaccharomyces pombe, cellulase, [beta]-glucosidase, and xylose isomerase

DOEpatents

Lastick, S.M.; Mohagheghi, A.; Tucker, M.P.; Grohmann, K.

1994-12-13

A process for producing ethanol from mixed sugar streams from pretreated biomass comprising xylose and cellulose using enzymes to convert these substrates to fermentable sugars; selecting and isolating a yeast Schizosaccharomyces pombe ATCC No. 2476, having the ability to ferment these sugars as they are being formed to produce ethanol; loading the substrates with the fermentation mix composed of yeast, enzymes and substrates; fermenting the loaded substrates and enzymes under anaerobic conditions at a pH range of between about 5.0 to about 6.0 and at a temperature range of between about 35 C to about 40 C until the fermentation is completed, the xylose being isomerized to xylulose, the cellulose being converted to glucose, and these sugars being concurrently converted to ethanol by yeast through means of the anaerobic fermentation; and recovering the ethanol. 2 figures.

Dipeptidyl peptidase-4 inhibitor induced angioedema - an overlooked and potentially lethal adverse drug reaction?

PubMed

Scott, Susanne Irene; Andersen, Michelle Fog; Aagaard, Lise; Buchwald, Christian Von; Rasmussen, Eva Rye

2017-02-14

Introduction Angioedema is a potentially fatal adverse drug reaction of some medications, as swellings of the upper airways can cause death by asphyxiation. Angiotensin converting enzyme-inhibitors are widely known to cause angioedema but less is known about the association between dipeptidyl peptidase-4 inhibitors (gliptins) and angioedema. Dipeptidyl peptidase-4 inhibitors are anti-diabetic drugs used to improve glycaemic control. They, as a class effect, inadvertently affect the degradation of the vasoactive kinins bradykinin and substance P, both of which can cause angioedema due to vasodilatation and increase in vascular permeability in the capillaries. Objective To assess the risk and pathomechanism of angioedema due to inhibition of dipeptidyl peptidase-4 inhibitors when used as monotherapy and in combination with angiotensin converting enzyme-inhibitors. Method PubMed, Embase, the Cochrane Library, PubMed Central, Web of Science, Google Scholar and clinicaltrials.gov were searched using different combinations of keywords "angioedema", "dipeptidyl peptidase 4", "dipeptidyl peptidase 4 inhibitors", "gliptins", "bradykinin", "substance P" and "angiotensin converting enzyme-inhibitors". Original research papers were preferably used as references and their bibliographies were used to further the search for original research results. Results Both angiotensin converting enzyme and dipeptidyl peptidase-4 are major enzymes in the degradation pathway of bradykinin and substance P, and when inhibited pharmacologically - especially at the same time - the theoretical risk of angioedema is increased due to accumulation of vasoactive kinins. Conclusion Treatment with dipeptidyl peptidase-4 inhibitors must be carefully considered and monitored especially during concurrent treatment with angiotensin converting enzyme-inhibitors or when treating patients with a known predisposition to angioedema. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

Angiotensin-converting enzyme-inhibitory activity in protein hydrolysates from normal and anthracnose disease-damaged Phaseolus vulgaris seeds.

PubMed

Hernández-Álvarez, Alan Javier; Carrasco-Castilla, Janet; Dávila-Ortiz, Gloria; Alaiz, Manuel; Girón-Calle, Julio; Vioque-Peña, Javier; Jacinto-Hernández, Carmen; Jiménez-Martínez, Cristian

2013-03-15

Bean seeds are an inexpensive source of protein. Anthracnose disease caused by the fungus Colletotrichum lindemuthianum results in serious losses in common bean (Phaseolus vulgaris L.) crops worldwide, affecting any above-ground plant part, and protein dysfunction, inducing the synthesis of proteins that allow plants to improve their stress tolerance. The aim of this study was to evaluate the use of beans damaged by anthracnose disease as a source of peptides with angiotensin-converting enzyme (ACE-I)-inhibitory activity. Protein concentrates from beans spoiled by anthracnose disease and from regular beans as controls were prepared by alkaline extraction and precipitation at isolelectric pH and hydrolysed using Alcalase 2.4 L. The hydrolysates from spoiled beans had ACE-I-inhibitory activity (IC(50) 0.0191 mg protein mL(-1)) and were very similar to those from control beans in terms of ACE-I inhibition, peptide electrophoretic profile and kinetics of hydrolysis. Thus preparation of hydrolysates using beans affected by anthracnose disease would allow for revalorisation of this otherwise wasted product. The present results suggest the use of spoiled bean seeds, e.g. anthracnose-damaged beans, as an alternative for the isolation of ACE-I-inhibitory peptides to be further introduced as active ingredients in functional foods. © 2012 Society of Chemical Industry.

Antioxidant and Angiotensin-Converting Enzyme Inhibitory Activity of Eucalyptus camaldulensis and Litsea glaucescens Infusions Fermented with Kombucha Consortium.

PubMed

Gamboa-Gómez, Claudia I; González-Laredo, Rubén F; Gallegos-Infante, José Alberto; Pérez, Mş Del Mar Larrosa; Moreno-Jiménez, Martha R; Flores-Rueda, Ana G; Rocha-Guzmán, Nuria E

2016-09-01

Physicochemical properties, consumer acceptance, antioxidant and angiotensin-converting enzyme (ACE) inhibitory activities of infusions and fermented beverages of Eucalyptus camaldulensis and Litsea glaucescens were compared. Among physicochemical parameters, only the pH of fermented beverages decreased compared with the unfermented infusions. No relevant changes were reported in consumer preference between infusions and fermented beverages. Phenolic profile measured by UPLC MS/MS analysis demonstrated significant concentration changes of these compounds in plant infusions and fermented beverages. Fermentation induced a decrease in the concentration required to stabilize 50% of DPPH radical ( i . e . lower IC 50 ). Additionally, it enhanced the antioxidant activity measured by the nitric oxide scavenging assay (14% of E. camaldulensis and 49% of L. glaucescens ); whereas relevant improvements in the fermented beverage were not observed in the lipid oxidation assay compared with unfermented infusions. The same behaviour was observed in the inhibitory activity of ACE; however, both infusions and fermented beverages had lower IC 50 than positive control (captopril). The present study demonstrated that fermentation has an influence on the concentration of phenolics and their potential bioactivity. E. camaldulensis and L. glaucescens can be considered as natural sources of biocompounds with antihypertensive potential used either as infusions or fermented beverages.

Antioxidant and Angiotensin-Converting Enzyme Inhibitory Activity of Eucalyptus camaldulensis and Litsea glaucescens Infusions Fermented with Kombucha Consortium

PubMed Central

Gamboa-Gómez, Claudia I.; González-Laredo, Rubén F.; Gallegos-Infante, José Alberto; Pérez, MŞ del Mar Larrosa; Moreno-Jiménez, Martha R.; Flores-Rueda, Ana G.

2016-01-01

Summary Physicochemical properties, consumer acceptance, antioxidant and angiotensin-converting enzyme (ACE) inhibitory activities of infusions and fermented beverages of Eucalyptus camaldulensis and Litsea glaucescens were compared. Among physicochemical parameters, only the pH of fermented beverages decreased compared with the unfermented infusions. No relevant changes were reported in consumer preference between infusions and fermented beverages. Phenolic profile measured by UPLC MS/MS analysis demonstrated significant concentration changes of these compounds in plant infusions and fermented beverages. Fermentation induced a decrease in the concentration required to stabilize 50% of DPPH radical (i.e. lower IC50). Additionally, it enhanced the antioxidant activity measured by the nitric oxide scavenging assay (14% of E. camaldulensis and 49% of L. glaucescens); whereas relevant improvements in the fermented beverage were not observed in the lipid oxidation assay compared with unfermented infusions. The same behaviour was observed in the inhibitory activity of ACE; however, both infusions and fermented beverages had lower IC50 than positive control (captopril). The present study demonstrated that fermentation has an influence on the concentration of phenolics and their potential bioactivity. E. camaldulensis and L. glaucescens can be considered as natural sources of biocompounds with antihypertensive potential used either as infusions or fermented beverages. PMID:27956869

The Role of Plasminogen Activator Inhibitor-1 and Angiotensin-Converting Enzyme Gene Polymorphisms in Bronchopulmonary Dysplasia

PubMed Central

Atac, Fatma Belgin; Ozkiraz, Servet; Dilmen, Ugur; Gulcan, Hande; Tarcan, Aylin; Ozbek, Namik

2010-01-01

Background: Bronchopulmonary dysplasia (BPD) is a multifactorial disease of preterm infants that is characterized by airway injury, inflammation, and parencymal remodeling. Activation of the coagulation cascade leads to intraalveolar fibrin deposition in many inflammatory pulmonary disorders. Increased fibrin formation or decreased fibrinolysis may cause extravascular fibrin deposition. Extravascular fibrin deposits in septae and alveoli due to the altered fibrin turnover are the pathological hallmarks of BPD, which strongly indicate the importance of the imbalance in the competing activities of coagulation and fibrinolysis. Objective: We investigated the predictive value of variations in plasminogen activator inhibitor-1 (PAI-1) and angiotensin-converting enzyme (ACE) genes as molecular determinants for BPD in neonates. Methods: The study group comprised 98 preterm infants with BPD and a control group including 94 preterm infants without BPD. Restriction fragment size analyses were performed by visualizing digested polymerase chain reaction products for ACE and PAI-1 genotypes. Results: No significant associations were found between ACE, PAI-1 gene polymorphisms, and BPD phenotype in our population. Conclusions: The two gene polymorphisms (PAI-1 and ACE) had no role in the development of BPD in our study. Further studies with other genes are required for the identification of molecular predisposing factors for BPD that may help in the development of new treatments. PMID:20818980

Investigation of lactic acid bacterial strains for meat fermentation and the product's antioxidant and angiotensin-I-converting-enzyme inhibitory activities.

PubMed

Takeda, Shiro; Matsufuji, Hisashi; Nakade, Koji; Takenoyama, Shin-Ichi; Ahhmed, Abdulatef; Sakata, Ryoichi; Kawahara, Satoshi; Muguruma, Michio

2017-03-01

In the lactic acid bacteria (LAB) strains screened from our LAB collection, Lactobacillus (L.) sakei strain no. 23 and L. curvatus strain no. 28 degraded meat protein and tolerated salt and nitrite in vitro. Fermented sausages inoculated strains no. 23 and no. 28 showed not only favorable increases in viable LAB counts and reduced pH, but also the degradation of meat protein. The sausages fermented with these strains showed significantly higher antioxidant activity than those without LAB or fermented by each LAB type strain. Angiotensin-I-converting-enzyme (ACE) inhibitory activity was also significantly higher in the sausages fermented with strain no. 23 than in those fermented with the type strain. Higher ACE inhibitory activity was also observed in the sausages fermented with strain no. 28, but did not differ significantly from those with the type strain. An analysis of the proteolysis and degradation products formed by each LAB in sausages suggested that those bioactivities yielded fermentation products such as peptides. Therefore, LAB starters that can adequately ferment meat, such as strains no. 23 and no. 28, should contribute to the production of bioactive compounds in meat products. © 2016 Japanese Society of Animal Science.

Fermentation characteristics and angiotensin I-converting enzyme-inhibitory activity of Lactobacillus helveticus isolate H9 in cow milk, soy milk, and mare milk.

PubMed

Wang, Jicheng; Li, Changkun; Xue, Jiangang; Yang, Jie; Zhang, Qing; Zhang, Heping; Chen, Yongfu

2015-06-01

Lactobacillus helveticus isolate H9 demonstrated high angiotensin I-converting enzyme (ACE)-inhibitory activity in previous research. Here, we evaluated the fermentation characteristics (pH, titratable acidity, free amino nitrogen, and viable bacterial counts), ACE-inhibitory activity, and contents of Val-Pro-Pro (VPP) and Ile-Pro-Pro (IPP) peptides of stored yogurt (4°C for 28 d) fermented by L. helveticus isolate H9 (initially inoculated at 4 concentrations), from cow, mare, and soy milks. During storage, the pH and titratable acidity remained stable in yogurts produced from all milk types and all inoculation concentrations. The viable bacterial counts in all stored yogurts ranged between 10(6.72) and 10(8.59) cfu/g. The highest ACE-inhibitory activity (70.9-74.5%) was achieved at inoculation concentrations of 5×10(6) cfu/mL. The ACE-inhibitory tripeptides VPP and IPP as determined by ultra-performance liquid chromatography-tandem mass spectrometry were not produced in yogurt made from soy milk or mare milk. These evaluations indicate that L. helveticus H9 has good probiotic properties and would be a promising candidate for production of fermented food with probiotic properties. Copyright © 2015 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.

Pomegranate peel extract attenuates oxidative stress by decreasing coronary angiotensin-converting enzyme (ACE) activity in hypertensive female rats.

PubMed

Dos Santos, Roger L; Dellacqua, Lais O; Delgado, Nathalie T B; Rouver, Wender N; Podratz, Priscila L; Lima, Leandro C F; Piccin, Mariela P C; Meyrelles, Silvana S; Mauad, Helder; Graceli, Jones B; Moyses, Margareth R

2016-01-01

Based on the antioxidant properties of pomegranate, this study was designed to investigate the effects of pomegranate peel extract on damage associated with hypertension and aging in a spontaneously hypertensive rat (SHR) model. The influence of pomegranate consumption was examined on systolic blood pressure (SBP), angiotensin-converting enzyme (ACE) coronary activity, oxidative stress, and vascular morphology. Four- or 28-wk-old SHR model rats were treated for 30 d, with terminal experimental animal age being 8 and 32 wk, respectively, with either pomegranate extract (SHR-PG) or filtered water (SHR). Data showed significant reduction in SBP and coronary ACE activity in both age groups. The levels of superoxide anion, a measure of oxidative stress, were significantly lower in animals in the SHR-PG group compared to SHR alone. Coronary morphology demonstrated total increases in vascular wall areas were in the SHR group, and pomegranate peel extract diminished this effect. Pomegranate peel extract consumption conferred protection against hypertension in the SHR model. This finding was demonstrated by marked reduction in coronary ACE activity, oxidative stress, and vascular remodelling. In addition, treatment was able to reduce SBP in both groups. Evidence indicates that the use of pomegranate peel extract may prove beneficial in alleviating coronary heart disease.

The periplasmic transaminase PtaA of Pseudomonas fluorescens converts the glutamic acid residue at the pyoverdine fluorophore to α-ketoglutaric acid.

PubMed

Ringel, Michael T; Dräger, Gerald; Brüser, Thomas

2017-11-10

The periplasmic conversion of ferribactin to pyoverdine is essential for siderophore biogenesis in fluorescent pseudomonads, such as pathogenic Pseudomonas aeruginosa or plant growth-promoting Pseudomonas fluorescens The non-ribosomal peptide ferribactin undergoes cyclizations and oxidations that result in the fluorophore, and a strictly conserved fluorophore-bound glutamic acid residue is converted to a range of variants, including succinamide, succinic acid, and α-ketoglutaric acid residues. We recently discovered that the pyridoxal phosphate-containing enzyme PvdN is responsible for the generation of the succinamide, which can be hydrolyzed to succinic acid. Based on this, a distinct unknown enzyme was postulated to be responsible for the conversion of the glutamic acid to α-ketoglutaric acid. Here we report the identification and characterization of this enzyme in P. fluorescens strain A506. In silico analyses indicated a periplasmic transaminase in fluorescent pseudomonads and other proteobacteria that we termed PtaA for " p eriplasmic t ransaminase A " An in-frame-deleted ptaA mutant selectively lacked the α-ketoglutaric acid form of pyoverdine, and recombinant PtaA complemented this phenotype. The ptaA / pvdN double mutant produced exclusively the glutamic acid form of pyoverdine. PtaA is homodimeric and contains a pyridoxal phosphate cofactor. Mutation of the active-site lysine abolished PtaA activity and affected folding as well as Tat-dependent transport of the enzyme. In pseudomonads, the occurrence of ptaA correlates with the occurrence of α-ketoglutaric acid forms of pyoverdines. As this enzyme is not restricted to pyoverdine-producing bacteria, its catalysis of periplasmic transaminations is most likely a general tool for specific biosynthetic pathways. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

Effects of cocoa extract and dark chocolate on angiotensin-converting enzyme and nitric oxide in human endothelial cells and healthy volunteers--a nutrigenomics perspective.

PubMed

Persson, Ingrid A L; Persson, Karin; Hägg, Staffan; Andersson, Rolf G G

2011-01-01

Evidence suggests that cocoa from the bean of Theobroma cacao L. has beneficial effects on cardiovascular disease. The aim of this study was to investigate if cocoa extract and dark chocolate influence angiotensin-converting enzyme (ACE) and nitric oxide (NO) in human endothelial cells (in vitro) and in healthy volunteers (in vivo). ACE activity was analyzed with a commercial radioenzymatic assay and measured in human endothelial cells from umbilical veins (HUVEC) after 10 minutes of incubation with cocoa extract. NO was measured after 24 hours of incubation. ACE activity and NO were measured at baseline and after 30, 60, and 180 minutes in 16 healthy volunteers after a single intake of 75 g of dark chocolate containing 72% cocoa. Significant inhibition of ACE activity (P < 0.01) and significant increase of NO (P < 0.001) were seen in HUVEC. In the study subjects, a significant inhibition of ACE activity (mean 18%) 3 hours after intake of dark chocolate was seen, but no significant change in NO was seen. According to ACE genotype, significant inhibition of ACE activity was seen after 3 hours in individuals with genotype insertion/insertion and deletion/deletion (mean 21% and 28%, respectively). Data suggest that intake of dark chocolate containing high amount of cocoa inhibits ACE activity in vitro and in vivo.

Engineering acidic Streptomyces rubiginosus D-xylose isomerase by rational enzyme design.

PubMed

Waltman, Mary Jo; Yang, Zamin Koo; Langan, Paul; Graham, David E; Kovalevsky, Andrey

2014-02-01

To maximize bioethanol production from lignocellulosic biomass, all sugars must be utilized. Yeast fermentation can be improved by introducing the d-xylose isomerase enzyme to convert the pentose sugar d-xylose, which cannot be fermented by Saccharomyces cerevisiae, into the fermentable ketose d-xylulose. The low activity of d-xylose isomerase, especially at the low pH required for optimal fermentation, limits its use. A rational enzyme engineering approach was undertaken, and seven amino acid positions were replaced to improve the activity of Streptomyces rubiginosus d-xylose isomerase towards its physiological substrate at pH values below 6. The active-site design was guided by mechanistic insights and the knowledge of amino acid protonation states at low pH obtained from previous joint X-ray/neutron crystallographic experiments. Tagging the enzyme with 6 or 12 histidine residues at the N-terminus resulted in a significant increase in the active-site affinity towards substrate at pH 5.8. Substituting an asparagine at position 215, which hydrogen bonded to the metal-bound Glu181 and Asp245, with an aspartate gave a variant with almost an order of magnitude lower KM than measured for the native enzyme, with a 4-fold increase in activity. Other studied variants showed similar (Asp57Asn, Glu186Gln/Asn215Asp), lower (Asp57His, Asn247Asp, Lys289His, Lys289Glu) or no (Gln256Asp, Asp287Asn, ΔAsp287) activity in acidic conditions relative to the native enzyme.

Angiotensin converting enzyme inhibitors and aortic arch obstructive malformations.

PubMed

Maliheh, Kadivar; Abdorrazagh, Kiani; Armen, Kocharian; Reza, Shabanian

2006-10-01

We describe two newborn infants with aortic arch obstructive malformations who became anuric after initiation of captopril. Since angiotensin converting enzyme inhibitors can alter renal blood flow by reduction in angiotensin II and blocking autoregulation phenomenon, it is important to use them with great caution in neonates with aortic arch obstructive malformations, while monitoring their renal function closely.

Characteristic Variations and Similarities in Biochemical, Molecular, and Functional Properties of Glyoxalases across Prokaryotes and Eukaryotes.

PubMed

Kaur, Charanpreet; Sharma, Shweta; Hasan, Mohammad Rokebul; Pareek, Ashwani; Singla-Pareek, Sneh L; Sopory, Sudhir K

2017-03-30

The glyoxalase system is the ubiquitous pathway for the detoxification of methylglyoxal (MG) in the biological systems. It comprises two enzymes, glyoxalase I (GLYI) and glyoxalase II (GLYII), which act sequentially to convert MG into d-lactate, thereby helping living systems get rid of this otherwise cytotoxic byproduct of metabolism. In addition, a glutathione-independent GLYIII enzyme activity also exists in the biological systems that can directly convert MG to d-lactate. Humans and Escherichia coli possess a single copy of GLYI (encoding either the Ni- or Zn-dependent form) and GLYII genes, which through MG detoxification provide protection against various pathological and disease conditions. By contrast, the plant genome possesses multiple GLYI and GLYII genes with a role in abiotic stress tolerance. Plants possess both Ni 2+ - and Zn 2+ -dependent forms of GLYI, and studies on plant glyoxalases reveal the various unique features of these enzymes distinguishing them from prokaryotic and other eukaryotic glyoxalases. Through this review, we provide an overview of the plant glyoxalase family along with a comparative analysis of glyoxalases across various species, highlighting similarities as well as differences in the biochemical, molecular, and physiological properties of these enzymes. We believe that the evolution of multiple glyoxalases isoforms in plants is an important component of their robust defense strategies.

Circulating angiotensin-converting enzyme 2 activity in patients with chronic kidney disease without previous history of cardiovascular disease.

PubMed

Anguiano, Lidia; Riera, Marta; Pascual, Julio; Valdivielso, José Manuel; Barrios, Clara; Betriu, Angels; Mojal, Sergi; Fernández, Elvira; Soler, María José

2015-07-01

Patients with cardiovascular (CV) disease have an increased circulating angiotensin-converting enzyme 2 (ACE2) activity, but there is little information about changes in ACE2 in chronic kidney disease (CKD) patients without history of CV disease. We examined circulating ACE2 activity in CKD patients at stages 3-5 (CKD3-5) and in dialysis (CKD5D) without any history of CV disease. Circulating ACE2 activity was measured in human ethylenediamine-tetraacetic acid (EDTA)-plasma samples from the NEFRONA study (n = 2572): control group (CONT) (n = 568), CKD3-5 (n = 1458) and CKD5D (n = 546). Different clinical and analytical variables such as gender; age; history of diabetes mellitus (DM), dyslipidemia and hypertension; glycaemic, renal, lipid and anaemia profiles; vitamin D analogues treatment and antihypertensive treatments (angiotensin-converting enzyme inhibitor and angiotensin receptor blockade) were analysed. Circulating ACE2 and ACE activities were measured using modified fluorimetric assay for EDTA-plasma samples, where zinc chloride was added to recover enzymatic activity. In CKD3-5 and CKD5D, significant decrease in circulating ACE2 activity was observed when compared with CONT, but no differences were found between CKD3-5 and CKD5 when performing paired case-control studies. By multivariate linear regression analysis, male gender and advanced age were identified as independent predictors of ACE2 activity in all groups. Diabetes was identified as independent predictor of ACE2 activity in CKD3-5. Significant increase in the activity of circulating ACE was found in CKD3-5 and CKD5D when compared with CONT and in CKD5D when compared with CKD3-5. By multiple regression analysis, female gender and younger age were identified as independent predictors of ACE activity in CONT and CKD3-5. Diabetes was also identified as an independent predictor of ACE activity in CKD3-5 patients. Circulating ACE2 and ACE activities can be measured in human EDTA-plasma samples with zinc added to recover enzymatic activity. In a CKD population without previous history of CV disease, ACE2 activity from human EDTA-plasma samples directly correlated with the classical CV risk factors namely older age, diabetes and male gender. Our data suggest that circulating ACE2 is altered in CKD patients at risk for CV event. © The Author 2015. Published by Oxford University Press on behalf of ERA-EDTA. All rights reserved.

Novel biological properties of Oenothera paradoxa defatted seed extracts: effects on metallopeptidase activity.

PubMed

Kiss, Anna K; Derwińska, Małgorzata; Dawidowska, Anna; Naruszewicz, Marek

2008-09-10

In this study, for the first time, we used the in vitro metallopeptidase model for the identification of a potential novel activity of defatted evening primrose seed extracts. Prepared extracts of different polarity (aqueous, 60% ethanolic, isopropanolic, and 30% isopropanolic) at concentrations of 1.5-100 microg/mL exhibited a significant and dose dependent inhibition of three tested enzymes. The 50% inhibition of enzymes activity showed that aminopeptidase N (APN) was the enzyme affected to the greatest extent with IC50 at the level of 2.8 microg/mL and 2.9 microg/mL for aqueous and 30% isopropanolic extracts, respectively. The activity of neutral endopeptidase (NEP) was quite strongly inhibited by the extracts as well. The HPLC-DAD analysis and bioguided fractionation led to the identification of four active compounds: (-)-epicatechin gallate, proanthocyanidin B3, oenothein B, and penta-O-galloyl-beta-D-glucose (PGG). Oenothein B has been shown previously to inhibit metallopeptidases. The three other compounds are known to inhibit angiotensin-converting enzyme (ACE), but they have not been previously reported to inhibit the NEP and APN activity. PGG and procyanidins with different degrees of polymerization, as the dominating compounds in O. paradoxa seeds, seemed to play a role in the crude extract activity.

Inhibitory activity on type 2 diabetes and hypertension key-enzymes, and antioxidant capacity of Veronica persica phenolic-rich extracts.

PubMed

Sharifi-Rad, M; Tayeboon, G S; Sharifi-Rad, J; Iriti, M; Varoni, E M; Razazi, S

2016-05-30

Veronica genus (Plantaginaceae) is broadly distributed in different habitats. In this study, the inhibitory activity of free soluble and conjugated phenolic extracts of Veronica persica on key enzymes associated to type 2 diabetes (α-glucosidase and α-amylase) and hypertension (angiotensin I converting enzyme, ACE) was assessed, as well as their antioxidant power. Our results showed that both the extracts inhibited α-amylase, α-glucosidase and ACE in a dose-dependent manner. In particular, free phenolic extract significantly (P<0.05) inhibited α-glucosidase (IC50 532.97 µg/mL), whereas conjugated phenolic extract significantly (P<0.05) inhibited α-amylase (IC50 489.73 µg/mL) and ACE (290.06 µg/mL). The enzyme inhibitory activities of the extracts were not associated with their phenolic content. Anyway, the inhibition of α-amylase, α-glucosidase and ACE, along with the antioxidant capacity of the phenolic-rich extracts, could represent a putative mechanism through which V. persica exerts its antidiabetes and antihypertension effects.

The dimerization domain in DapE enzymes is required for catalysis.

PubMed

Nocek, Boguslaw; Starus, Anna; Makowska-Grzyska, Magdalena; Gutierrez, Blanca; Sanchez, Stephen; Jedrzejczak, Robert; Mack, Jamey C; Olsen, Kenneth W; Joachimiak, Andrzej; Holz, Richard C

2014-01-01

The emergence of antibiotic-resistant bacterial strains underscores the importance of identifying new drug targets and developing new antimicrobial compounds. Lysine and meso-diaminopimelic acid are essential for protein production and bacterial peptidoglycan cell wall remodeling and are synthesized in bacteria by enzymes encoded within dap operon. Therefore dap enzymes may serve as excellent targets for developing a new class of antimicrobial agents. The dapE-encoded N-succinyl-L,L-diaminopimelic acid desuccinylase (DapE) converts N-succinyl-L,L-diaminopimelic acid to L,L-diaminopimelic acid and succinate. The enzyme is composed of catalytic and dimerization domains, and belongs to the M20 peptidase family. To understand the specific role of each domain of the enzyme we engineered dimerization domain deletion mutants of DapEs from Haemophilus influenzae and Vibrio cholerae, and characterized these proteins structurally and biochemically. No activity was observed for all deletion mutants. Structural comparisons of wild-type, inactive monomeric DapE enzymes with other M20 peptidases suggest that the dimerization domain is essential for DapE enzymatic activity. Structural analysis and molecular dynamics simulations indicate that removal of the dimerization domain increased the flexibility of a conserved active site loop that may provide critical interactions with the substrate.

The prokaryotic FAD synthetase family: a potential drug target.

PubMed

Serrano, Ana; Ferreira, Patricia; Martínez-Júlvez, Marta; Medina, Milagros

2013-01-01

Disruption of cellular production of the flavin cofactors, flavin adenine mononucleotide (FMN) and flavin adenine dinucleotide(FAD) will prevent the assembly of a large number of flavoproteins and flavoenzymes involved in key metabolic processes in all types of organisms. The enzymes responsible for FMN and FAD production in prokaryotes and eukaryotes exhibit various structural characteristics to catalyze the same chemistry, a fact that converts the prokaryotic FAD synthetase (FADS) in a potential drug target for the development of inhibitors endowed with anti-pathogenic activity. The first step before searching for selective inhibitors of FADS is to understand the structural and functional mechanisms for the riboflavin kinase and FMN adenylyltransferase activities of the prokaryotic enzyme, and particularly to identify their differential functional characteristics with regard to the enzymes performing similar functions in other organisms, particularly humans. In this paper, an overview of the current knowledge of the structure-function relationships in prokaryotic FADS has been presented, as well as of the state of the art in the use of these enzymes as drug targets.

Penicillium subrubescens is a promising alternative for Aspergillus niger in enzymatic plant biomass saccharification.

PubMed

Mäkelä, Miia R; Mansouri, Sadegh; Wiebenga, Ad; Rytioja, Johanna; de Vries, Ronald P; Hildén, Kristiina S

2016-12-25

In industrial applications, efficient mixtures of polysaccharide-degrading enzymes are needed to convert plant biomass into fermentable sugars. Most of the commercially produced lignocellulolytic enzymes are from a limited number of filamentous fungi, such as Trichoderma and Aspergillus species. In contrast, the plant biomass-degrading capacity of Penicillia has been less explored. We performed growth profiling of several Penicillia on diverse plant biomass-related substrates demonstrating the capacity particularly of Penicillium subrubescens to degrade crude lignocellulose feedstock, as well as polysaccharides, and metabolise their monomeric components. We focussed on the lignocellulolytic potential of P. subrubescens FBCC1632, which produced a variable set of (hemi-)cellulolytic activities on plant biomass substrates with activity levels comparable to those of Aspergillus niger. The good ability of the extracellular enzyme mixtures produced by P. subrubescens to saccharify complex plant biomasses, wheat bran and sugar beet pulp, indicated a high potential for this strain as a producer of industrial enzyme cocktails. Copyright © 2016 Elsevier B.V. All rights reserved.

Substrate Tunnels in Enzymes: Structure-Function Relationships and Computational Methodology

PubMed Central

Kingsley, Laura J.; Lill, Markus A.

2015-01-01

In enzymes, the active site is the location where incoming substrates are chemically converted to products. In some enzymes, this site is deeply buried within the core of the protein and in order to access the active site, substrates must pass through the body of the protein via a tunnel. In many systems, these tunnels act as filters and have been found to influence both substrate specificity and catalytic mechanism. Identifying and understanding how these tunnels exert such control has been of growing interest over the past several years due to implications in fields such as protein engineering and drug design. This growing interest has spurred the development of several computational methods to identify and analyze tunnels and how ligands migrate through these tunnels. The goal of this review is to outline how tunnels influence substrate specificity and catalytic efficiency in enzymes with tunnels and to provide a brief summary of the computational tools used to identify and evaluate these tunnels. PMID:25663659

Potential and utilization of thermophiles and thermostable enzymes in biorefining

PubMed Central

Turner, Pernilla; Mamo, Gashaw; Karlsson, Eva Nordberg

2007-01-01

In today's world, there is an increasing trend towards the use of renewable, cheap and readily available biomass in the production of a wide variety of fine and bulk chemicals in different biorefineries. Biorefineries utilize the activities of microbial cells and their enzymes to convert biomass into target products. Many of these processes require enzymes which are operationally stable at high temperature thus allowing e.g. easy mixing, better substrate solubility, high mass transfer rate, and lowered risk of contamination. Thermophiles have often been proposed as sources of industrially relevant thermostable enzymes. Here we discuss existing and potential applications of thermophiles and thermostable enzymes with focus on conversion of carbohydrate containing raw materials. Their importance in biorefineries is explained using examples of lignocellulose and starch conversions to desired products. Strategies that enhance thermostablity of enzymes both in vivo and in vitro are also assessed. Moreover, this review deals with efforts made on developing vectors for expressing recombinant enzymes in thermophilic hosts. PMID:17359551

Design and Properties of an Immobilization Enzyme System for Inulin Conversion.

PubMed

Hang, Hua; Wang, Changbao; Cheng, Yiqun; Li, Ning; Song, Liuli

2018-02-01

A commercial inulinase could convert inulin into fructose, which was optimized to be entrapped in the calcium alginate-gelatin beads with the immobilization yield of 86% for free inulinase activities. The optimum pH values and temperatures were 4.5 and 40 °C for the free enzyme and 5.0-5.5 and 45-50 °C for the immobilized enzyme. The kinetic parameters of V max and K m were 5.24 μmol/min and 57.6 mg/mL for the free inulinase and 4.32 μmol/min and 65.8 mg/mL for the immobilized inulinase, respectively. The immobilized enzyme retained 80% of its initial activities at 45 °C for 4 days, which could exhibit better thermal stability. The reuse of immobilized inulinase throughout the continuous batch operations was explored, which had better reusability of the immobilized biocatalyst. At the same time, the stability of immobilized enzyme in the continuous packed-bed bioreactor was estimated, which showed the better results and had its potential scale-up fructose production for inulin conversion.

Amine oxidation by d-arginine dehydrogenase in Pseudomonas aeruginosa.

PubMed

Ouedraogo, Daniel; Ball, Jacob; Iyer, Archana; Reis, Renata A G; Vodovoz, Maria; Gadda, Giovanni

2017-10-15

d-Arginine dehydrogenase from Pseudomonas aeruginosa (PaDADH) is a flavin-dependent oxidoreductase, which is part of a novel two-enzyme racemization system that functions to convert d-arginine to l-arginine. PaDADH contains a noncovalently linked FAD that shows the highest activity with d-arginine. The enzyme exhibits broad substrate specificity towards d-amino acids, particularly with cationic and hydrophobic d-amino acids. Biochemical studies have established the structure and the mechanistic properties of the enzyme. The enzyme is a true dehydrogenase because it displays no reactivity towards molecular oxygen. As established through solvent and multiple kinetic isotope studies, PaDADH catalyzes an asynchronous CH and NH bond cleavage via a hydride transfer mechanism. Steady-state kinetic studies with d-arginine and d-histidine are consistent with the enzyme following a ping-pong bi-bi mechanism. As shown by a combination of crystallography, kinetic and computational data, the shape and flexibility of loop L1 in the active site of PaDADH are important for substrate capture and broad substrate specificity. Copyright © 2017 Elsevier Inc. All rights reserved.

Utility of angiotensin-converting enzyme activity in aqueous humor in the diagnosis of ocular sarcoidosis.

PubMed

Mihailovic-Vucinic, Violeta; Popevic, Ljubica; Popevic, Spasoje; Stjepanovic, Mihailo; Aleksic, Andjelka; Stanojevic-Paovic, Anka

2017-10-01

Many studies include elevated activity of angiotensin-converting enzyme (ACE) in serum in sarcoidosis and in ocular sarcoidosis as well, but there are only a few analyzing ACE activities in aqueous humor. The aim of this study is to illuminate the diagnostic value of ACE in aqueous humor in patients with ocular sarcoidosis. We analyzed twenty patients with ocular sarcoidosis and 18 patients with nonocular involvement. All patients have biopsy-positive sarcoidosis of the lungs and/or mediastinal lymph nodes. Blood samples for ACE serum levels were obtained from all patients. Aqueous humor samples were taken by paracentesis with a 25-gauge needle in local anesthesia. With appropriate statistical tests, we compared ACE activity in serum and aqueous humor in patients with and without ocular sarcoidosis. The majority of our patients with ocular sarcoidosis were female (12/20), also in the group with systemic sarcoidosis and without ocular involvement (12/6). Mean age of the whole analyzed group of sarcoidosis patients was 45 ± 6 years. There is no statistically significant difference in ACE activity in serum between two groups of patients (with and without ocular sarcoidosis). There is statistically significant difference in ACE activity in aqueous humor among patients with ocular and nonocular sarcoidosis. ACE activity in aqueous humor is significantly higher in patients with ocular sarcoidosis. Increased ACE activity in aqueous humor can point to a diagnosis of ocular sarcoidosis, without the need for ocular biopsy.

Proteomic analysis of nitrate-dependent acetone degradation by Alicycliphilus denitrificans strain BC.

PubMed

Oosterkamp, Margreet J; Boeren, Sjef; Atashgahi, Siavash; Plugge, Caroline M; Schaap, Peter J; Stams, Alfons J M

2015-06-01

Alicycliphilus denitrificans strain BC grows anaerobically on acetone with nitrate as electron acceptor. Comparative proteomics of cultures of A. denitrificans strain BC grown on either acetone or acetate with nitrate was performed to study the enzymes involved in the acetone degradation pathway. In the proposed acetone degradation pathway, an acetone carboxylase converts acetone to acetoacetate, an AMP-dependent synthetase/ligase converts acetoacetate to acetoacetyl-CoA, and an acetyl-CoA acetyltransferase cleaves acetoacetyl-CoA to two acetyl-CoA. We also found a putative aldehyde dehydrogenase associated with acetone degradation. This enzyme functioned as a β-hydroxybutyrate dehydrogenase catalyzing the conversion of surplus acetoacetate to β-hydroxybutyrate that may be converted to the energy and carbon storage compound, poly-β-hydroxybutyrate. Accordingly, we confirmed the formation of poly-β-hydroxybutyrate in acetone-grown cells of strain BC. Our findings provide insight in nitrate-dependent acetone degradation that is activated by carboxylation of acetone. This will aid studies of similar pathways found in other microorganisms degrading acetone with nitrate or sulfate as electron acceptor. © FEMS 2015. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

Omapatrilat, a dual angiotensin-converting enzyme and neutral endopeptidase inhibitor, prevents fatty streak deposit in apolipoprotein E-deficient mice.

PubMed

Arnal, J F; Castano, C; Maupas, E; Mugniot, A; Darblade, B; Gourdy, P; Michel, J B; Bayard, F

2001-04-01

Angiotensin-converting enzyme (ACE) is mainly responsible for converting angiotensin I (AI) to angiotensin II (AII), and ACE inhibitors prevent atherosclerosis in animal models. Neutral endopeptidase 24.11 (NEP) degrades substance P, kinins and atrial natriuretic peptide (ANP), and aortic wall NEP activity was found to be increased in atherosclerosis. In the present study, we have evaluated the effect of candoxatril, a NEP inhibitor, and of omapatrilat, a dual ACE and NEP inhibitor, on the development of fatty streak in apolipoprotein E (apoE)-deficient mice. Groups of ten male apoE-deficient mice were given either placebo, candoxatril 50 mg/kg per day, or omapatrilat 10, or 100 mg/kg per day for 4 months. None of the treatments influenced body weight, serum total or HDL-cholesterol. Compared with the placebo, candoxatril did not protect the mice from fatty streak deposit. In contrast, omapatrilat dose dependently inhibited the constitution of fatty streak in apoE-deficient mice. The precise advantages of the dual ACE and NEP inhibition versus the inhibition of only ACE should now be considered in the prevention of atherosclerosis as well as in the occurrence of its complications.

Insights into the Hypertensive Effects of Tityus serrulatus Scorpion Venom: Purification of an Angiotensin-Converting Enzyme-Like Peptidase

PubMed Central

Cajado-Carvalho, Daniela; Kuniyoshi, Alexandre Kazuo; Duzzi, Bruno; Iwai, Leo Kei; de Oliveira, Úrsula Castro; Junqueira de Azevedo, Inácio de Loiola Meirelles; Kodama, Roberto Tadashi; Portaro, Fernanda Vieira

2016-01-01

The number of cases of envenomation by scorpions has grown significantly in Brazil since 2007, with the most severe cases being caused by the Tityus serrulatus scorpion. Although envenomed patients mostly suffer neurotoxic manifestations, other symptoms, such as hypertension, cannot be exclusively attributed to neurotoxins. Omics analyses have detected plentiful amounts of metalloproteases in T. serrulatus venom. However, the roles played by these enzymes in envenomation are still unclear. Endeavoring to investigate the functions of scorpion venom proteases, we describe here for the first time an Angiotensin I-Converting Enzyme-like peptidase (ACE-like) purified from T. serrulatus venom. The crude venom cleaved natural and fluorescent substrates and these activities were inhibited by captopril. Regarding the serum neutralization, the scorpion antivenom was more effective at blocking the ACE-like activity than arachnid antivenom, although neither completely inhibited the venom cleavage action, even at higher doses. ACE-like was purified from the venom after three chromatographic steps and its identity was confirmed by mass spectrometric and transcriptomic analyses. Bioinformatics analysis showed homology between the ACE-like transcript sequences from Tityus spp. and human testis ACE. These findings advance our understanding of T. serrulatus venom components and may improve treatment of envenomation victims, as ACE-like may contribute to envenomation symptoms, especially the resulting hypertension. PMID:27886129

Getting a Handle on the Role of Coenzyme M in Alkene Metabolism

PubMed Central

Krishnakumar, Arathi M.; Sliwa, Darius; Endrizzi, James A.; Boyd, Eric S.; Ensign, Scott A.; Peters, John W.

2008-01-01

Summary: Coenzyme M (2-mercaptoethanesulfonate; CoM) is one of several atypical cofactors discovered in methanogenic archaea which participate in the biological reduction of CO2 to methane. Elegantly simple, CoM, so named for its role as a methyl carrier in all methanogenic archaea, is the smallest known organic cofactor. It was thought that this cofactor was used exclusively in methanogenesis until it was recently discovered that CoM is a key cofactor in the pathway of propylene metabolism in the gram-negative soil microorganism Xanthobacter autotrophicus Py2. A four-step pathway requiring CoM converts propylene and CO2 to acetoacetate, which feeds into central metabolism. In this process, CoM is used to activate and convert highly electrophilic epoxypropane, formed from propylene epoxidation, into a nucleophilic species that undergoes carboxylation. The unique properties of CoM provide a chemical handle for orienting compounds for site-specific redox chemistry and stereospecific catalysis. The three-dimensional structures of several of the enzymes in the pathway of propylene metabolism in defined states have been determined, providing significant insights into both the enzyme mechanisms and the role of CoM in this pathway. These studies provide the structural basis for understanding the efficacy of CoM as a handle to direct organic substrate transformations at the active sites of enzymes. PMID:18772284

Effects of Cordyceps militaris (L.) Fr. fermentation on the nutritional, physicochemical, functional properties and angiotensin I converting enzyme inhibitory activity of red bean (Phaseolus angularis [Willd.] W.F. Wight.) flour.

PubMed

Xiao, Yu; Sun, Mingmei; Zhang, Qiuqin; Chen, Yulian; Miao, Junqing; Rui, Xin; Dong, Mingsheng

2018-04-01

The effects of solid-state fermentation with Cordyceps militaris (L.) Fr. on the nutritional, physicochemical, and functional properties as well as angiotensin I converting enzyme (ACE) inhibitory activity of red bean ( Phaseolus angularis [Willd.] W.F. Wight.) flour were determined. Fermentation increased the amount of small peptides but significantly decreased large peptides. Fermentation also increased proteins and essential amino acids (by 9.31 and 13.89%, respectively) and improved the in vitro protein digestibility (6.54%) of red beans. Moreover, fermentation increased the water holding capacity (from 2.36 to 2.59 mL/g), fat absorption capacity (from 84.65 to 114.55%), emulsion activity (from 10.96 to 52.77%), emulsion stability (from 5.43 to 53.82%), and foaming stability (from 11.95 to 20.68%). Fermented red bean flour achieved a lower least gelation concentration of 14% than that of the control (18%). In contrast to the non-fermented red bean, the fermented red bean showed ACE inhibitory activity, with IC 50 value of 0.63 mg protein/mL. Overall, fermentation improved the nutritional, physicochemical, and functional properties as well as the biological activity of red bean flour. Thus, fermented red bean flour may serve as a novel nutritional and functional ingredient for applications in food design.

Inhibition of the renin-angiotensin-aldosterone system: is there room for dual blockade in the cardiorenal continuum?

PubMed

Volpe, Massimo; Danser, A H Jan; Menard, Joël; Waeber, Bernard; Mueller, Dominik N; Maggioni, Aldo P; Ruilope, Luis M

2012-04-01

Antagonism of renin-angiotensin-aldosterone system is exerted through angiotensin-converting enzyme inhibitors, angiotensin receptor antagonists, renin inhibitors and mineralocorticoid receptor antagonists. These drugs have been successfully tested in numerous trials and in different clinical settings. The original indications of renin-angiotensin-aldosterone system blockers have progressively expanded from the advanced stages to the earlier stages of cardiorenal continuum. To optimize the degree of blockade of renin-angiotensin-aldosterone system, dose uptitrations of angiotensin-converting enzyme inhibitors and angiotensin receptor antagonists or the use of a dual blockade, initially identified with the combination of angiotensin-converting enzyme inhibitors and angiotensin receptor antagonists, have been proposed. The data from the Ongoing Telmisartan Alone and in Combination with Ramipril Global Endpoint Trial (ONTARGET) study do not support this specific dual blockade approach. However, the dual blockade of angiotensin-converting enzyme inhibitors/angiotensin receptor antagonists with direct renin inhibitors is currently under investigation while that based on an aldosterone blocker with any of the previous three drugs requires more evidence beyond heart failure. In this review, we revisited potential advantages of dual blockade of renin-angiotensin-aldosterone system in arterial hypertension and diabetes.

Wheat Bread with Pumpkin (Cucurbita maxima L.) Pulp
as a Functional Food Product.

PubMed

Różyło, Renata; Gawlik-Dziki, Urszula; Dziki, Dariusz; Jakubczyk, Anna; Karaś, Monika; Różyło, Krzysztof

2014-12-01

In this study, a new application of pumpkin pulp in bread production is shown. The aim of this work is to determine the influence of the addition of fresh pumpkin pulp directly into wheat flour on physical, sensorial and biological properties of bread. The bioaccessibility of active compounds was also studied. An increase in the addition of pumpkin pulp from 5 to 20% (converted to dry matter) caused a decrease of bread volume and increase of crumb hardness and cohesiveness. The sensory characteristics of the bread showed that a partial replacement of wheat flour with up to 10% of pumpkin pulp gave satisfactory results. The taste, aroma and overall acceptability of control bread and bread containing 5 or 10% of pulp had the highest degree of liking. The addition of higher levels of pumpkin pulp caused an unpleasant aroma and taste. Pumpkin pulp is a good material to complement the bread with potentially bioaccessible phenolics (including flavonoids) and, especially, with peptides. The highest antioxidant activity was observed, in most cases, of the samples with added 10 and 15% of pumpkin pulp. The addition of the pulp significantly enriched the bread with potentially bioaccessible angiotensin-converting enzyme (ACE) inhibitors. The highest activity was determined in the bread with 15 and 20% pumpkin pulp. ACE inhibitors from the tested bread were highly bioaccessible in vitro . Pumpkin pulp seems to be a valuable source of active compounds to complement the wheat bread. Adding the pulp directly to the wheat flour gives satisfactory baking results and reduces the cost of production. Additionally, pumpkin pulp is sometimes treated as waste material after the acquisition of seeds, thus using it as bread supplement also has environmental and economic benefits. Key words : pumpkin, bread, texture, antioxidants, bioaccessibility in vitro, angiotensin-converting enzyme (ACE) inhibition.

Exquisite Modulation of the Active Site of Methanocaldococcus jannaschii Adenylosuccinate Synthetase in Forward Reaction Complexes.

PubMed

Karnawat, Vishakha; Mehrotra, Sonali; Balaram, Hemalatha; Puranik, Mrinalini

2016-05-03

In enzymes that conduct complex reactions involving several substrates and chemical transformations, the active site must reorganize at each step to complement the transition state of that chemical step. Adenylosuccinate synthetase (ADSS) utilizes a molecule each of guanosine 5'-monophosphate (GTP) and aspartate to convert inosine 5'-monophosphate (IMP) into succinyl adenosine 5'-monophosphate (sAMP) through several kinetic intermediates. Here we followed catalysis by ADSS through high-resolution vibrational spectral fingerprints of each substrate and intermediate involved in the forward reaction. Vibrational spectra show differential ligand distortion at each step of catalysis, and band positions of substrates are influenced by binding of cosubstrates. We found that the bound IMP is distorted toward its N1-deprotonated form even in the absence of any other ligands. Several specific interactions between GTP and active-site amino acid residues result in large Raman shifts and contribute substantially to intrinsic binding energy. When both IMP and GTP are simultaneously bound to ADSS, IMP is converted into an intermediate 6-phosphoryl inosine 5'-monophosphate (6-pIMP). The 6-pIMP·ADSS complex was found to be stable upon binding of the third ligand, hadacidin (HDA), an analogue of l-aspartate. We find that in the absence of HDA, 6-pIMP is quickly released from ADSS, is unstable in solution, and converts back into IMP. HDA allosterically stabilizes ADSS through local conformational rearrangements. We captured this complex and determined the spectra and structure of 6-pIMP in its enzyme-bound state. These results provide important insights into the exquisite tuning of active-site interactions with changing substrate at each kinetic step of catalysis.

Angiotensin Converting Enzyme Regulates Cell Proliferation and Migration

PubMed Central

Carvalho, Clarissa Coelho; Florentino, Rodrigo Machado; França, Andressa; Matias, Eveline; Guimarães, Paola Bianchi; Batista, Carolina; Freire, Valder; Carmona, Adriana Karaoglanovic; Pesquero, João Bosco; de Paula, Ana Maria; Foureaux, Giselle; Leite, Maria de Fatima

2016-01-01

Background The angiotensin-I converting enzyme (ACE) plays a central role in the renin-angiotensin system, acting by converting the hormone angiotensin-I to the active peptide angiotensin-II (Ang-II). More recently, ACE was shown to act as a receptor for Ang-II, and its expression level was demonstrated to be higher in melanoma cells compared to their normal counterparts. However, the function that ACE plays as an Ang-II receptor in melanoma cells has not been defined yet. Aim Therefore, our aim was to examine the role of ACE in tumor cell proliferation and migration. Results We found that upon binding to ACE, Ang-II internalizes with a faster onset compared to the binding of Ang-II to its classical AT1 receptor. We also found that the complex Ang-II/ACE translocates to the nucleus, through a clathrin-mediated process, triggering a transient nuclear Ca2+ signal. In silico studies revealed a possible interaction site between ACE and phospholipase C (PLC), and experimental results in CHO cells, demonstrated that the β3 isoform of PLC is the one involved in the Ca2+ signals induced by Ang-II/ACE interaction. Further studies in melanoma cells (TM-5) showed that Ang-II induced cell proliferation through ACE activation, an event that could be inhibited either by ACE inhibitor (Lisinopril) or by the silencing of ACE. In addition, we found that stimulation of ACE by Ang-II caused the melanoma cells to migrate, at least in part due to decreased vinculin expression, a focal adhesion structural protein. Conclusion ACE activation regulates melanoma cell proliferation and migration. PMID:27992423

Zeaxanthin epoxidation - an in vitro approach.

PubMed

Kuczyńska, Paulina; Latowski, Dariusz; Niczyporuk, Sylvia; Olchawa-Pajor, Monika; Jahns, Peter; Gruszecki, Wiesław I; Strzałka, Kazimierz

2012-01-01

Zeaxanthin epoxidase (ZE) is an enzyme operating in the violaxanthin cycle, which is involved in photoprotective mechanisms. In this work model systems to study zeaxanthin (Zx) epoxidation were developed. Two assay systems are presented in which epoxidation of Zx was observed. In these assays two mutants of Arabidopsis thaliana which have active only one of the two xanthophyll cycle enzymes were used. The npq1 mutant possesses an active ZE and is thus able to convert Zx to violaxanthin (Vx) but the violaxanthin de-epoxidase (VDE) is inactive, so that Vx cannot be converted to Zx. The other mutant, npq2, possesses an active VDE and can convert exogenous Vx to Zx under strong light conditions but reverse reaction is not possible. The first assay containing thylakoids from npq1 and npq2 mutants of A. thaliana gave positive results and high efficiency of epoxidation reaction was observed. The amount of Zx was reduced by 25%. To optimize high efficiency of epoxidation reaction additional factors facilitating both fusion of the two types of thylakoids and incorporation of Zx to their membranes were also studied. The second kind of assay contained npq1 mutant thylakoids of A. thaliana supplemented with exogenous Zx and monogalactosyldiacylglycerol (MGDG). Experiments with different proportions of Zx and MGDG showed that their optimal ratio is 1:60. In such system, due to epoxidation, the amount of Zx was reduced by 38% of its initial level. The in vitro systems of Zx epoxidation described in this paper enable analysis some properties of the ZE without necessity of its isolation.

Biostable beta-Amino Acid PK/PBAN Analogs: Agonist and Antagonist Properties

DTIC Science & Technology

2009-01-01

peptidases neprilysin and angiotensin converting enzyme that are shown to degrade the natural peptides . Despite the changes to the PK core, analog PK-bA-4...bound peptidase - resistant analogs of the insect allatostatins. Peptides 1999;20:23–9. [21] Nachman RJ, Holman GM, Cook BJ. Active fragments and...pheromonotropic activity of peptidase -resistant topical amphiphilic analogs of pyrokinin/PBAN insect neuropeptides. Peptides 2002;23:2035–43. [28] Nachman RJ

Molecular characterization of an enzyme that degrades neuromodulatory fatty-acid amides.

PubMed

Cravatt, B F; Giang, D K; Mayfield, S P; Boger, D L; Lerner, R A; Gilula, N B

1996-11-07

Endogenous neuromodulatory molecules are commonly coupled to specific metabolic enzymes to ensure rapid signal inactivation. Thus, acetylcholine is hydrolysed by acetylcholine esterase and tryptamine neurotransmitters like serotonin are degraded by monoamine oxidases. Previously, we reported the structure and sleep-inducing properties of cis-9-octadecenamide, a lipid isolated from the cerebrospinal fluid of sleep-deprived cats. cis-9-Octadecenamide, or oleamide, has since been shown to affect serotonergic systems and block gap-junction communication in glial cells (our unpublished results). We also identified a membrane-bound enzyme activity that hydrolyses oleamide to its inactive acid, oleic acid. We now report the mechanism-based isolation, cloning and expression of this enzyme activity, originally named oleamide hydrolase, from rat liver plasma membranes. We also show that oleamide hydrolase converts anandamide, a fatty-acid amide identified as the endogenous ligand for the cannabinoid receptor, to arachidonic acid, indicating that oleamide hydrolase may serve as the general inactivating enzyme for a growing family of bioactive signalling molecules, the fatty-acid amides. Therefore we will hereafter refer to oleamide hydrolase as fatty-acid amide hydrolase, in recognition of the plurality of fatty-acid amides that the enzyme can accept as substrates.

Design, Synthesis and Biological Evaluation of Oxindole-Based Chalcones as Small-Molecule Inhibitors of Melanogenic Tyrosinase.

PubMed

Suthar, Sharad Kumar; Bansal, Sumit; Narkhede, Niteen; Guleria, Manju; Alex, Angel Treasa; Joseph, Alex

2017-01-01

The enzyme tyrosinase regulates melanogenesis and skin hyperpigmentation by converting L-3,4-dihydroxyphenylalanine (L-DOPA) into dopaquinone, a key step in the melanin biosynthesis. The present work deals with design and synthesis of various oxindole-based chalcones as monophenolase and diphenolase activity inhibitors of tyrosinase. Among the screened compounds, 4-hydroxy-3-methoxybenzylidene moiety bearing chalcone (7) prepared by one pot reaction of oxindole and vanillin displayed the highest activity against tyrosinase with IC 50 s of 63.37 and 59.71 µM in monophenolase and diphenolase activity assays, respectively. In molecular docking studies, chalcone 7 also showed the highest binding affinity towards the enzyme tyrosinase while exhibiting the lowest estimated free energy of binding, among all the ligands docked.

Angiotensin converting enzyme (ACE) gene expression in experimentally induced liver cirrhosis in rats.

PubMed

Shahid, Syed Muhammad; Fatima, Syeda Nuzhat; Mahboob, Tabassum

2013-09-01

Angiotensin converting enzyme (ACE) is a key player of Renin Angiotensin System (RAS), involved in conversion of active product, angiotensin-II. Alterations in RAS have been implicated in the pathophysiology of various diseases involving heart, kidney, lung and liver. This study is designed to investigate the association of ACE gene expression in induction of liver cirrhosis in rats. Total 12 male albino Wistar rats were selected and divided in two groups. Control group received 0.9% NaCl, where as Test group received thioacidamide (TAA), dissolved in 0.9%NaCl, injected intraperitoneally at a dosage of 200mg/Kg of body weight, twice a week for 12 weeks. The rats were decapitated and blood sample was collected at the end of experimental period and used for liver functions, enzyme activity, antioxidant enzymes and lipid peroxidation estimations. Genomic DNA was isolated from excised tissue determine the ACE genotypes using specific primers. The ACE gene expression in liver tissue was assessed using the quantitative RT-PCR method. The activity of ALT, total and direct bilirubin, SOD and CAT levels were significantly high (p<0.05) and level of MDA was significantly low (p<0.05) in TAA treated rats as compared to control rats. The ACE gene expression after 12 weeks TAA treatment in cirrhotic rats was significantly increased (p<0.05) in comparison to controls. This study describes the importance of RAS in the development of hepatic fibrosis and the benefits of modulation of this system ACE gene expression. The finding of major up-regulation of ACE in the experimental rat liver provides further insight into the complexities of the RAS and its regulation in liver injury. The development of specific modulators of ACE activity and function, in future, will help determine the role of ACE and its genetic variants in the pathophysiology of liver disease.

Relationship of quadriceps muscle power and optimal shortening velocity with angiotensin-converting enzyme activity in older women.

PubMed

Kostka, Joanna; Sikora, Joanna; Kostka, Tomasz

2017-01-01

The goal of this study was to assess whether angiotensin-converting enzyme (ACE) activity is related to muscle function (strength, power and velocity), as well as to assess if ACE inhibitors (ACEIs) and other angiotensin system blocking medications (ASBMs) influence muscle performance in elderly women. Ninety-five community-dwelling elderly women took part in this study. Anthropometric data, blood ACE activity analysis, maximum power (P max ) and optimal shortening velocity (υ opt ) of the knee extensor muscles, handgrip strength, physical activity (PA) and functional performance were measured. Women taking ACEI were on average almost 2 years older than the women who did not take ACEI. They took more medicines and were also characterized by significantly lower level of ACE, but they did not differ in terms of PA level, results of functional performance and parameters characterizing muscle functions. No correlations of ACE activity with P max and handgrip strength, as well as with PA or functional performance were found. Higher ACE activity was connected with lower υ opt for women who did not take any ASBMs (rho =-0.37; p =0.01). Serum ACE activity was not associated with muscle strength, power and functional performance in both ASBM users and nonusers, but was associated with optimal shortening velocity of quadriceps muscles in older women. Further prospective studies are needed to assess if ACEIs or other ASBMs may slow down the decline in muscle function and performance.

Redirection of the Reaction Specificity of a Thermophilic Acetolactate Synthase toward Acetaldehyde Formation

PubMed Central

Cheng, Maria; Yoshiyasu, Hayato; Okano, Kenji; Ohtake, Hisao; Honda, Kohsuke

2016-01-01

Acetolactate synthase and pyruvate decarboxylase are thiamine pyrophosphate-dependent enzymes that convert pyruvate into acetolactate and acetaldehyde, respectively. Although the former are encoded in the genomes of many thermophiles and hyperthermophiles, the latter has been found only in mesophilic organisms. In this study, the reaction specificity of acetolactate synthase from Thermus thermophilus was redirected to catalyze acetaldehyde formation to develop a thermophilic pyruvate decarboxylase. Error-prone PCR and mutant library screening led to the identification of a quadruple mutant with 3.1-fold higher acetaldehyde-forming activity than the wild-type. Site-directed mutagenesis experiments revealed that the increased activity of the mutant was due to H474R amino acid substitution, which likely generated two new hydrogen bonds near the thiamine pyrophosphate-binding site. These hydrogen bonds might result in the better accessibility of H+ to the substrate-cofactor-enzyme intermediate and a shift in the reaction specificity of the enzyme. PMID:26731734

Structures of Arg- and Gln-type bacterial cysteine dioxygenase homologs: Arg- and Gln-type Bacterial CDO Homologs

DOE PAGES

Driggers, Camden M.; Hartman, Steven J.; Karplus, P. Andrew

2015-01-01

In some bacteria, cysteine is converted to cysteine sulfinic acid by cysteine dioxygenases (CDO) that are only ~15–30% identical in sequence to mammalian CDOs. Among bacterial proteins having this range of sequence similarity to mammalian CDO are some that conserve an active site Arg residue (“Arg-type” enzymes) and some having a Gln substituted for this Arg (“Gln-type” enzymes). Here, we describe a structure from each of these enzyme types by analyzing structures originally solved by structural genomics groups but not published: a Bacillus subtilis “Arg-type” enzyme that has cysteine dioxygenase activity (BsCDO), and a Ralstonia eutropha “Gln-type” CDO homolog ofmore » uncharacterized activity (ReCDOhom). The BsCDO active site is well conserved with mammalian CDO, and a cysteine complex captured in the active site confirms that the cysteine binding mode is also similar. The ReCDOhom structure reveals a new active site Arg residue that is hydrogen bonding to an iron-bound diatomic molecule we have interpreted as dioxygen. Notably, the Arg position is not compatible with the mode of Cys binding seen in both rat CDO and BsCDO. As sequence alignments show that this newly discovered active site Arg is well conserved among “Gln-type” CDO enzymes, we conclude that the “Gln-type” CDO homologs are not authentic CDOs but will have substrate specificity more similar to 3-mercaptopropionate dioxygenases.« less

ADAM-17: The Enzyme That Does It All

PubMed Central

Gooz, Monika

2010-01-01

This review focuses on the role of ADAM-17 in disease. Since its debut as the tumor necrosis factor converting enzyme or TACE, ADAM-17 has been reported to be an indispensible regulator of almost every cellular event from proliferation to migration. The central role of ADAM-17 in cell regulation is rooted in its diverse array of substrates: cytokines, growth factors, and their receptors as well as adhesion molecules are activated or inactivated by their cleavage with ADAM-17. It is therefore not surprising that ADAM-17 is implicated in numerous human diseases including cancer, heart disease, diabetes, rheumatoid arthritis, kidney fibrosis, Alzheimer’s disease, and is a promising target for future treatments. The specific role of ADAM-17 in the pathophysiology of these diseases is very complex and depends on the cellular context. To exploit the therapeutic potential of ADAM-17, it is important to understand how its activity is regulated and how specific organs and cells can be targeted to inactivate or activate the enzyme. PMID:20184396

Purification and characterization of substance P endopeptidase activities in the rat spinal cord.

PubMed

Karlsson, K; Eriksson, U; Andrén, P; Nyberg, F

1997-02-01

Two enzymes with substance P degrading activity were purified from the membrane bound fraction of the rat spinal cord. The purified enzymes were characterized with regard to biochemical and kinetic properties. One of the enzymes exhibited close similarity to neutral endopeptidase 24.11 (NEP, EC 3.4.24.11), while the other resembled a substance P converting endopeptidase (SPE), which has previously been identified and purified from human cerebrospinal fluid (CSF). Detergent treated spinal cord homogenates from male Sprague Dawley rats were purified by anion-exchange chromatography (DEAE-sepharose CL-6B), hydrophobic-interaction chromatography (phenyl-sepharose CL-4B) and molecular sieving (Sephadex G-50). Two fractions with enzymes differing in size were recovered and allowed for further purification to apparent homogeneity by ion-exchange chromatography and molecular sieving on a micro-purification system (SMART). The enzyme activities were monitored by following the conversion of synthetic substance P using a radioimmunoassay specific for the heptapeptide product, substance P (1-7). By SDS-polyacrylamide gel electrophoresis of the purified enzymes molecular weights of 43 and 70 kDa were estimated for the SPE-like and NEP-like activity, respectively. A K(m) of 5 microM was determined for the conversion of substance P to its (1-7) fragment by the SPE-like activity. Reversed-phase HPLC together with mass spectrometry permitted identification of all fragments released from substance P by the peptidases. The released fragments were for both enzymes identified as substance P (1-7), substance P (8-11), substance P (1-8), substance P (9-11). The NEP-like enzyme preparation also gave substance P (1-6) as a major product.

Binding of Cimetidine to Balb/C Mouse Liver Catalase; Kinetics and Conformational Studies.

PubMed

Jahangirvand, Mahboubeh; Minai-Tehrani, Dariush; Yazdi, Fatemeh; Minai-Tehrani, Arash; Razmi, Nematollah

2016-01-01

Catalase is responsible for converting hydrogen peroxide (H2O2) into water and oxygen in cells. This enzyme has high affinity for hydrogen peroxide and can protect the cells from oxidative stress damage. Catalase is a tetramer protein and each monomer contains a heme group. Cimetidine is a histamine H2 receptor blocker which inhibits acid release from stomach and is used for gasterointestinal diseases. In this research, effect of cimetidine on the activity of liver catalase was studied and the kinetic parameters of this enzyme and its conformational changes were investigated. Cell free extract of mouse liver was used for the catalase assay. The activity of the catalase was detected in the absence and presence of cimetidine by monitoring hydrogen peroxide reduction absorbance at 240 nm. The purified enzyme was used for conformational studies by Fluorescence spectrophotometry. The data showed that cimetidine could inhibit the enzyme in a non-competitive manner. Ki and IC50 values of the drug were determined to be about 0.75 and 0.85 uM, respectively. The Arrhenius plot showed that activation energy was 6.68 and 4.77 kJ/mol in the presence and absence of the drug, respectively. Fluorescence spectrophotometry revealed that the binding of cimetidine to the purified enzyme induced hyperchromicity and red shift which determined the conformational change on the enzyme. Cimetidine could non-competitively inhibit the liver catalase with high affinity. Binding of cimetidine to the enzyme induced conformational alteration in the enzyme.

Activities of purine converting enzymes in heart, liver and kidney mice LDLR-/- and Apo E-/.

PubMed

Rybakowska, I M; Kutryb-Zając, B; Milczarek, R; Łukasz, B; Slominska, E M; Smolenski, R T

2018-05-21

Nucleotide metabolism plays a major role in a number of vital cellular processes such as energetics. This, in turn, is important in pathologies such as atherosclerosis. Three month old atherosclerotic mice with knock outs for LDLR and apolipoprotein E (ApoE) were used for the experiments. Activities of AMP-deaminase (AMPD), ecto5'-nucleotidase (e5NT), adenosine deaminase (ADA), purine nucleoside phosphorylase (PNP) were measured in heart, liver and kidney cortex and medulla by analysing conversion of substrates into products using HPLC. The activity of ecto5'-nucleotidase differ in hearts of LDLR -/- and ApoE -/- mice with no differences in ADA and AMPD activity. We noticed highest activity of e5NT in kidney medulla of the models. This model of atherosclerosis characterize with an inhibition of enzyme responsible for production of protective adenosine in heart but not in other organs and different metabolism of nucleotides in kidney medulla.

Production of the angiotensin I converting enzyme inhibitory peptides and isolation of four novel peptides from jellyfish (Rhopilema esculentum) protein hydrolysate.

PubMed

Liu, Xin; Zhang, Miansong; Shi, Yaping; Qiao, Ruojin; Tang, Wei; Sun, Zhenliang

2016-07-01

Angiotensin I converting enzyme (ACE) plays an important role in regulating blood pressure in the human body. ACE inhibitory peptides derived from food proteins could exert antihypertensive effects without side effects. Jellyfish (Rhopilema esculentum) is an important fishery resource suitable for production of ACE inhibitory peptides. The objective of this study was to optimize the hydrolysis conditions for production of protein hydrolysate from R. esculentum (RPH) with ACE inhibitory activity, and to isolate and identify the ACE inhibitory peptides from RPH. Rhopilema esculentum protein was hydrolyzed with Compound proteinase AQ to produce protein hydrolysate with ACE inhibitory activity, and the hydrolysis conditions were optimized using response surface methodology. The optimum parameters for producing peptides with the highest ACE inhibitory activity were as follows: hydrolysis time 3.90 h, hydrolysis temperature 58 °C, enzyme:substrate ratio 2.8% and pH 7.60. Under these conditions, the ACE inhibitory rate reached 32.21%. In addition, four novel ACE inhibitory peptides were isolated, and their amino acids sequences were identified as Val-Gly-Pro-Tyr, Phe-Thr-Tyr-Val-Pro-Gly, Phe-Thr-Tyr-Val-Pro-Gly-Ala and Phe-Gln-Ala-Val-Trp-Ala-Gly, respectively. The IC50 value of the purified peptides for ACE inhibitory activity was 8.40, 23.42, 21.15 and 19.11 µmol L(-1) . These results indicate that the protein hydrolysate prepared from R. esculentum might be a commercial competitive source of ACE inhibitory ingredients to be used in functional foods. © 2015 Society of Chemical Industry. © 2015 Society of Chemical Industry.

Addition of vitamin D reverses the decline in GFR following treatment with ACE inhibitors/angiotensin receptor blockers in patients with chronic kidney disease.

PubMed

Soares, Abel Esteves; Maes, Michael; Godeny, Paula; Matsumoto, Andressa Keiko; Barbosa, Décio Sabbatini; da Silva, Taysa Antonia F; Souza, Flávio Henrique M O; Delfino, Vinicius Daher Alvares

2017-12-15

Vitamin D has anti-inflammatory, anti-fibrotic effect, and may block the intrarenal renin-angiotensin system. Adequate vitamin D levels in conjunction with the use of Angiotensin-converting Enzyme Inhibitors/Angiotensin Receptor Blockers may help to slow down chronic kidney disease progression. To study a possible beneficial effect of vitamin D supplementation in chronic kidney disease patients using angiotensin-converting enzyme inhibitors/angiotensin receptor blockers on chronic kidney disease progression we performed a clinical study involving vitamin D supplementation in patients with deficiency of this vitamin. This study was conducted in two chronic kidney disease clinics in the city of Londrina, Brazil, from October 2010 to December 2012. It was involved stage 3 and 4 chronic kidney disease (estimated glomerular filtration rate between 60 and 15mL/min/1.73m 2 ) patients with and without vitamin D deficiency. The patients ingested six-month cholecalciferol 50,000IU oral supplementation to chronic kidney disease patients with vitamin D deficiency. We hypothesize changes in estimated glomerular filtration rate over study period. Our data demonstrate reservation of estimated glomerular filtration with cholecalciferol supplementation to chronic kidney disease patients taking angiotensin-converting enzyme inhibitors/angiotensin receptor blockers. The combination treatment of angiotensin converting enzyme inhibitors/angiotensin receptor blockers with cholecalciferol prevents the decline in estimated glomerular filtration in patients with chronic kidney disease following treatment with angiotensin-converting enzyme inhibitors/angiotensin receptor blockers and may represent a valid approach to reduce renal disease progression in chronic kidney disease patients with vitamin D deficiency. This result needs confirmation in prospective controlled clinical trials. Copyright © 2017. Published by Elsevier Inc.

Demonstration of extrapulmonary activity of angiotensin converting enzyme in intact tissue preparations.

PubMed

Lembeck, F; Griesbacher, T; Eckhardt, M

1990-05-01

1. The activity of angiotensin converting enzyme (ACE) has been studied on functional parameters of intact isolated preparations of extrapulmonary tissues. The conversion of angiotensin I (A I) to angiotensin II (A II) and the cleavage of bradykinin (BK) were used as indicators of ACE activity. Captopril was employed as a specific inhibitor of ACE. 2. Captopril augmented the BK-induced contractions of the rat isolated uterus, the BK- and substance P-induced contractions of the guinea-pig ileum, and the BK-induced venoconstriction in the isolated perfused ear of the rabbit. Degradation of BK by ACE was calculated to be 52% in the rat uterus and 75% in the rabbit perfused ear. 3. Captopril inhibited the A I-induced contractions of the rat isolated colon, the A I-induced vasoconstriction in the isolated perfused ear of the rabbit and the rise in blood pressure induced by i.a. injections of A I in pithed rats. Conversion of A I to A II was calculated to be 13% in the rat colon and 26% in the rabbit perfused ear. 4. From estimations of the A II activity (bioassay on the rat colon) in the effluent of the perfused ear of the rabbit after injections of A I into the arterial inflow cannula it was calculated that approximately one tenth of A I was converted to A II during a single passage through the ear (less than 15 s). 5. The present experiments suggest that the high activity of ACE in endothelium of blood vessels of extrapulmonary tissues may provide an additional (endothelium-dependent) local vasoconstrictor mechanism by the rapid formation of A II and inactivation of BK. The ACE activity in non-vascular smooth muscles, other than those of blood vessels, may also affect the physiological functions of these tissues.

NREL Explains the Higher Cellulolytic Activity of a Vital Microorganism

DOE Office of Scientific and Technical Information (OSTI.GOV)

The discovery of a new mode of action by C. thermocellum to convert biomass to biofuels is significant because the bacterium is already recognized as one of the most effective in the biosphere. Researchers found that, in addition to using common cellulase degradation mechanisms attached to cells, C. thermocellum also uses a new category of cell-free scaffolded enzymes. The new discovery will influence the strategies used to improve the cellulolytic activity of biomass degrading microbes going forward. Better understanding of this bacterium could lead to cheaper production of ethanol and drop-in fuels. Also, this discovery demonstrates that nature's biomass conversionmore » behaviors are not fully understood and remain as opportunities for future microbial/enzyme engineering efforts.« less

Expression of apical Na(+)-L-glutamine co-transport activity, B(0)-system neutral amino acid co-transporter (B(0)AT1) and angiotensin-converting enzyme 2 along the jejunal crypt-villus axis in young pigs fed a liquid formula

USDA-ARS?s Scientific Manuscript database

Gut apical amino acid (AA) transport activity is high at birth and during suckling, thus being essential to maintain luminal nutrient-dependent mucosal growth through providing AA as essential metabolic fuel, substrates and nutrient stimuli for cellular growth. Because system-B(0) Na(+)-neutral AA c...

The influence of angiotensin-converting enzyme inhibitors on the aorta elastin metabolism in diet-induced hypercholesterolaemia in rabbits.

PubMed

Wojakowski, W; Gminski, J; Siemianowicz, K; Goss, M; Machalski, M

2001-03-01

Aortic elastin turnover is significantly accelerated in atherosclerosis, partly because of activation of the renin-angiotensin-aldosterone system caused by hypercholesterolaemia. We postulated that angiotensin-converting enzyme inhibitors (ACE-I) prevent the aortic elastin loss in experimental hypercholesterolaemia. Two doses of ACE-I (captopril, enalapril and quinapril) were used: a dose equivalent to that applied to human subjects and a dose 10 times higher. We found that the increase in serum and aortic elastolytic activity in cholesterol-fed rabbits was prevented by high-dose captopril. The elastin content in aorta homogenates from cholesterol-fed rabbits was significantly decreased. The higher dose of captopril, but no other ACE-I, prevented this decrease in aortic elastin content. In cholesterol-fed rabbits the elastin-bound calcium content was significantly elevated. The higher doses of captopril and enalapril lowered the elastin-bound calcium content. In serum and aortic homogenates of cholesterol-fed rabbits, ACE activity was elevated by 15% and 77%, respectively. Both doses of captopril, enalapril and quinapril prevented this cholesterol-induced increase in serum and aortic ACE activity. We conclude that: 1) administration of captopril at doses 10 times higher than those used in humans prevents hypercholesterolaemia increased aortic elastin loss. 2) higher doses of captopril and enalapril prevent the hypercholesterolaemia-induced increase in aortic elastin-bound calcium.

Isolation of angiotensin converting enzyme (ACE) inhibiting triterpenes from Schinus molle.

PubMed

Olafsson, K; Jaroszewski, J W; Smitt, U W; Nyman, U

1997-08-01

Bioactivity-guided fractionation of extracts of Schinus molle leaves, using an in vitro assay, led to the isolation of ACE-inhibitory steroidal triterpenes of the euphane type, identified by means of NMR spectroscopic methods. One of the triterpenes was isolated as an equilibrium mixture of epimeric aldehydes. The triterpenes showed moderate ACE-inhibitory activity (IC(50) about 250 microM).

The discovery of novel tartrate-based TNF-[alpha] converting enzyme (TACE) inhibitors

DOE Office of Scientific and Technical Information (OSTI.GOV)

Rosner, Kristin E.; Guo, Zhuyan; Orth, Peter

2010-09-17

A novel series of TNF-{alpha} convertase (TACE) inhibitors which are non-hydroxamate have been discovered. These compounds are bis-amides of L-tartaric acid (tartrate) and coordinate to the active site zinc in a tridentate manner. They are selective for TACE over other MMP's. We report the first X-ray crystal structure for a tartrate-based TACE inhibitor.

Chronic alcohol intake up-regulates hepatic expressions of carotenoid cleavage enzymes and peroxisomal proliferator-activated receptors in rats

USDA-ARS?s Scientific Manuscript database

Excessive and chronic alcohol intake leads to a lower hepatic vitamin A status by interfering with vitamin A metabolism.Dietary provitamin A carotenoids can be converted into vitamin A mainly by carotenoid 15,15’-monooxygenase 1 (CMO1) and, to a lesser degree, carotenoid 9910’-monooxygenase 2 (CMO2)...

Inhibition of Angiotensin-Converting Enzyme Activity by Flavonoids: Structure-Activity Relationship Studies

PubMed Central

Guerrero, Ligia; Castillo, Julián; Quiñones, Mar; Garcia-Vallvé, Santiago; Arola, Lluis; Pujadas, Gerard; Muguerza, Begoña

2012-01-01

Previous studies have demonstrated that certain flavonoids can have an inhibitory effect on angiotensin-converting enzyme (ACE) activity, which plays a key role in the regulation of arterial blood pressure. In the present study, 17 flavonoids belonging to five structural subtypes were evaluated in vitro for their ability to inhibit ACE in order to establish the structural basis of their bioactivity. The ACE inhibitory (ACEI) activity of these 17 flavonoids was determined by fluorimetric method at two concentrations (500 µM and 100 µM). Their inhibitory potencies ranged from 17 to 95% at 500 µM and from 0 to 57% at 100 µM. In both cases, the highest ACEI activity was obtained for luteolin. Following the determination of ACEI activity, the flavonoids with higher ACEI activity (i.e., ACEI >60% at 500 µM) were selected for further IC50 determination. The IC50 values for luteolin, quercetin, rutin, kaempferol, rhoifolin and apigenin K were 23, 43, 64, 178, 183 and 196 µM, respectively. Our results suggest that flavonoids are an excellent source of functional antihypertensive products. Furthermore, our structure-activity relationship studies show that the combination of sub-structures on the flavonoid skeleton that increase ACEI activity is made up of the following elements: (a) the catechol group in the B-ring, (b) the double bond between C2 and C3 at the C-ring, and (c) the cetone group in C4 at the C-ring. Protein-ligand docking studies are used to understand the molecular basis for these results. PMID:23185345

Exploration of the molecular interactions between angiotensin-I-converting enzyme (ACE) and the inhibitory peptides derived from hazelnut (Corylus heterophylla Fisch.).

PubMed

Liu, Chunlei; Fang, Li; Min, Weihong; Liu, Jingsheng; Li, Hongmei

2018-04-15

The mechanism of action of food-derived angiotensin-I-converting enzyme (ACE) inhibitory peptides has not been completely elucidated. In the present study, ion-exchange chromatography, gel filtration chromatography, reverse phase-high performance liquid chromatography, and liquid chromatography-electrospray ionization-tandem mass (LC-ESI-MS/MS) were employed for purifying and identifying the ACE inhibitory peptides from hazelnut. To understand the mode of action of these peptides, ACE inhibition kinetics, in vitro and in vivo bioavailability assays, active site analysis, and interaction between the inhibitory peptides and ACE were investigated. The results identified novel ACE inhibitory peptides Ala-Val-Lys-Val-Leu (AVKVL), Tyr-Leu-Val-Arg (YLVR), and Thr-Leu-Val-Gly-Arg (TLVGR) with IC 50 values of 73.06, 15.42, and 249.3 μM, respectively. All peptides inhibited the ACE activity via a non-competitive mode. The binding free energies of AVKVL, YLVR, and TLVGR for ACE were -3.46, -6.48, and -7.37 kcal/mol, respectively. The strong inhibition of ACE by YLVR may be attributed to the formation of cation-pi interactions. Copyright © 2017 Elsevier Ltd. All rights reserved.

Endotoxin reduces specific pulmonary uptake of radiolabeled monoclonal antibody to angiotensin-converting enzyme

DOE Office of Scientific and Technical Information (OSTI.GOV)

Muzykantov, V.R.; Puchnina, E.A.; Atochina, E.N.

The biodistribution of radiolabeled monoclonal antibody (Mab) to angiotensin-converting enzyme (ACE) was examined in normal and endotoxin-treated rats. Endotoxin administration at a dose of 4 mg/kg induced mild or middle pulmonary edema. The ACE activity in lung homogenate remained virtually unchanged, while the activity of serum ACE increased 15 hr after endotoxin infusion. In normal rats, anti-ACE Mab accumulates specifically in the lung after i.v. injection. Endotoxin injection induces reduction of specific pulmonary uptake of this antibody. Even in non-edematous endotoxemia, the accumulation of anti-ACE Mab antibody (Mab 9B9) decreased from 19.02 to 11.91% of ID/g of tissue without anymore » change in accumulation of control nonspecific IgG. The antibody distribution in other organs and its blood level were almost the same as in the control. In a case of endotoxemia accompanied by increased microvascular permeability, the lung accumulation of Mab 9B9 was reduced to 9.17% of ID/g of tissue, while the accumulation of nonspecific IgG increased to 1.44% versus 0.89% in the control.« less

Angiotensin-converting enzyme inhibitor (enalapril maleate) accelerates recovery of mouse skin from UVB-induced wrinkles.

PubMed

Matsuura-Hachiya, Yuko; Arai, Koji Y; Ozeki, Rieko; Kikuta, Ayako; Nishiyama, Toshio

2013-12-06

Angiotensin-converting enzyme (ACE) activity and angiotensin II signaling regulate cell proliferation, differentiation, and tissue remodeling, as well as blood pressure, while in skin, angiotensin II signaling is involved in wound healing, inflammation, and pathological scar formation. Therefore, we hypothesized that angiotensin II is also involved in photoaging of skin. In this study, we examined the effect of enalapril maleate, an ACE inhibitor, on recovery of wrinkled skin of hairless mice exposed to long-term UVB irradiation. Immunohistochemical observation revealed that expression of ACE, angiotensin II, and angiotensin II type 1 (AT1) and type 2 (AT2) receptors in the skin was increased after UVB irradiation (3 times/week at increasing intensities for 8 weeks). Administration of enalapril maleate (5 times/week for 6 weeks, starting 1 week after 10-week irradiation) accelerated recovery from UVB-induced wrinkles, epidermal hyperplasia and epidermal barrier dysfunction, as compared with the vehicle control. Our results indicate that ACE and angiotensin II activity are involved in skin photoaging, and suggest that ACE inhibitor such as enalapril maleate may have potential for improvement of photoaged skin. Copyright © 2013 Elsevier Inc. All rights reserved.

Phosphorylation of TNF-alpha converting enzyme by gastrin-releasing peptide induces amphiregulin release and EGF receptor activation.

PubMed

Zhang, Qing; Thomas, Sufi M; Lui, Vivian Wai Yan; Xi, Sichuan; Siegfried, Jill M; Fan, Huizhou; Smithgall, Thomas E; Mills, Gordon B; Grandis, Jennifer Rubin

2006-05-02

G protein-coupled receptors induce EGF receptor (EGFR) signaling, leading to the proliferation and invasion of cancer cells. Elucidation of the mechanism of EGFR activation by G protein-coupled receptors may identify new signaling paradigms. A gastrin-releasing peptide (GRP)/GRP receptor-mediated autocrine pathway was previously described in squamous cell carcinoma of head and neck. In the present study, we demonstrate that TNF-alpha converting enzyme (TACE), a disintegrin and metalloproteinse-17, undergoes a Src-dependent phosphorylation that regulates release of the EGFR ligand amphiregulin upon GRP treatment. Further investigation reveals the phosphatidylinositol 3-kinase (PI3-K) as the intermediate of c-Src and TACE, contributing to their association and TACE phosphorylation. Phosphoinositide-dependent kinase 1 (PDK1), a downstream target of PI3-K, has been identified as the previously undescribed kinase to directly phosphorylate TACE upon GRP treatment. These findings suggest a signaling cascade of GRP-Src-PI3-K-PDK1-TACE-amphiregulin-EGFR with multiple points of interaction, translocation, and phosphorylation. Furthermore, knockdown of PDK1 augmented the antitumor effects of the EGFR inhibitor erlotinib, indicating PDK1 as a therapeutic target to improve the clinical response to EGFR inhibitors.

Neuropeptide-hydrolysing activities in synaptosomal fractions from dog ileum myenteric, deep muscular and submucous plexi. Their participation in neurotensin inactivation.

PubMed

Barelli, H; Ahmad, S; Kostka, P; Fox, J A; Daniel, E E; Vincent, J P; Checler, F

1989-01-01

The mapping of neuropeptidases in synaptosomal fractions prepared from dog ileum myenteric, deep muscular and submucous plexus was established by means of fluorigenic substrates and specific inhibitors. Endopeptidase 24.11, angiotensin-converting enzyme and aminopeptidases were found in all tissues, the highest amounts being recovered in the submucous preparation. Post-proline dipeptidyl aminopeptidase was obtained in high quantities whatever the tissue source while proline endopeptidase was detected in low amounts and pyroglutamyl-peptide hydrolase was never detectable. The above peptidases were examined for their putative participation in the inactivation of neurotensin by monitoring the effect of specific inhibitors on the formation of the metabolites of labeled neurotensin separated by HPLC. Endopeptidases 24.11, 24.15 and 24.16 were respectively responsible for the formation of neurotensin(1-11), neurotensin(1-8) and neurotensin(1-10) that are devoid of biological activity. The secondary attacks occurring on neurotensin degradation products were the following: cleavage of neurotensin(1-10) into neurotensin(1-8) by angiotensin-converting enzyme; conversion of neurotensin(9-13) into neurotensin(11-13) by post-proline dipeptidyl aminopeptidase; hydrolysis of neurotensin(11-13) into free tyrosine by aminopeptidase(s).

Capillary liquid chromatography-ion trap-mass spectrometry methodology for the simultaneous quantification of four angiotensin-converting enzyme-inhibitory peptides in Prunus seed hydrolysates.

PubMed

González-García, Estefanía; García, María Concepción; Marina, María Luisa

2018-03-09

Prunus genus fruit seeds are sources of highly angiotensin-I-converting enzyme (ACE)-inhibitory peptides. The presence of peptides IYSPH, IYTPH, IFSPR, and VAIP seems to be related to this activity but no previous work has demonstrated the direct relationship between the concentration of these peptides and the antihypertensive activity of hydrolysates. This work describes the development of a method for the quantification of these peptides in Prunus seeds hydrolysates based on capillary liquid chromatography-IT-MS/MS. The analytical characteristics of the method were evaluated through the study of the linearity, LOD, LOQ, presence of matrix interferences, precision, and recovery. The developed methodology was applied to the determination of the four peptides in seed hydrolysates from different Prunus genus fruits: peaches (7 varieties), plums (2 varieties), nectarines (3 varieties), apricots (2 varieties), cherry, and paraguayo. Peaches and plums seed hydrolysates yielded the highest concentrations of these peptides while paraguayo one showed the lowest concentrations. A high correlation between peptides concentrations was demonstrated suggesting that the four peptides could be released from the same seed proteins. Copyright © 2018 Elsevier B.V. All rights reserved.

Phenolic compounds, antioxidant activity and in vitro inhibitory potential against key enzymes relevant for hyperglycemia and hypertension of commonly used medicinal plants, herbs and spices in Latin America.

PubMed

Ranilla, Lena Galvez; Kwon, Young-In; Apostolidis, Emmanouil; Shetty, Kalidas

2010-06-01

Traditionally used medicinal plants, herbs and spices in Latin America were investigated to determine their phenolic profiles, antioxidant activity and in vitro inhibitory potential against key enzymes relevant for hyperglycemia and hypertension. High phenolic and antioxidant activity-containing medicinal plants and spices such as Chancapiedra (Phyllantus niruri L.), Zarzaparrilla (Smilax officinalis), Yerba Mate (Ilex paraguayensis St-Hil), and Huacatay (Tagetes minuta) had the highest anti-hyperglycemia relevant in vitro alpha-glucosidase inhibitory activities with no effect on alpha-amylase. Molle (Schinus molle), Maca (Lepidium meyenii Walp), Caigua (Cyclanthera pedata) and ginger (Zingiber officinale) inhibited significantly the hypertension relevant angiotensin I-converting enzyme (ACE). All evaluated pepper (Capsicum) genus exhibited both anti-hyperglycemia and anti-hypertension potential. Major phenolic compounds in Matico (Piper angustifolium R.), Guascas (Galinsoga parviflora) and Huacatay were chlorogenic acid and hydroxycinnamic acid derivatives. Therefore, specific medicinal plants, herbs and spices from Latin America have potential for hyperglycemia and hypertension prevention associated with Type 2 diabetes. (c) 2010 Elsevier Ltd. All rights reserved.

Enhanced activity of an angiotensin-(1-7) neuropeptidase in glucocorticoid-induced fetal programming.

PubMed

Marshall, Allyson C; Shaltout, Hossam A; Pirro, Nancy T; Rose, James C; Diz, Debra I; Chappell, Mark C

2014-02-01

We previously identified angiotensin converting enzyme (ACE) and an endopeptidase activity that degraded angiotensin-(1-7) [Ang-(1-7)] to Ang-(1-5) and Ang-(1-4), respectively, in the cerebrospinal fluid (CSF) of 6-month old male sheep. The present study undertook a more comprehensive analysis of the CSF peptidase that converts Ang-(1-7) to Ang-(1-4) in control and in utero betamethasone-exposed sheep (BMX). Characterization of the Ang-(1-7) peptidase revealed that the thiol agents 4-aminophenylmercuric acetate (APMA) and p-chloromercuribenzoic acid (PCMB), as well as the metallo-chelators o-phenanthroline and EDTA essentially abolished the enzyme activity. Additional inhibitors for serine, aspartyl, and cysteine proteases, as well as selective inhibitors against the endopeptidases neprilysin, neurolysin, prolyl and thimet oligopeptidases did not attenuate enzymatic activity. Competition studies against the peptidase revealed similar IC50s for Ang-(1-7) (5μM) and Ang II (3μM), but lower values for Ala(1)-Ang-(1-7) and Ang-(2-7) of 1.8 and 2.0μM, respectively. In contrast, bradykinin exhibited a 6-fold higher IC50 (32μM) than Ang-(1-7) while neurotensin was a poor competitor. Mean arterial pressure (78±1 vs. 94±2mmHg, N=4-5, P<0.01) and Ang-(1-7) peptidase activity (14.2±1 vs 32±1.5fmol/min/ml CSF, N=5, P<0.01) were higher in the BMX group, and enzyme activity inversely correlated with Ang-(1-7) content in CSF. Lower Ang-(1-7) expression in brain is linked to baroreflex impairment in hypertension and aging, thus, increased activity of an Ang-(1-7) peptidase may contribute to lower CSF Ang-(1-7) levels, elevated blood pressure and impaired reflex function in this model of fetal programming. Copyright © 2013 Elsevier Inc. All rights reserved.

Characteristic Variations and Similarities in Biochemical, Molecular, and Functional Properties of Glyoxalases across Prokaryotes and Eukaryotes

PubMed Central

Kaur, Charanpreet; Sharma, Shweta; Hasan, Mohammad Rokebul; Pareek, Ashwani; Singla-Pareek, Sneh L.; Sopory, Sudhir K.

2017-01-01

The glyoxalase system is the ubiquitous pathway for the detoxification of methylglyoxal (MG) in the biological systems. It comprises two enzymes, glyoxalase I (GLYI) and glyoxalase II (GLYII), which act sequentially to convert MG into d-lactate, thereby helping living systems get rid of this otherwise cytotoxic byproduct of metabolism. In addition, a glutathione-independent GLYIII enzyme activity also exists in the biological systems that can directly convert MG to d-lactate. Humans and Escherichia coli possess a single copy of GLYI (encoding either the Ni- or Zn-dependent form) and GLYII genes, which through MG detoxification provide protection against various pathological and disease conditions. By contrast, the plant genome possesses multiple GLYI and GLYII genes with a role in abiotic stress tolerance. Plants possess both Ni2+- and Zn2+-dependent forms of GLYI, and studies on plant glyoxalases reveal the various unique features of these enzymes distinguishing them from prokaryotic and other eukaryotic glyoxalases. Through this review, we provide an overview of the plant glyoxalase family along with a comparative analysis of glyoxalases across various species, highlighting similarities as well as differences in the biochemical, molecular, and physiological properties of these enzymes. We believe that the evolution of multiple glyoxalases isoforms in plants is an important component of their robust defense strategies. PMID:28358304

Enzymes processing somatostatin precursors: an Arg-Lys esteropeptidase from the rat brain cortex converting somatostatin-28 into somatostatin-14.

PubMed Central

Gluschankof, P; Morel, A; Gomez, S; Nicolas, P; Fahy, C; Cohen, P

1984-01-01

The post-translational proteolytic conversion of somatostatin-14 precursors was studied to characterize the enzyme system responsible for the production of the tetradecapeptide either from its 15-kDa precursor protein or from its COOH-terminal fragment, somatostatin-28. A synthetic undecapeptide Pro-Arg-Glu-Arg-Lys-Ala-Gly-Ala-Lys-Asn-Tyr(NH2), homologous to the amino acid sequence of the octacosapeptide at the putative Arg-Lys cleavage locus, was used as substrate, after 125I labeling on the COOH-terminal tyrosine residue. A 90-kDa proteolytic activity was detected in rat brain cortex extracts after molecular sieve fractionation followed by ion exchange chromatography. The protease released the peptide 125I-Ala-Gly-Ala-Lys-Asn-Tyr(NH2) from the synthetic undecapeptide substrate and converted somatostatin-28 into somatostatin-14 under similar conditions (pH 7.0). Under these experimental conditions, the product tetradecapeptide was not further degraded by the enzyme. In contrast, the purified 15-kDa hypothalamic precursor remained unaffected when exposed to the proteolytic enzyme under identical conditions. It is concluded that this Arg-Lys esteropeptidase from the brain cortex may be involved in the in vivo processing of the somatostatin-28 fragment of prosomatostatin into somatostatin-14, the former species being an obligatory intermediate in a two-step proteolytic mechanism leading to somatostatin-14. PMID:6149550

Molecular recognition and regulation of human angiotensin-I converting enzyme (ACE) activity by natural inhibitory peptides

PubMed Central

Masuyer, Geoffrey; Schwager, Sylva L. U.; Sturrock, Edward D.; Isaac, R. Elwyn; Acharya, K. Ravi

2012-01-01

Angiotensin-I converting enzyme (ACE), a two-domain dipeptidylcarboxypeptidase, is a key regulator of blood pressure as a result of its critical role in the renin-angiotensin-aldosterone and kallikrein-kinin systems. Hence it is an important drug target in the treatment of cardiovascular diseases. ACE is primarily known for its ability to cleave angiotensin I (Ang I) to the vasoactive octapeptide angiotensin II (Ang II), but is also able to cleave a number of other substrates including the vasodilator bradykinin and N-acetyl-Ser-Asp-Lys-Pro (Ac-SDKP), a physiological modulator of hematopoiesis. For the first time we provide a detailed biochemical and structural basis for the domain selectivity of the natural peptide inhibitors of ACE, bradykinin potentiating peptide b and Ang II. Moreover, Ang II showed selective competitive inhibition of the carboxy-terminal domain of human somatic ACE providing evidence for a regulatory role in the human renin-angiotensin system (RAS). PMID:23056909

Apricot and other seed stones: amygdalin content and the potential to obtain antioxidant, angiotensin I converting enzyme inhibitor and hypocholesterolemic peptides.

PubMed

García, M C; González-García, E; Vásquez-Villanueva, R; Marina, M L

2016-11-09

Stones from olives and Prunus genus fruits are cheap and sustainable sources of proteins and could be potential sources of bioactive peptides. The main limitation to the use of these seeds is the presence of amygdalin. This work proposes to determine amygdalin in olive and Prunus seeds and in protein isolates obtained from them. Moreover, antioxidant, angiotensin I converting enzyme (ACE) inhibitor, and hypocholesterolemic properties will be evaluated in hydrolysates obtained from these seeds. Despite some seeds contained amygdalin, all protein isolates were free of this substance. Two different procedures to obtain bioactive peptides from protein isolates were examined: gastrointestinal digestion and processing with Alcalase, Flavourzyme or Thermolysin. Higher antioxidant, ACE inhibitor and hypocholesterolemic activities were observed when proteins were processed with Alcalase, Flavourzyme or Thermolysin. The highest antioxidant and ACE inhibitor capacities were observed for the Prunus genus seed hydrolysates while the highest capacity to reduce micellar cholesterol solubility was observed for the apricot and olive seed hydrolysates.

Preparation of lisinopril-capped gold nanoparticles for molecular imaging of angiotensin-converting enzyme

NASA Astrophysics Data System (ADS)

Li, Yuan; Baeta, Cesar; Aras, Omer; Daniel, Marie-Christine

2009-05-01

Overexpression of angiotensin-converting enzyme (ACE) has been associated with the pathophysiology of cardiac and pulmonary fibrosis. Moreover, the prescription of ACE inhibitors, such as lisinopril, has shown a favorable effect on patient outcome for patients with heart failure or systemic hypertension. Thus targeted imaging of the ACE would be of crucial importance for monitoring tissue ACE activity as well as the treatment efficacy in heart failure. In this respect, lisinopril-coated gold nanoparticles were prepared to provide a new type of probe for targeted molecular imaging of ACE by tuned K-edge computed tomography (CT) imaging. The preparation involved non-modified lisinopril, using its primary amine group as the anchoring function on the gold nanoparticles surface. The stable lisinopril-coated gold nanoparticles obtained were characterized by UV-vis spectroscopy, dynamic light scattering (DLS), transmission electron microscopy (TEM). Their zeta potential was also measured in order to assess the charge density on the modified gold nanoparticles (GNPs).

Angiotensin-I converting enzyme inhibitory peptides from antihypertensive skate (Okamejei kenojei) skin gelatin hydrolysate in spontaneously hypertensive rats.

PubMed

Ngo, Dai-Hung; Kang, Kyong-Hwa; Ryu, BoMi; Vo, Thanh-Sang; Jung, Won-Kyo; Byun, Hee-Guk; Kim, Se-Kwon

2015-05-01

The aim of this study was to investigate antihypertensive effect of bioactive peptides from skate (Okamejei kenojei) skin gelatin. The Alcalase/protease gelatin hydrolysate below 1 kDa (SAP) exhibited the highest angiotensin-I converting enzyme (ACE) inhibition compared to other hydrolysates. SAP can decrease systolic blood pressure significantly in spontaneously hypertensive rats. SAP inhibited vasoconstriction via PPAR-γ expression, activation and phosphorylation of eNOS in lungs. Moreover, the expression levels of endothelin-1, RhoA, α-smooth muscle actin, cleaved caspase 3 and MAPK were decreased by SAP in lungs. Vascularity, muscularization and cellular proliferation in lungs were detected by immunohistochemical staining. Finally, two purified peptides (LGPLGHQ, 720Da and MVGSAPGVL, 829Da) showed potent ACE inhibition with IC50 values of 4.22 and 3.09 μM, respectively. These results indicate that bioactive peptides isolated from skate skin gelatin may serve as candidates against hypertension and could be used as functional food ingredients. Copyright © 2014 Elsevier Ltd. All rights reserved.

Reducing Enzyme Costs Increases Market Potential of Biofuels; The Spectrum of Clean Energy Innovation (Fact Sheet)

DOE Office of Scientific and Technical Information (OSTI.GOV)

Not Available

Fact sheet describing NREL's work with enzyme producers Novozymes and Genencor to engineer new cellulase enzymes to breakdown cellulosic ethanol into fermentable sugars that can be converted into biofuels.

Exercise training modulates the hepatic renin-angiotensin system in fructose-fed rats.

PubMed

Frantz, Eliete Dalla Corte; Medeiros, Renata Frauches; Giori, Isabele Gomes; Lima, Juliana Bittencourt Silveira; Bento-Bernardes, Thais; Gaique, Thaiane Gadioli; Fernandes-Santos, Caroline; Fernandes, Tiago; Oliveira, Edilamar Menezes; Vieira, Carla Paulo; Conte-Junior, Carlos Adam; Oliveira, Karen Jesus; Nobrega, Antonio Claudio Lucas

2017-09-01

What is the central question of this study? What are the effects of exercise training on the hepatic renin-angiotensin system and their contribution to damage resulting from fructose overload in rats? What is the main finding and its importance? Exercise training attenuated the deleterious actions of the angiotensin-converting enzyme/angiotensin II/angiotensin II type 1 receptor axis and increased expression of the counter-regulatory (angiotensin-converting enzyme 2/angiotensin (1-7)/Mas receptor) axis in the liver. Therefore, our study provides evidence that exercise training modulates the hepatic renin-angiotensin system, which contributes to reducing the progression of metabolic dysfunction and non-alcoholic fatty liver disease in fructose-fed rats. The renin-angiotensin system (RAS) has been implicated in the development of metabolic syndrome. We investigated whether the hepatic RAS is modulated by exercise training and whether this modulation improves the deleterious effects of fructose overload in rats. Male Wistar rats were divided into (n = 8 each) control (CT), exercise control (CT-Ex), high-fructose (HFr) and exercise high-fructose (HFr-Ex) groups. Fructose-drinking rats received d-fructose (100 g l -1 ). After 2 weeks, CT-Ex and HFr-Ex rats were assigned to a treadmill training protocol at moderate intensity for 8 weeks (60 min day -1 , 4 days per week). We assessed body mass, glucose and lipid metabolism, hepatic histopathology, angiotensin-converting enzyme (ACE) and angiotensin-converting enzyme 2 (ACE2) activity, the angiotensin concentration and the expression profile of proteins affecting the hepatic RAS, gluconeogenesis and inflammation. Neither fructose overload nor exercise training influenced body mass gain and serum ACE and ACE2 activity. The HFr group showed hyperinsulinaemia, but exercise training normalized this parameter. Exercise training was effective in preventing hepatic steatosis and in preventing triacylglycerol and glycogen accumulation. Furthermore, exercise improved the response to the deleterious effects of HFr overload by normalizing the gluconeogenesis pathway and the protein levels of interleukin-6 and tumour necrosis factor-α. The HFr rats displayed increased hepatic ACE activity and protein expression and angiotensin II concentration, which were attenuated by exercise training. Exercise training restored the ACE2/angiotensin-(1-7)/Mas receptor axis. Exercise training may favour the counter-regulatory ACE2/angiotensin-(1-7)/Mas receptor axis over the classical RAS (ACE/angiotensin II/angiotensin II type 1 receptor axis), which could be responsible for the reduction of metabolic dysfunction and the prevention of non-alcoholic fatty liver disease. © 2017 The Authors. Experimental Physiology © 2017 The Physiological Society.

Computational multiscale modeling in protein--ligand docking.

PubMed

Taufer, Michela; Armen, Roger; Chen, Jianhan; Teller, Patricia; Brooks, Charles

2009-01-01

In biological systems, the binding of small molecule ligands to proteins is a crucial process for almost every aspect of biochemistry and molecular biology. Enzymes are proteins that function by catalyzing specific biochemical reactions that convert reactants into products. Complex organisms are typically composed of cells in which thousands of enzymes participate in complex and interconnected biochemical pathways. Some enzymes serve as sequential steps in specific pathways (such as energy metabolism), while others function to regulate entire pathways and cellular functions [1]. Small molecule ligands can be designed to bind to a specific enzyme and inhibit the biochemical reaction. Inhibiting the activity of key enzymes may result in the entire biochemical pathways being turned on or off [2], [3]. Many small molecule drugs marketed today function in this generic way as enzyme inhibitors. If research identifies a specific enzyme as being crucial to the progress of disease, then this enzyme may be targeted with an inhibitor, which may slow down or reverse the progress of disease. In this way, enzymes are targeted from specific pathogens (e.g., virus, bacteria, fungi) for infectious diseases [4], [5], and human enzymes are targeted for noninfectious diseases such as cardiovascular disease, cancer, diabetes, and neurodegenerative diseases [6].

Rediscovering ACE: Novel insights into the many roles of the angiotensin-converting enzyme

PubMed Central

Gonzalez-Villalobos, Romer A.; Shen, Xiao Z.; Bernstein, Ellen A.; Janjulia, Tea; Taylor, Brian; Giani, Jorge F.; Blackwell, Wendell-Lamar B.; Shah, Kandarp H.; Shi, Peng D.; Fuchs, Sebastien; Bernstein, Kenneth E.

2013-01-01

Angiotensin converting enzyme (ACE) is best known for the catalytic conversion of angiotensin I to angiotensin II. However, the use of gene-targeting techniques has led to mouse models highlighting many other biochemical properties and actions of this enzyme. This review discusses recent studies examining the functional significance of ACE tissue-specific expression and the presence in ACE of two independent catalytic sites with distinct substrates and biological effects. It is these features which explain why ACE makes important contributions to many different physiological processes including renal development, blood pressure control, inflammation and immunity. PMID:23686164

Activation mechanism of erythrocyte cathepsin E. evidence for the occurrence of the membrane-associated active enzyme.

PubMed

Ueno, E; Sakai, H; Kato, Y; Yamamoto, K

1989-06-01

Activation of the erythrocyte cathepsin E located on the cytoplasmic surface of the membrane in a latent form was studied in stripped inside-out membrane vesicles prepared from human erythrocyte membranes. Incubation of the vesicles at 40 degrees C at pH 4 resulted in increased degradation of the membrane proteins, especially band 3. This proteolysis was selectively inhibited by the inclusion of pepstatin (isovaleryl-Val-Val-statyl-Ala-statine) or H 297 [Pro-Thr-Glu-Phe(CH2-NH)Nle-Arg-Leu] in the incubation mixtures, indicating that cathepsin E, as the only aspartic proteinase in erythrocytes, is responsible for the proteolysis. Two potential active-site-directed inhibitors of aspartic proteinases, pepstatin and H 297, were used to prove the occurrence of the membrane-associated active enzyme. To minimize potential errors arising from non-specific binding, the concentrations of the inhibitors used in the binding assay (pepstatin, 5 x 10(-8) M; H 297, 1 x 10(-5) M) were determined by calibration for purified and membrane-associated cathepsin E. The inhibition of the membrane-associated cathepsin E by each inhibitor, which showed the binding of the inhibitor to the activated enzyme, was temperature- and time-dependent. The binding of each inhibitor to the enzyme on the exposed surface of the membrane at pH 4 was highly specific, saturable, and reversible. The present study thus provides the first evidence that cathepsin E tightly bound to the membrane is converted to the active enzyme in the membrane-associated form, and suggests that this enzyme may be responsible for the degradation of band 3.

Doppler laser imaging predicts response to topical minoxidil in the treatment of female pattern hair loss.

PubMed

McCoy, J; Kovacevic, M; Situm, M; Stanimirovic, A; Bolanca, Z; Goren, A

2016-01-01

Topical minoxidil is the only drug approved by the US FDA for the treatment of female pattern hair loss. Unfortunately, following 16 weeks of daily application, less than 40% of patients regrow hair. Several studies have demonstrated that sulfotransferase enzyme activity in plucked hair follicles predicts topical minoxidil response in female pattern hair loss patients. However, due to patients’ discomfort with the procedure, and the time required to perform the enzymatic assay it would be ideal to develop a rapid, non-invasive test for sulfotransferase enzyme activity. Minoxidil is a pro-drug converted to its active form, minoxidil sulfate, by sulfotransferase enzymes in the outer root sheath of hair. Minoxidil sulfate is the active form required for both the promotion of hair regrowth and the vasodilatory effects of minoxidil. We thus hypothesized that laser Doppler velocimetry measurement of scalp blood perfusion subsequent to the application of topical minoxidil would correlate with sulfotransferase enzyme activity in plucked hair follicles. In this study, plucked hair follicles from female pattern hair loss patients were analyzed for sulfotransferase enzyme activity. Additionally, laser Doppler velocimetry was used to measure the change in scalp perfusion at 15, 30, 45, and 60 minutes, after the application of minoxidil. In agreement with our hypothesis, we discovered a correlation (r=1.0) between the change in scalp perfusion within 60 minutes after topical minoxidil application and sulfotransferase enzyme activity in plucked hairs. To our knowledge, this is the first study demonstrating the feasibility of using laser Doppler imaging as a rapid, non-invasive diagnostic test to predict topical minoxidil response in the treatment of female pattern hair loss.

Dependence of PERT endpoint on endogenous lipase activity.

PubMed

Gao, Wen-Yi; Mulberg, Andrew E

2014-11-01

To clarify and to understand the potential for misinterpretation of change in fecal fat quantitation during pancreatic enzyme replacement therapy (PERT) trials for treatment of exocrine pancreatic insufficiency. Analysis of clinical trials submitted to the U.S. Food and Drug Administration (FDA) for approval of PERT that enrolled 123 cystic fibrosis adult and pediatric patients treated with Creon, Pertzye, Ultresa, and Zenpep. The CFA% defines lipase activity as a percentage of converting substrate of "Total Daily Dietary Fat Intake." PERT trials performed to date have modified the definition to converting the "Shared Daily Fat Intake," generating "Partial CFA" for the exogenous lipase: the higher the activity of coexisting endogenous lipase, the lower the "Partial CFA" of exogenous measured. This review shows that "Partial CFA" is not CFA. Enrollment of patients with low HPLA during treatment may improve the interpretability of "Partial CFA" measured by PERT trials.

Association between angiotensin II receptor gene polymorphism and serum angiotensin converting enzyme (SACE) activity in patients with sarcoidosis.

PubMed

Takemoto, Y; Sakatani, M; Takami, S; Tachibana, T; Higaki, J; Ogihara, T; Miki, T; Katsuya, T; Tsuchiyama, T; Yoshida, A; Yu, H; Tanio, Y; Ueda, E

1998-06-01

Serum angiotensin converting enzyme (SACE) is considered to reflect disease activity in sarcoidosis. SACE activity is increased in many patients with active sarcoid lesions. The mechanism for the increased SACE activity in this disease has not been clarified. ACE insertion/deletion (I/D) gene polymorphism has been reported to have an association with SACE levels in sarcoidosis, but no evidence of an association between angiotensin II receptor gene polymorphism and SACE in this disease has been found. A study of the association of angiotensin II receptor gene polymorphisms with sarcoidosis was therefore undertaken. ACE (I/D), angiotensin II type 1 receptor (AGTR1), and angiotensin II type 2 receptor (AGTR2) gene polymorphisms were investigated by polymerase chain reaction (PCR) and SACE levels were measured in three groups of patients: those with sarcoidosis or tuberculosis and normal controls. There was no difference in allele frequency of AGTR1 and AGTR2 polymorphism among the three groups. Neither AGTR1 nor AGTR2 polymorphisms were associated with sarcoidosis. SACE activity was higher in patients with sarcoidosis with the AGTR1 A/C genotype than in others. However, this tendency was not detected in patients with tuberculosis. The AGTR1 allele C is associated with high activity of SACE in patients with sarcoidosis. It is another predisposing factor for high levels of SACE in patients with sarcoidosis and is considered to be an independent factor from the ACE D allele for high levels of SACE in sarcoidosis. This fact could be one of the explanations for the increased SACE activity in sarcoidosis.

Respiratory epithelial cells convert inactive vitamin D to its active form: potential effects on host defense.

PubMed

Hansdottir, Sif; Monick, Martha M; Hinde, Sara L; Lovan, Nina; Look, Dwight C; Hunninghake, Gary W

2008-11-15

The role of vitamin D in innate immunity is increasingly recognized. Recent work has identified a number of tissues that express the enzyme 1alpha-hydroxylase and are able to activate vitamin D. This locally produced vitamin D is believed to have important immunomodulatory effects. In this paper, we show that primary lung epithelial cells express high baseline levels of activating 1alpha-hydroxylase and low levels of inactivating 24-hydroxylase. The result of this enzyme expression is that airway epithelial cells constitutively convert inactive 25-dihydroxyvitamin D(3) to the active 1,25-dihydroxyvitamin D(3). Active vitamin D that is generated by lung epithelium leads to increased expression of vitamin D-regulated genes with important innate immune functions. These include the cathelicidin antimicrobial peptide gene and the TLR coreceptor CD14. dsRNA increases the expression of 1alpha-hydroxylase, augments the production of active vitamin D, and synergizes with vitamin D to increase expression of cathelicidin. In contrast to induction of the antimicrobial peptide, vitamin D attenuates dsRNA-induced expression of the NF-kappaB-driven gene IL-8. We conclude that primary epithelial cells generate active vitamin D, which then influences the expression of vitamin D-driven genes that play a major role in host defense. Furthermore, the presence of vitamin D alters induction of antimicrobial peptides and inflammatory cytokines in response to viruses. These observations suggest a novel mechanism by which local conversion of inactive to active vitamin D alters immune function in the lung.

Alpha 2-macroglobulin capture allows detection of mast cell chymase in serum and creates a reservoir of angiotensin II-generating activity.

PubMed

Raymond, Wilfred W; Su, Sharon; Makarova, Anastasia; Wilson, Todd M; Carter, Melody C; Metcalfe, Dean D; Caughey, George H

2009-05-01

Human chymase is a highly efficient angiotensin II-generating serine peptidase expressed by mast cells. When secreted from degranulating cells, it can interact with a variety of circulating antipeptidases, but is mostly captured by alpha(2)-macroglobulin, which sequesters peptidases in a cage-like structure that precludes interactions with large protein substrates and inhibitors, like serpins. The present work shows that alpha(2)-macroglobulin-bound chymase remains accessible to small substrates, including angiotensin I, with activity in serum that is stable with prolonged incubation. We used alpha(2)-macroglobulin capture to develop a sensitive, microtiter plate-based assay for serum chymase, assisted by a novel substrate synthesized based on results of combinatorial screening of peptide substrates. The substrate has low background hydrolysis in serum and is chymase-selective, with minimal cleavage by the chymotryptic peptidases cathepsin G and chymotrypsin. The assay detects activity in chymase-spiked serum with a threshold of approximately 1 pM (30 pg/ml), and reveals native chymase activity in serum of most subjects with systemic mastocytosis. alpha(2)-Macroglobulin-bound chymase generates angiotensin II in chymase-spiked serum, and it appears in native serum as chymostatin-inhibited activity, which can exceed activity of captopril-sensitive angiotensin-converting enzyme. These findings suggest that chymase bound to alpha(2)-macroglobulin is active, that the complex is an angiotensin-converting enzyme inhibitor-resistant reservoir of angiotensin II-generating activity, and that alpha(2)-macroglobulin capture may be exploited in assessing systemic release of secreted peptidases.

α2-Macroglobulin Capture Allows Detection of Mast Cell Chymase in Serum and Creates a Circulating Reservoir of Angiotensin II-generating Activity1

PubMed Central

Raymond, Wilfred W.; Su, Sharon; Makarova, Anastasia; Wilson, Todd M.; Carter, Melody C.; Metcalfe, Dean D.; Caughey, George H.

2009-01-01

Human chymase is a highly efficient angiotensin II-generating serine peptidase expressed by the MCTC subset of mast cells. When secreted from degranulating cells, it can interact with a variety of circulating anti-peptidases, but is mostly captured by α2-macroglobulin, which sequesters peptidases in a cage-like structure that precludes interactions with large protein substrates and inhibitors, like serpins. The present work shows that α2-macroglobulin-bound chymase remains accessible to small substrates, including angiotensin I, with activity in serum that is stable with prolonged incubation. We used α2-macroglobulin capture to develop a sensitive, microtiter plate-based assay for serum chymase, assisted by a novel substrate synthesized based on results of combinatorial screening of peptide substrates. The substrate has low background hydrolysis in serum and is chymase-selective, with minimal cleavage by the chymotryptic peptidases cathepsin G and chymotrypsin. The assay detects activity in chymase-spiked serum with a threshold of ~1 pM (30 pg/ml), and reveals native chymase activity in serum of most subjects with systemic mastocytosis. α2-Macroglobulin-bound chymase generates angiotensin II in chymase-spiked serum, and appears in native serum as chymostatin-inhibited activity, which can exceed activity of captopril-sensitive angiotensin converting enzyme. These findings suggest that chymase bound to α2-macroglobulin is active, that the circulating complex is an angiotensin-converting enzyme inhibitor-resistant reservoir of angiotensin II-generating activity, and that α2-macroglobulin capture may be exploited in assessing systemic release of secreted peptidases. PMID:19380825

Elevated plasma angiotensin converting enzyme 2 activity is an independent predictor of major adverse cardiac events in patients with obstructive coronary artery disease.

PubMed

Ramchand, Jay; Patel, Sheila K; Srivastava, Piyush M; Farouque, Omar; Burrell, Louise M

2018-01-01

Angiotensin converting enzyme 2 (ACE2) is an endogenous regulator of the renin angiotensin system. Increased circulating ACE2 predicts adverse outcomes in patients with heart failure (HF), but it is unknown if elevated plasma ACE2 activity predicts major adverse cardiovascular events (MACE) in patients with obstructive coronary artery disease (CAD). We prospectively recruited patients with obstructive CAD (defined as ≥50% stenosis of the left main coronary artery and/or ≥70% stenosis in ≥ 1 other major epicardial vessel on invasive coronary angiography) and measured plasma ACE2 activity. Patients were followed up to determine if circulating ACE2 activity levels predicted the primary endpoint of MACE (cardiovascular mortality, HF or myocardial infarction). We recruited 79 patients with obstructive coronary artery disease. The median (IQR) plasma ACE2 activity was 29.3 pmol/ml/min [21.2-41.2]. Over a median follow up of 10.5 years [9.6-10.8years], MACE occurred in 46% of patients (36 events). On Kaplan-Meier analysis, above-median plasma ACE2 activity was associated with MACE (log-rank test, p = 0.035) and HF hospitalisation (p = 0.01). After Cox multivariable adjustment, log ACE2 activity remained an independent predictor of MACE (hazard ratio (HR) 2.4, 95% confidence interval (CI) 1.24-4.72, p = 0.009) and HF hospitalisation (HR: 4.03, 95% CI: 1.42-11.5, p = 0.009). Plasma ACE2 activity independently increased the hazard of adverse long-term cardiovascular outcomes in patients with obstructive CAD.

Serum angiotensin-converting enzyme is elevated in association with underground coal mining

DOE Office of Scientific and Technical Information (OSTI.GOV)

Thompson, A.B.; Cale, W.F.; Lapp, N.L.

1991-10-01

Serum angiotensin-converting enzyme activity (SACE) and lysozyme activity were measured in a group of 40 underground coal miners and two control groups, 20 subjects with sarcoidosis and 15 normal non-dust-exposed volunteers. The miners were grouped first according to whether they had recent exposure (still actively mining or retired three years or less prior to measurement) or temporally more distant exposure (retired more than three years prior to measurement). Secondly, they were grouped as to whether or not they had coal workers' pneumoconiosis (CWP). The subjects with sarcoidosis were grouped according to disease activity. As expected, the subjects with active sarcoidosismore » had elevated SACE activity compared with normal subjects. The coal miners as a group did not have elevation of their SACE activity. However, the coal miners with recent exposure had elevated SACE activity (57.1 {plus minus} 3.9 U/ml) compared with normal controls (43.8 {plus minus} 1.5 U/ml, p = 0.007). The SACE activity in miners without recent exposure was not elevated (39.8 {plus minus} 1.3 U/ml) compared with the normal controls. No increase in SACE activity was found when the miners were grouped according to the presence or absence of CWP. In contrast, the miners' serum lysozyme activity was not elevated. Since alveolar macrophages are a potential source of SACE, elevation of SACE activity in underground coal miners may reflect alveolar macrophage activation caused by increased pulmonary mixed coal mine dust burden. Furthermore, since both SACE and serum lysozyme are elevated in association with silicosis, these findings may confirm that the macrophage responses to inhaled silica and coal dust differ.« less

Two enzymatic reaction pathways in the formation of pyropheophorbide a.

PubMed

Suzuki, Yasuyo; Doi, Michio; Shioi, Yuzo

2002-01-01

The demethoxycarbonyl reaction of pheophorbide a in plants and algae was investigated. Two types of enzyme that catalyze alternative reactions in the formation of pyropheophorbide a were found. One enzyme, designated 'pheophorbidase (Phedase)', was purified nearly to homogeneity from cotyledons of radish (Raphanus sativus). This enzyme catalyzes the conversion of pheophorbide a to a precursor of pyropheophorbide a, C-13(2)-carboxylpyropheophorbide a, by demethylation, and then the precursor is decarboxylated non-enzymatically to yield pyropheophorbide a. The activity of Phedase was inhibited by the reaction product, methanol. The other enzyme, termed 'pheophorbide demethoxycarbonylase (PDC)', was highly purified from the Chl b-less mutant NL-105 of Chlamydomonas reinhardtii. This enzyme had produced no intermediate as shown in the Phedase reaction, indicating that it converts pheophorbide a directly into pyropheophorbide a, probably by nucleophilic reaction. Phedase and PDC consisted of both senescence-induced and constitutive enzymes. The molecular weight of both Phedases was 113 000 and of senescence-induced PDC was 170 000. The K (m) values against pheophorbide a for both Phedases were 14-15 muM and 283 muM for senescence-induced PDC. The activity of both Phedases was inhibited by the reaction product, methanol, whereas methanol had no specific effect on senescence-induced PDC. Phenylmethylsulfonic fluoride and N-ethylmaleimide inhibited the senescence-induced Phedase and PDC, respectively. Among the 23 species from 15 different families tested, Phedase activity was found in 10 species from three families. PDC activity was not detected in plants lacking Phedase activity, except for Chlamydomonas. Based on these findings, a likely conclusion is that at least two alternative pathways that are catalyzed by two different enzymes, Phedase and PDC, exist for the formation of pyropheophorbide a.

New Perspectives in the Renin-Angiotensin-Aldosterone System (RAAS) I: Endogenous Angiotensin Converting Enzyme (ACE) Inhibition

PubMed Central

Fagyas, Miklós; Úri, Katalin; Siket, Ivetta M.; Daragó, Andrea; Boczán, Judit; Bányai, Emese; Édes, István; Papp, Zoltán; Tóth, Attila

2014-01-01

Angiotensin-converting enzyme (ACE) inhibitors represent the fifth most often prescribed drugs. ACE inhibitors decrease 5-year mortality by approximately one-fifth in cardiovascular patients. Surprisingly, there are reports dating back to 1979 suggesting the existence of endogenous ACE inhibitors, which endogenous inhibitory effects are much less characterized than that for the clinically administered ACE inhibitors. Here we aimed to investigate this endogenous ACE inhibition in human sera. It was hypothesized that ACE activity is masked by an endogenous inhibitor, which dissociates from the ACE when its concentration decreases upon dilution. ACE activity was measured by FAPGG hydrolysis first. The specific (dilution corrected) enzyme activities significantly increased by dilution of human serum samples (23.2±0.7 U/L at 4-fold dilution, 51.4±0.3 U/L at 32-fold dilution, n = 3, p = 0.001), suggesting the presence of an endogenous inhibitor. In accordance, specific enzyme activities did not changed by dilution when purified renal ACE was used, where no endogenous inhibitor was present (655±145 U/L, 605±42 U/L, n = 3, p = 0.715, respectively). FAPGG conversion strongly correlated with angiotensin I conversion suggesting that this feature is not related to the artificial substrate. Serum samples were ultra-filtered to separate ACE (MW: 180 kDa) and the hypothesized inhibitor. Filtering through 50 kDa filters was without effect, while filtering through 100 kDa filters eliminated the inhibiting factor (ACE activity after <100 kDa filtering: 56.4±2.4 U/L, n = 4, control: 26.4±0.7 U/L, n = 4, p<0.001). Lineweaver-Burk plot indicated non-competitive inhibition of ACE by this endogenous factor. The endogenous inhibitor had higher potency on the C-terminal active site than N-terminal active site of ACE. Finally, this endogenous ACE inhibition was also present in mouse, donkey, goat, bovine sera besides men (increasing of specific ACE activity from 4-fold to 32-fold dilution: 2.8-fold, 1.7-fold, 1.5-fold, 1.8-fold, 2.6-fold, respectively). We report here the existence of an evolutionary conserved mechanism suppressing circulating ACE activity, in vivo, similarly to ACE inhibitory drugs. PMID:24691160

Substrate metabolism, hormone interaction, and angiotensin-converting enzyme inhibitors in left ventricular hypertrophy.

PubMed

Zhu, Y C; Zhu, Y Z; Spitznagel, H; Gohlke, P; Unger, T

1996-01-01

Left ventricular hypertrophy is considered to be an independent risk factor giving rise to ischemia, arrhythmias, and left ventricular dysfunction. Slow movement of intracellular calcium contributes to the impaired contraction and relaxation function of hypertrophied myocardium. Myofibril content may also be shifted to fetal-type isoforms with decreased contraction and relaxation properties in left ventricular hypertrophy. Myocyte hypertrophy and interstitial fibrosis are regulated independently by mechanical and neurohumoral mechanisms. In severely hypertrophied myocardium, capillary density is reduced, the diffusion distance for oxygen, nutrients, and metabolites is increased, and the ratio of energy-production sites to energy-consumption sites is decreased. The metabolic state of severely hypertrophied myocardium is anaerobic, as indicated by the shift of lactate dehydrogenase marker enzymes. Therefore, the hypertrophied myocardium is more vulnerable to ischemic events. As a compensatory response to severe cardiac hypertrophy and congestive heart failure, the ADP/ATP carrier is activated and atrial natriuretic peptide is released to increase high-energy phosphate production and reduce cardiac energy consumption by vasodilation and sodium and fluid elimination. However, in severely hypertrophied and failing myocardium, vasoconstrictor and sodium- and fluid-retaining factors, such as the renin-angiotensin system, aldosterone, and sympathetic nerve activity, play an overwhelming role. Angiotensin-converting enzyme inhibitors (ACEIs) are able to prevent cardiac hypertrophy and improve cardiac function and metabolism. Under experimental conditions, these beneficial effects can be ascribed mainly to bradykinin potentiation, although a contribution of the ACEI-induced angiotensin II reduction cannot be excluded.

Angiotensin-converting enzyme inhibition studies by natural leech inhibitors by capillary electrophoresis and competition assay.

PubMed

Deloffre, Laurence; Sautiere, Pierre-Eric; Huybrechts, Roger; Hens, Korneel; Vieau, Didier; Salzet, Michel

2004-06-01

A protocol to follow the processing of angiotensin I into angiotensin II by rabbit angiotensin-converting enzyme (ACE) and its inhibition by a novel natural antagonist, the leech osmoregulator factor (LORF) using capillary zonal electrophoresis is described. The experiment was carried out using the Beckman PACE system and steps were taken to determine (a) the migration profiles of angiotensin and its yielded peptides, (b) the minimal amount of angiotensin II detected, (c) the use of different electrolytes and (d) the concentration of inhibitor. We demonstrated that LORF (IPEPYVWD), a neuropeptide previously found in leech brain, is able to inhibit rabbit ACE with an IC(50) of 19.8 micro m. Interestingly, its cleavage product, IPEP exhibits an IC(50) of 11.5 micro m. A competition assay using p-benzoylglycylglycylglycine and insect ACE established that LORF and IPEP fragments are natural inhibitors for invertebrate ACE. Fifty-four percent of insect ACE activity is inhibited with 50 micro m IPEP and 35% inhibition with LORF (25 mm). Extending the peptide at both N- and C-terminus (GWEIPEPYVWDES) and the cleavage of IPEP in IP abolished the inhibitory activity of both peptides. Immunocytochemical data obtained with antisera raised against LORF and leech ACE showed a colocalization between the enzyme and its inhibitor in the same neurons. These results showed that capillary zonal electrophoresis is a useful technique for following enzymatic processes with small amounts of products and constitutes the first evidence of a natural ACE inhibitor in invertebrates.

Effect of Jatropha curcas Peptide Fractions on the Angiotensin I-Converting Enzyme Inhibitory Activity

PubMed Central

Segura-Campos, Maira R.; Peralta-González, Fanny; Castellanos-Ruelas, Arturo; Chel-Guerrero, Luis A.; Betancur-Ancona, David A.

2013-01-01

Hypertension is one of the most common worldwide diseases in humans. Angiotensin I-converting enzyme (ACE) plays an important role in regulating blood pressure and hypertension. An evaluation was done on the effect of Alcalase hydrolysis of defatted Jatropha curcas kernel meal on ACE inhibitory activity in the resulting hydrolysate and its purified fractions. Alcalase exhibited broad specificity and produced a protein hydrolysate with a 21.35% degree of hydrolysis and 34.87% ACE inhibition. Ultrafiltration of the hydrolysate produced peptide fractions with increased biological activity (24.46–61.41%). Hydrophobic residues contributed substantially to the peptides' inhibitory potency. The 5–10 and <1 kDa fractions were selected for further fractionation by gel filtration chromatography. ACE inhibitory activity (%) ranged from 22.66 to 45.96% with the 5–10 kDa ultrafiltered fraction and from 36.91 to 55.83% with the <1 kDa ultrafiltered fraction. The highest ACE inhibitory activity was observed in F2 (IC50 = 6.7 μg/mL) from the 5–10 kDa fraction and F1 (IC50 = 4.78 μg/mL) from the <1 kDa fraction. ACE inhibitory fractions from Jatropha kernel have potential applications in alternative hypertension therapies, adding a new application for the Jatropha plant protein fraction and improving the financial viability and sustainability of a Jatropha-based biodiesel industry. PMID:24224169

Two Novel Bioactive Peptides from Antarctic Krill with Dual Angiotensin Converting Enzyme and Dipeptidyl Peptidase IV Inhibitory Activities.

PubMed

Ji, Wei; Zhang, Chaohua; Ji, Hongwu

2017-07-01

Inhibition of dipeptidyl peptidase IV (DPP-IV) and angiotensin converting enzyme (ACE) are considered useful in managing 2 often associated conditions: diabetes and hypertension. In this study, corolase PP was used to hydrolyze Antarctic krill protein. The hydrolysate (AKH) was isolated by ultrafiltration and purified by size-exclusion chromatography, ion exchange chromatography and reversed-phase high-performance liquid chromatography (RP-HPLC) sequentially. The in vitro inhibitory activities of all AKHs and several fractions obtained against ACE and DPP-IV were assessed. Two peptides, purified with dual-strength inhibitory activity against ACE and DPP-IV, were identified by TOF-MS/MS. Results indicated that not all fractions exhibited dual inhibitory activities of ACE and DPP-IV. The purified peptide Lys-Val-Glu-Pro-Leu-Pro had half-maximal inhibitory concentrations (IC 50 ) of 0.93±0.05 and 0.73±0.04 mg/mL against ACE and DPP-IV, respectively. The other peptide Pro-Ala-Leu had IC 50 values of 0.64±0.05 and 0.88±0.03 mg/mL against ACE and DPP-IV, respectively. This study firstly reported the sequences of dual bioactive peptides from Antarctic krill proteins, further provided new insights into the bioactive peptides responsible for the ACE and DPP-IV inhibitory activities from the Antarctic krill protein hydrolysate to manage hypertension and diabetes. © 2017 Institute of Food Technologists®.

The inhibitory activity of HL-7 and HL-10 peptide from scorpion venom (Hemiscorpius lepturus) on angiotensin converting enzyme: Kinetic and docking study.

PubMed

Setayesh-Mehr, Zahra; Asoodeh, Ahmad

2017-12-01

The hypertension is one of the highest risk factors for stroke, myocardial infarction, vascular disease and chronic kidney disease. Angiotensin converting enzyme (ACE) has an important role in the physiological regulation of cardiovascular system. ACE inhibition is a key purpose for hypertension treatment. In this study, two peptides named HL-7 with the sequence of YLYELAR (MW: 927.07Da) and HL-10 with the sequence of AFPYYGHHLG (MW: 1161.28Da) were identified from scorpion venom of H. lepturus. The inhibitory activity of HL-7 and HL-10 was examined on rabbit ACE. The inhibition mechanisms were assayed by kinetic and docking studies. The IC 50 values for ACE inhibition of HL-7 and HL-10 were 9.37µM and 17.22µM, respectively. Lineweaver-Burk plots showed that two peptides inhibited rabbit ACE with competitive manner. The molecular docking conformed experimental results and showed that the two peptides interacted with N-domain and C-domain active sites. Also, docking study revealed that the two peptides can form hydrogen and hydrophobic bonds at their binding sites. Both peptides had higher affinity to N-domain. Our results showed that HL-7 exhibited more strong interactions with amino acids at active site. It seems that HL-10 peptide could occupy more space, thereby inhibiting the substrate entrance to active site. Copyright © 2017 Elsevier Inc. All rights reserved.

Chemical composition, angiotensin-converting enzyme-inhibitory activity and antioxidant activities of few-flower wild rice (Zizania latifolia Turcz.).

PubMed

Qian, Bingjun; Luo, Yali; Deng, Yun; Cao, Linkui; Yang, Hongshun; Shen, Yongpei; Ping, Jian

2012-01-15

The chemical compositions of the stem and leaf sheath of few-flower wild rice were analysed. In addition, their extracts were evaluated for diphenylpicrylhydrazyl (DPPH) free radical-scavenging activity, ferric-reducing antioxidant power and angiotensin-converting enzyme (ACE)-inhibitory activity, since these are important properties of sources of nutraceuticals or functional foods. The stems contained more ascorbic acid (0.06 g kg(-1) fresh weight), protein (28.18 g kg(-1) dry weight (DW)), reducing sugars (308.54 g kg(-1) DW), water-soluble pectin (20.63 g kg(-1) DW), Na(2) CO(3) -soluble pectin (44.14 g kg(-1) DW), K (8 g kg(-1) dry matter (DM), S (6 g kg(-1) DM) and P (5 g kg(-1) DM) but less starch, total dietary fibre, Si, Na and Ca than the leaf sheaths. The DPPH free radical-scavenging IC(50) values of the stem and leaf sheath extracts were 19.28 and 21.22 mg mL(-1) respectively. In addition, the ACE-inhibitory IC(50) value of the stem extracts was 38.54 mg mL(-1). Both the stem and leaf sheath extracts exhibited good antioxidant properties, while good ACE-inhibitory activity was detected only in the phosphate buffer solution extracts of the stem. Few-flower wild rice could be processed into formula feeds for fish, poultry, etc. or functional foods for persons with high blood pressure. Copyright © 2011 Society of Chemical Industry.

Isolation, purification and characterization of β-amylase from Dioscorea hispida Dennst

NASA Astrophysics Data System (ADS)

Oktiarni, Dwita; Lusiana, Simamora, Febri Yanti; Gaol, Jusni M. Lumban

2015-09-01

β-amylase (E.C 3.2.1.2) is an enzyme commonly found in plants and bacteria. The enzyme is an exo-acting carbohydrolase which hydrolyzes α-1.4-glucosidic linkages of starch, removing maltose units from the non-reducing end of the polysaccharide chain, producing β-maltose and β-limit dextrin as the final product. β-amylase is widely distributed in the higher plants such as sweet potato. Besides the use in starch hydrolysis, starch-converting enzymes are also used in a number of other industrial applications, such as laundry and porcelain detergents or as anti-stalling agents in baking. This enzyme was extracted from Dioscorea hispida Dennst in 0.05 M acetate buffer pH 4.8 and followed by ammonium sulfate fractionation at cold temperature (10°C). Ammonium sulfate fractionation was shared into fraction of 0-60%, 60-70%, 70-80% and 80-100%. The fraction containing high of specific activity (determined by Somogyi-Nelson and Lowry methods) was futher purified by dialysis. Fraction with high enzyme activity of β-amylase were fraction 60-70% and 70-80%, with specific activity of Dioscorea hispida Dennst were 1.32 and 1.55 mg sugar.mg protein-1.minute-1, whereas specific activity of crude extract enzyme was 0.21 mg sugar.mg protein-1.minute-1. After purified with dialysis, fraction with high enzyme activity of β-amylase were fraction of 60-70% and 70-80%, with specific activity of Dioscorea hispida Dennst was 2.72 and 4.24 mg sugar.mg protein-1.minute-1. The purified Dioscorea hispida Dennst β-amylase from dialysis showed increasing in spesific activity the crude enzyme as much as 24 folds. The characterization of enzyme showed that Dioscorea hispida Dennst derived enzyme had optimum pH of 5.5 and temperature of 70°C. The kinetic parameters of purified Dioscorea hispida Dennst β-amylase showed that the KMapp, Vmaxapp value and Hill constant were 0.0211 mg/ml, 9.63 mg sugar.minute-1 and 1.34, respectively.

Activation of angiotensin-converting enzyme 2 (ACE2) attenuates allergic airway inflammation in rat asthma model

DOE Office of Scientific and Technical Information (OSTI.GOV)

Dhawale, Vaibhav Shrirang; Amara, Venkateswara Rao

Angiotensin-I converting enzyme (ACE) is positively correlated to asthma, chronic obstructive pulmonary disease (COPD), acute respiratory distress syndrome (ARDS) and is highly expressed in lungs. ACE2, the counteracting enzyme of ACE, was proven to be protective in pulmonary, cardiovascular diseases. In the present study we checked the effect of ACE2 activation in animal model of asthma. Asthma was induced in male wistar rats by sensitization and challenge with ovalbumin and then treated with ACE2 activator, diminazene aceturate (DIZE) for 2 weeks. 48 h after last allergen challenge, animals were anesthetized, blood, BALF, femoral bone marrow lavage were collected for leucocytemore » count; trachea for measuring airway responsiveness to carbachol; lungs and heart were isolated for histological studies and western blotting. In our animal model, the characteristic features of asthma such as altered airway responsiveness to carbachol, eosinophilia and neutrophilia were observed. Western blotting revealed the increased pulmonary expression of ACE1, IL-1β, IL-4, NF-κB, BCL2, p-AKT, p-p38 and decreased expression of ACE2 and IκB. DIZE treatment prevented these alterations. Intraalveolar interstitial thickening, inflammatory cell infiltration, interstitial fibrosis, oxidative stress and right ventricular hypertrophy in asthma control animals were also reversed by DIZE treatment. Activation of ACE2 by DIZE conferred protection against asthma as evident from biochemical, functional, histological and molecular parameters. To the best of our knowledge, we report for the first time that activation of ACE2 by DIZE prevents asthma progression by altering AKT, p38, NF-κB and other inflammatory markers. - Highlights: • Diminazene aceturate (DIZE), an ACE2 activator prevents ovalbumin-induced asthma. • DIZE acted by upregulating ACE2, downregulating ACE1, MAPKs, markers of inflammation, apoptosis. • DIZE reduced airway inflammation, fibrosis, right ventricular hypertrophy and restored airway responsiveness.« less

Murine recombinant angiotensin-converting enzyme 2 attenuates kidney injury in experimental Alport syndrome.

PubMed

Bae, Eun Hui; Fang, Fei; Williams, Vanessa R; Konvalinka, Ana; Zhou, Xiaohua; Patel, Vaibhav B; Song, Xuewen; John, Rohan; Oudit, Gavin Y; Pei, York; Scholey, James W

2017-06-01

Angiotensin-converting enzyme 2 (ACE2) is a monocarboxypeptidase in the renin-angiotensin system that catalyzes the breakdown of angiotensin II to angiotensin 1-7. We have reported that ACE2 expression in the kidney is reduced in experimental Alport syndrome but the impact of this finding on disease progression has not been studied. Accordingly, we evaluated effects of murine recombinant ACE2 treatment in Col4a3 knockout mice, a model of Alport syndrome characterized by proteinuria and progressive renal injury. Murine recombinant ACE2 (0.5 mg/kg/day) was administered from four to seven weeks of age via osmotic mini-pump. Pathological changes were attenuated by murine recombinant ACE2 treatment which ameliorated kidney fibrosis as shown by decreased expression of COL1α1 mRNA, less accumulation of extracellular matrix proteins, and inhibition of transforming growth factor-β signaling. Further, increases in proinflammatory cytokine expression, macrophage infiltration, inflammatory signaling pathway activation, and heme oxygenase-1 levels in Col4a3 knockout mice were also reduced by murine recombinant ACE2 treatment. Lastly, murine recombinant ACE2 influenced the turnover of renal ACE2, as it suppressed the expression of tumor necrosis factor-α converting enzyme, a negative regulator of ACE2. Thus, treatment with exogenous ACE2 alters angiotensin peptide metabolism in the kidneys of Col4a3 knockout mice and attenuates the progression of Alport syndrome nephropathy. Copyright © 2017 International Society of Nephrology. Published by Elsevier Inc. All rights reserved.

Carotenoid biosynthesis in bacteria: In vitro studies of a crt/bch transcription factor from Rhodobacter capsulatus and carotenoid enzymes from Erwinia herbicola

DOE Office of Scientific and Technical Information (OSTI.GOV)

O'Brien, D.A.

1992-11-01

A putative transcription factor in Rhodobactor capsulatus which binds upstream of the crt and bch pigment biosynthesis operons and appears to play a role in the adaptation of the organism from the aerobic to the anaerobic-photosynthetic growth mode was characterized. Chapter 2 describes the identification of this factor through an in vitro mobility shift assay, as well as the determination of its binding properties and sequence specificity. Chapter 3 focuses on the isolation of this factor. Biochemistry of later carotenoid biosynthesis enzymes derived from the non-photosynthetic bacterium, Erwinia herbicola. Chapter 4 describes the separate overexpression and in vitro analysis ofmore » two enzymes involved in the main sequence of the carotenoid biosynthesis pathway, lycopene cyclase and 5-carotene hydroxylase. Chapter 5 examines the overexpression and enzymology of functionally active zeaxanthin glucosyltransferase, an enzyme which carries out a more unusual transformation, converting a carotenoid into its more hydrophilic mono- and diglucoside derivatives. In addition, amino acid homology with other glucosyltransferases suggests a putative binding site for the UDP-activated glucose substrate.« less

Phytochemicals as Anticancer and Chemopreventive Topoisomerase II Poisons

PubMed Central

Ketron, Adam C.

2013-01-01

Phytochemicals are a rich source of anticancer drugs and chemopreventive agents. Several of these chemicals appear to exert at least some of their effects through interactions with topoisomerase II, an essential enzyme that regulates DNA supercoiling and removes knots and tangles from the genome. Topoisomerase II-active phytochemicals function by stabilizing covalent protein-cleaved DNA complexes that are intermediates in the catalytic cycle of the enzyme. As a result, these compounds convert topoisomerase II to a cellular toxin that fragments the genome. Because of their mode of action, they are referred to as topoisomerase II poisons as opposed to catalytic inhibitors. The first sections of this article discuss DNA topology, the catalytic cycle of topoisomerase II, and the two mechanisms (interfacial vs. covalent) by which different classes of topoisomerase II poisons alter enzyme activity. Subsequent sections discuss the effects of several phytochemicals on the type II enzyme, including demethyl-epipodophyllotoxins (semisynthetic anticancer drugs) as well as flavones, flavonols, isoflavones, catechins, isothiocyanates, and curcumin (dietary chemopreventive agents). Finally, the leukemogenic potential of topoisomerase II-targeted phytochemicals is described. PMID:24678287

Selective Activation of N,N'-Diacyl Rhodamine Pro-fluorophores Paired with Releasing Enzyme, Porcine Liver Esterase (PLE).

PubMed

Abney, Kristopher K; Ramos-Hunter, Susan J; Romaine, Ian M; Godwin, J Shawn; Sulikowski, Gary A; Weaver, Charles David

2018-04-21

This study reports the synthesis and testing of a family of rhodamine pro-fluorophores and an enzyme capable of converting pro-fluorophores to Rhodamine 110. We prepared a library of simple N,N'-diacyl rhodamines and investigated Porcine Liver Esterase (PLE) as an enzyme to activate rhodamine-based pro-fluorophores. A PLE-expressing cell line generated an increase in fluorescence rapidly upon pro-fluorophore addition demonstrating the rhodamine pro-fluorophores are readily taken up and fluorescent upon PLE-mediated release. Rhodamine pro-fluorophore amides trifluoroacetamide (TFAm) and proponamide (PAm) appeared to be the best substrates using a cell-based assay using PLE expressing HEK293. Our pro-fluorophore series showed diffusion into live cells and resisted endogenous hydrolysis. The use of our engineered cell line containing the exogenous enzyme PLE demonstrated the rigorousness of amide masking when compared to cells not containing PLE. This simple and selective pro-fluorophore rhodamine pair with PLE offers the potential to be used in vitro and in vivo fluorescence based assays. © 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Ochratoxin degradation and adsorption caused by astaxanthin-producing yeasts.

PubMed

Péteri, Z; Téren, J; Vágvölgyi, C; Varga, J

2007-05-01

Ochratoxin degrading and adsorbing activities of Phaffia rhodozyma and Xanthophyllomyces dendrorhous isolates were tested. P. rhodozyma CBS 5905 degraded more than 90% of ochratoxin A (OTA) in 15 days at 20 degrees C. The data presented indicate that P. rhodozyma is able to convert OTA to ochratoxin alpha, and this conversion is possibly mediated by an enzyme related to carboxypeptidases. Chelating agents like EDTA and 1,10-phenanthroline inhibited OTA degradation caused by P. rhodozyma indicating that the carboxypeptidase is a metalloprotease, similarly to carboxypeptidase A. The temperature optimum of this enzyme was found to be above 30 degrees C, which is much higher than the temperature optimum for growth of P. rhodozyma cells, which is around 20 degrees C. The enzyme responsible for ochratoxin degradation was found to be cell-bound. Besides, both viable and heat-treated (dead) P. rhodozyma cells were also able to adsorb significant amounts (up to 250 ng ml(-1)) of OTA. Heat treatment enhanced OTA adsorbing activities of the cells. Further studies are in progress to identify the enzyme responsible for OTA degradation in P. rhodozyma.

Efficient conversion of phenylpyruvic acid to phenyllactic acid by using whole cells of Bacillus coagulans SDM.

PubMed

Zheng, Zhaojuan; Ma, Cuiqing; Gao, Chao; Li, Fengsong; Qin, Jiayang; Zhang, Haiwei; Wang, Kai; Xu, Ping

2011-04-20

Phenyllactic acid (PLA), a novel antimicrobial compound with broad and effective antimicrobial activity against both bacteria and fungi, can be produced by many microorganisms, especially lactic acid bacteria. However, the concentration and productivity of PLA have been low in previous studies. The enzymes responsible for conversion of phenylpyruvic acid (PPA) into PLA are equivocal. A novel thermophilic strain, Bacillus coagulans SDM, was isolated for production of PLA. When the solubility and dissolution rate of PPA were enhanced at a high temperature, whole cells of B. coagulans SDM could effectively convert PPA into PLA at a high concentration (37.3 g l(-1)) and high productivity (2.3 g l(-1) h(-1)) under optimal conditions. Enzyme activity staining and kinetic studies identified NAD-dependent lactate dehydrogenases as the key enzymes that reduced PPA to PLA. Taking advantage of the thermophilic character of B. coagulans SDM, a high yield and productivity of PLA were obtained. The enzymes involved in PLA production were identified and characterized, which makes possible the rational design and construction of microorganisms suitable for PLA production with metabolic engineering.

Characterization of an Agrobacterium tumefaciens d-Psicose 3-Epimerase That Converts d-Fructose to d-Psicose

PubMed Central

Kim, Hye-Jung; Hyun, Eun-Kyung; Kim, Yeong-Su; Lee, Yong-Joo; Oh, Deok-Kun

2006-01-01

The noncharacterized gene previously proposed as the d-tagatose 3-epimerase gene from Agrobacterium tumefaciens was cloned and expressed in Escherichia coli. The expressed enzyme was purified by three-step chromatography with a final specific activity of 8.89 U/mg. The molecular mass of the purified protein was estimated to be 132 kDa of four identical subunits. Mn2+ significantly increased the epimerization rate from d-fructose to d-psicose. The enzyme exhibited maximal activity at 50°C and pH 8.0 with Mn2+. The turnover number (kcat) and catalytic efficiency (kcat/Km) of the enzyme for d-psicose were markedly higher than those for d-tagatose, suggesting that the enzyme is not d-tagatose 3-epimerase but d-psicose 3-epimerase. The equilibrium ratio between d-psicose and d-fructose was 32:68 at 30°C. d-Psicose was produced at 230 g/liter from 700-g/liter d-fructose at 50°C after 100 min, corresponding to a conversion yield of 32.9%. PMID:16461638

Physiological and biochemical effect of neem and other Meliaceae plants secondary metabolites against Lepidopteran insects

PubMed Central

Senthil-Nathan, Sengottayan

2013-01-01

This review described the physiological and biochemical effects of various secondary metabolites from Meliaceae against major Lepidopteran insect pest including, Noctuidae and Pyralidae. The biochemical effect of major Meliaceae secondary metabolites were discussed more in this review. Several enzymes based on food materials have critical roles in nutritional indices (food utilization) of the insect pest population. Several research work has been referred and the effect of Meliaceae secondary metabolites on feeding parameters of insects by demonstrating food consumption, approximate digestibility of consumed food, efficiency of converting the ingested food to body substance, efficiency of converting digested food to body substance and consumption index was reviewed in detail. Further how the digestive enzymes including a-Amylases, α and β-glucosidases (EC 3.2.1.1), lipases (EC 3.1.1) Proteases, serine, cysteine, and aspartic proteinases affected by the Meliaceae secondary metabolites was reviewed. Further effect of Meliaceae secondary metabolites on detoxifying enzymes have been found to react against botanical insecticides including general esterases (EST), glutathione S-transferase (GST) and phosphatases was reviewed. Alkaline phosphatase (ALP, E.C.3.1.3.1) and acid phosphatase (ACP, E.C.3.1.3.2) are hydrolytic enzymes, which hydrolyze phosphomonoesters under alkaline or acid conditions, respectively. These enzymes were affected by the secondary metabolites treatment. The detailed mechanism of action was further explained in this review. Acethylcholine esterase (AChE) is a key enzyme that terminates nerve impulses by catalyzing the hydrolysis of neurotransmitter, acetylcholine, in the nervous system of various organisms. How the AChE activity was altered by the Meliaceae secondary metabolites reviewed in detail. PMID:24391591

Metabolism of β-valine via a CoA-dependent ammonia lyase pathway.

PubMed

Otzen, Marleen; Crismaru, Ciprian G; Postema, Christiaan P; Wijma, Hein J; Heberling, Matthew M; Szymanski, Wiktor; de Wildeman, Stefaan; Janssen, Dick B

2015-11-01

Pseudomonas species strain SBV1 can rapidly grow on medium containing β-valine as a sole nitrogen source. The tertiary amine feature of β-valine prevents direct deamination reactions catalyzed by aminotransferases, amino acid dehydrogenases, and amino acid oxidases. However, lyase- or aminomutase-mediated conversions would be possible. To identify enzymes involved in the degradation of β-valine, a PsSBV1 gene library was prepared and used to complement the β-valine growth deficiency of a closely related Pseudomonas strain. This resulted in the identification of a gene encoding β-valinyl-coenzyme A ligase (BvaA) and two genes encoding β-valinyl-CoA ammonia lyases (BvaB1 and BvaB2). The BvaA protein demonstrated high sequence identity to several known phenylacetate CoA ligases. Purified BvaA enzyme did not convert phenyl acetic acid but was able to activate β-valine in an adenosine triphosphate (ATP)- and CoA-dependent manner. The substrate range of the enzyme appears to be narrow, converting only β-valine and to a lesser extent, 3-aminobutyrate and β-alanine. Characterization of BvaB1 and BvaB2 revealed that both enzymes were able to deaminate β-valinyl-CoA to produce 3-methylcrotonyl-CoA, a common intermediate in the leucine degradation pathway. Interestingly, BvaB1 and BvaB2 demonstrated no significant sequence identity to known CoA-dependent ammonia lyases, suggesting they belong to a new family of enzymes. BLAST searches revealed that BvaB1 and BvaB2 show high sequence identity to each other and to several enoyl-CoA hydratases, a class of enzymes that catalyze a similar reaction with water instead of amine as the leaving group.

Enhanced conversion of plant biomass into glucose using transgenic rice-produced endoglucanase for cellulosic ethanol.

PubMed

Oraby, Hesham; Venkatesh, Balan; Dale, Bruce; Ahmad, Rashid; Ransom, Callista; Oehmke, James; Sticklen, Mariam

2007-12-01

The catalytic domain of Acidothermus cellulolyticus thermostable endoglucanase gene (encoding for endo-1,4-beta-glucanase enzyme or E1) was constitutively expressed in rice. Molecular analyses of T1 plants confirmed presence and expression of the transgene. The amount of E1 enzyme accounted for up to 4.9% of the plant total soluble proteins, and its accumulation had no apparent deleterious effects on plant growth and development. Approximately 22 and 30% of the cellulose of the Ammonia Fiber Explosion (AFEX)-pretreated rice and maize biomass respectively was converted into glucose using rice E1 heterologous enzyme. As rice is the major food crop of the world with minimal use for its straw, our results suggest a successful strategy for producing biologically active hydrolysis enzymes in rice to help generate alcohol fuel, by substituting the wasteful and polluting practice of rice straw burning with an environmentally friendly technology.

The metabolic profile of acteoside produced by human or rat intestinal bacteria or intestinal enzyme in vitro employed UPLC-Q-TOF-MS.

PubMed

Cui, Qingling; Pan, Yingni; Xu, Xiaotong; Zhang, Wenjie; Wu, Xiao; Qu, Shouhe; Liu, Xiaoqiu

2016-03-01

Acteoside, the main and representative phenylethanoid glycosides of Herba Cistanches, possesses wide bioactivities but low oral bioavailability. It may serve as the prodrug and be converted into the active forms in gastrointestinal tract, which mainly occurred in intestinal tract composed of intestinal bacteria and intestinal enzyme. Intestinal bacteria, a new drug target, take a significant role on exerting pharmacological effects of drugs by oral administration. In this paper, acteoside was incubated with human or rat intestinal bacteria or rat intestinal enzyme for 36 h to seek metabolites responsible for pharmacodynamics. The samples were analyzed by ultra-performance liquid chromatography coupled with quadrupole time-of-flight mass spectrometry. Besides the parent compound, 14 metabolites were detected and identified based on their retention times and fragmentation patterns in their MS spectra including 8 degradation metabolites, 2 isomers in intestinal bacteria and intestinal enzyme samples and 4 parent metabolites only found in intestinal enzymes. The metabolic pathway of acteoside was thus proposed. Identification of these metabolites of acteoside by the intestinal bacteria or intestinal enzyme gave an insight to clarify pharmacological mechanism of traditional Chinese medicines and identify the real active molecules. Copyright © 2015 Elsevier B.V. All rights reserved.

Correlation of endothelin-1 concentration and angiotensin-converting enzyme activity with the staging of liver fibrosis.

PubMed

Kardum, Dusko; Fabijanić, Damir; Lukić, Anita; Romić, Zeljko; Petrovecki, Mladen; Bogdanović, Zoran; Jurić, Klara; Urek-Crncević, Marija; Banić, Marko

2012-06-01

Increased serum angiotensin-converting enzyme (SACE) activity and serum concentration of endothelin-1 (ET-1) were found in liver cirrhosis. We investigated a correlation between the different stages of liver fibrosis and SACE activity and serum ET-1 concentration. Seventy patients with pathohistologically established chronic liver disease were divided in three groups according to Ishak criteria for liver fibrosis: minimal fibrosis (Ishak score 0-1, n =20), medium fibrosis (Ishak score 2-5, n=20) and cirrhosis (Ishak score 6, n=30). SACE activity and ET-1 concentration were determined using commercial ELISA kits. SACE activity and ET-1 concentrations were proportional to the severity of disease, the highest being in patients with liver cirrhosis. Maximal increase in SACE activity was found between minimal and medium fibrosis while maximal increase in ET-1 concentration was revealed between medium fibrosis and cirrhosis. The analysis of the Receiver Operating Characteristic (ROC) curve for SACE activity suggested a cut-off value to separate minimal from medium fibrosis at 59.00 U/L (sensitivity 100%, specificity 64.7%). The cut-off value for serum ET-1 concentration to separate medium fibrosis from cirrhosis was 12.4 pg/mL (sensitivity 96.8%, specificity 94.4%). A positive correlation between SACE activity and ET-1 concentration was registered (Spearman's ñ = 0.438, p = 0.004). Both SACE activity and ET-1 concentration were increased in all stages of liver fibrosis. Cut-off points for SACE activity and ET-1 concentration could be a biochemical marker for the progression of fibrosis. Positive correlation between SACE activity and ET-1 concentration might indicate their interaction in the development of liver cirrhosis.

Structural insights into the recovery of aldolase activity in N-acetylneuraminic acid lyase by replacement of the catalytically active lysine with γ-thialysine by using a chemical mutagenesis strategy.

PubMed

Timms, Nicole; Windle, Claire L; Polyakova, Anna; Ault, James R; Trinh, Chi H; Pearson, Arwen R; Nelson, Adam; Berry, Alan

2013-03-04

Chemical modification has been used to introduce the unnatural amino acid γ-thialysine in place of the catalytically important Lys165 in the enzyme N-acetylneuraminic acid lyase (NAL). The Staphylococcus aureus nanA gene, encoding NAL, was cloned and expressed in E. coli. The protein, purified in high yield, has all the properties expected of a class I NAL. The S. aureus NAL which contains no natural cysteine residues was subjected to site-directed mutagenesis to introduce a cysteine in place of Lys165 in the enzyme active site. Subsequently chemical mutagenesis completely converted the cysteine into γ-thialysine through dehydroalanine (Dha) as demonstrated by ESI-MS. Initial kinetic characterisation showed that the protein containing γ-thialysine regained 17 % of the wild-type activity. To understand the reason for this lower activity, we solved X-ray crystal structures of the wild-type S. aureus NAL, both in the absence of, and in complex with, pyruvate. We also report the structures of the K165C variant, and the K165-γ-thialysine enzyme in the presence, or absence, of pyruvate. These structures reveal that γ-thialysine in NAL is an excellent structural mimic of lysine. Measurement of the pH-activity profile of the thialysine modified enzyme revealed that its pH optimum is shifted from 7.4 to 6.8. At its optimum pH, the thialysine-containing enzyme showed almost 30 % of the activity of the wild-type enzyme at its pH optimum. The lowered activity and altered pH profile of the unnatural amino acid-containing enzyme can be rationalised by imbalances of the ionisation states of residues within the active site when the pK(a) of the residue at position 165 is perturbed by replacement with γ-thialysine. The results reveal the utility of chemical mutagenesis for the modification of enzyme active sites and the exquisite sensitivity of catalysis to the local structural and electrostatic environment in NAL. Copyright © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Xanthine Oxidoreductase in Drug Metabolism: Beyond a Role as a Detoxifying Enzyme.

PubMed

Battelli, Maria Giulia; Polito, Letizia; Bortolotti, Massimo; Bolognesi, Andrea

2016-01-01

The enzyme xanthine oxidoreductase (XOR) catalyzes the last two steps of purine catabolism in the highest uricotelic primates. XOR is an enzyme with dehydrogenase activity that, in mammals, may be converted into oxidase activity under a variety of pathophysiologic conditions. XOR activity is highly regulated at the transcriptional and post-translational levels and may generate reactive oxygen and nitrogen species, which trigger different consequences, ranging from cytotoxicity to inflammation. The low specificity for substrates allows XOR to metabolize a number of endogenous metabolites and a variety of exogenous compounds, including drugs. The present review focuses on the role of XOR as a drug-metabolizing enzyme, specifically for drugs with anticancer, antimicrobial, antiviral, immunosuppressive or vasodilator activities, as well as drugs acting on metabolism or inducing XOR expression. XOR has an activating role that is essential to the pharmacological action of quinone drugs, cyadox, antiviral nucleoside analogues, allopurinol, nitrate and nitrite. XOR activity has a degradation function toward thiopurine nucleotides, pyrazinoic acid, methylxanthines and tolbutamide, whose half-life may be prolonged by the use of XOR inhibitors. In conclusion, to avoid potential drug interaction risks, such as a toxic excess of drug bioavailability or a loss of drug efficacy, caution is suggested in the use of XOR inhibitors, as in the case of hyperuricemic patients affected by gout or tumor lysis syndrome, when it is necessary to simultaneously administer therapeutic substances that are activated or degraded by the drug-metabolizing activity of XOR.

In vitro inhibitory activities of selected Australian medicinal plant extracts against protein glycation, angiotensin converting enzyme (ACE) and digestive enzymes linked to type II diabetes.

PubMed

Deo, Permal; Hewawasam, Erandi; Karakoulakis, Aris; Claudie, David J; Nelson, Robert; Simpson, Bradley S; Smith, Nicholas M; Semple, Susan J

2016-11-04

There is a need to develop potential new therapies for the management of diabetes and hypertension. Australian medicinal plants collected from the Kuuku I'yu (Northern Kaanju) homelands, Cape York Peninsula, Queensland, Australia were investigated to determine their therapeutic potential. Extracts were tested for inhibition of protein glycation and key enzymes relevant to the management of hyperglycaemia and hypertension. The inhibitory activities were further correlated with the antioxidant activities. Extracts of five selected plant species were investigated: Petalostigma pubescens, Petalostigma banksii, Memecylon pauciflorum, Millettia pinnata and Grewia mesomischa. Enzyme inhibitory activity of the plant extracts was assessed against α-amylase, α-glucosidase and angiotensin converting enzyme (ACE). Antiglycation activity was determined using glucose-induced protein glycation models and formation of protein-bound fluorescent advanced glycation endproducts (AGEs). Antioxidant activity was determined by measuring the scavenging effect of plant extracts against 1, 1-diphenyl-2-picryl hydrazyl (DPPH) and using the ferric reducing anti-oxidant potential assay (FRAP). Total phenolic and flavonoid contents were also determined. Extracts of the leaves of Petalostigma banksii and P. pubescens showed the strongest inhibition of α-amylase with IC 50 values of 166.50 ± 5.50 μg/mL and 160.20 ± 27.92 μg/mL, respectively. The P. pubescens leaf extract was also the strongest inhibitor of α-glucosidase with an IC 50 of 167.83 ± 23.82 μg/mL. Testing for the antiglycation potential of the extracts, measured as inhibition of formation of protein-bound fluorescent AGEs, showed that P. banksii root and fruit extracts had IC 50 values of 34.49 ± 4.31 μg/mL and 47.72 ± 1.65 μg/mL, respectively, which were significantly lower (p < 0.05) than other extracts. The inhibitory effect on α-amylase, α-glucosidase and the antiglycation potential of the extracts did not correlate with the total phenolic, total flavonoid, FRAP or DPPH. For ACE inhibition, IC 50 values ranged between 266.27 ± 6.91 to 695.17 ± 15.38 μg/mL. The tested Australian medicinal plant extracts inhibit glucose-induced fluorescent AGEs, α-amylase, α-glucosidase and ACE with extracts of Petalostigma species showing the most promising activity. These medicinal plants could potentially be further developed as therapeutic agents in the treatment of hyperglycaemia and hypertension.

Pharmacological Potential of Sea Cucumbers

PubMed Central

Khotimchenko, Yuri

2018-01-01

This review presents a detailed analysis of published research data focused on the pharmacological activity exerted by biologically active compounds isolated from sea cucumbers belonging to the class of Holothuroidea, phylum Echinodermata. The review contains descriptions of the structure, physico-chemical properties and pharmacological effects of these active substances. Particular attention is given to compounds with anticoagulant, antithrombotic, antioxidant, anticancer, anti-infectious, immune-stimulating and anti-ACE (angiotensin converting enzyme) activities as well as to the substances exerting a regulating influence on lipid and carbohydrate metabolism. All these compounds may be considered as prototypes for development of new pharmaceutical substances and medicines. PMID:29724051

The Effect Enzymes Have On Bodies Of Water

NASA Astrophysics Data System (ADS)

Igiebor, I. W.; Murray, P.; Montes, D. R.

2017-12-01

With our ongoing research and studies of extracellular enzymes, we have gathered that the enzymes have a process in which they convert organic matter into CO2. Therefore, one of our goals is to find out how much and at what rate the organic matter was being transformed into CO2 so that we or others may somehow make this into a tool to reduce CO2 levels in Earth's atmosphere. Although, to find an answer or learn more about it, we needed to continue to test different water samples and see which enzymes were active, under what conditions, and at what rates. Currently I am testing enzymes in new water sources in and around the Newark, NJ area. The ultimate goal is to find out why and how the conversion of organic matter to carbon occurs, still not having a clear answer on why it does happen, but realizing that continuing to accumulate data for the scientific community to examine is the path forward to solving the atmospheric CO2 problem.

Biomimicry Promotes the Efficiency of a 10-Step Sequential Enzymatic Reaction on Nanoparticles, Converting Glucose to Lactate.

PubMed

Mukai, Chinatsu; Gao, Lizeng; Nelson, Jacquelyn L; Lata, James P; Cohen, Roy; Wu, Lauren; Hinchman, Meleana M; Bergkvist, Magnus; Sherwood, Robert W; Zhang, Sheng; Travis, Alexander J

2017-01-02

For nanobiotechnology to achieve its potential, complex organic-inorganic systems must grow to utilize the sequential functions of multiple biological components. Critical challenges exist: immobilizing enzymes can block substrate-binding sites or prohibit conformational changes, substrate composition can interfere with activity, and multistep reactions risk diffusion of intermediates. As a result, the most complex tethered reaction reported involves only 3 enzymes. Inspired by the oriented immobilization of glycolytic enzymes on the fibrous sheath of mammalian sperm, here we show a complex reaction of 10 enzymes tethered to nanoparticles. Although individual enzyme efficiency was higher in solution, the efficacy of the 10-step pathway measured by conversion of glucose to lactate was significantly higher when tethered. To our knowledge, this is the most complex organic-inorganic system described, and it shows that tethered, multi-step biological pathways can be reconstituted in hybrid systems to carry out functions such as energy production or delivery of molecular cargo. © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

Biomimicry promotes the efficiency of a 10-step sequential enzymatic reaction on nanoparticles, converting glucose to lactate

PubMed Central

Mukai, Chinatsu; Gao, Lizeng; Nelson, Jacquelyn L.; Lata, James P.; Cohen, Roy; Wu, Lauren; Hinchman, Meleana M.; Bergkvist, Magnus; Sherwood, Robert W.; Zhang, Sheng; Travis, Alexander J.

2016-01-01

For nanobiotechnology to achieve its potential, complex organic-inorganic systems must grow to utilize the sequential functions of multiple biological components. Critical challenges exist: immobilizing enzymes can block substrate-binding sites or prohibit conformational changes, substrate composition can interfere with activity, and multistep reactions risk diffusion of intermediates. As a result, the most complex tethered reaction reported involves only 3 enzymes. Inspired by the oriented immobilization of glycolytic enzymes on the fibrous sheath of mammalian sperm, here we show a complex reaction of 10 enzymes tethered to nanoparticles. Although individual enzyme efficiency was higher in solution, the efficacy of the 10-step pathway measured by conversion of glucose to lactate was significantly higher when tethered. To our knowledge, this is the most complex organic-inorganic system described, and it shows that tethered, multi-step biological pathways can be reconstituted in hybrid systems to carry out functions such as energy production or delivery of molecular cargo. PMID:27901298

Effect of long-term treatment with urocortin on the activity of somatic angiotensin-converting enzyme in spontaneously hypertensive rats.

PubMed

Yang, Cui; Liu, Xiuxia; Li, Shengnan

2010-02-01

Our previous acute study on urocortin (Ucn) demonstrated that Ucn altered serum and tissue angiotensin-converting enzyme (ACE) activity in rats. Therefore, the present investigation was designed to explore the effect of long-term treatment with Ucn on somatic ACE (sACE) and other components of the renin-angiotensin system (RAS). After 8 weeks of intravenous administration of Ucn in spontaneously hypertensive rats (SHR), serum and tissue sACE, angiotensin II (Ang II), nitric oxide (NO), Ang-(1-7), and tissue chymase activities were evaluated. RT-PCR analysis was performed to determine the quantity of tissue sACE mRNA. Serum sACE activity was reduced by Ucn, although tissue sACE activity and tissue sACE mRNA were elevated. Chymase activity was observed to be enhanced by Ucn, whereas the ACE inhibitor enalapril failed to influence chymase. Serum and tissue Ang II activity was reduced, but NO and Ang-(1-7) production was increased in a concentration-dependent manner after Ucn treatment. Meanwhile, a significant decrease of the systolic blood pressure (SBP) was observed after the long-term Ucn administration, and there was a significant positive correlation (r2 = 0.6993) between serum ACE activity and SBP. Pretreatment with the corticotropin-releasing factor (CRF) blocker astressin and the extracellular signal-regulated kinase 1/2 (ERK1/2) pathway blocker PD98059 abolished these effects of Ucn. Our findings further support the hypothesis that the changes of sACE activity and the production of other RAS components may play roles in the vasodilatory property of Ucn via the activation of the ERK1/2 pathway.

Biological activity of silylated amino acid containing substance P analogues.

PubMed

Cavelier, F; Marchand, D; Martinez, J; Sagan, S

2004-03-01

The need to replace natural amino acids in peptides with nonproteinogenic counterparts to obtain new medicinal agents has stimulated a great deal of innovation on synthetic methods. Here, we report the incorporation of non-natural silylated amino acids in substance P (SP), the binding affinity for the two hNK-1 binding sites and, the potency to stimulate phospholipase C (PLC) and adenylate cyclase of the resulting peptide. We also assess the improvement of their stability towards enzyme degradation. Altogether, we found that replacing glycine with silaproline (Sip) in position 9 of SP leads to a potent analogue exhibiting an increased resistance to angiotensin-converting enzyme hydrolysis.

Conjugations with glutathione. The enzymic conjugation of some chlorocyclohexenes

PubMed Central

Sims, P.; Grover, P. L.

1965-01-01

1. α-3,4,5,6-Tetrachlorocyclohex-1-ene and γ-2,3,4,5,6-pentachlorocyclohex-1-ene are conjugated with glutathione in vitro by a rat-liver enzyme that is probably glutathione S-aryltransferase. 2. Chlorocyclohexane and the α-, β-, γ- and δ-isomers of hexachlorocyclohexane were not substrates for rat-liver glutathione S-aryltransferase. 3. Glutathione-S-aryltransferase activity was present in tissue preparations of houseflies of insecticide-resistant and -susceptible strains. More activity was found in a dieldrin-resistant strain of houseflies fed on dieldrin than in either a dieldrin-resistant strain not fed on dieldrin or a control strain of dieldrin-susceptible houseflies. 4. Housefly soluble supernatant preparations converted S-(2-chloro-4-nitrophenyl)glutathione into the corresponding cysteine and mercapturic acid derivatives. PMID:14333551

Enzymatic conversion of sucrose to glucose and its anomerization by quantitative NMR spectroscopy: Application of a simple consecutive reaction rates approach

NASA Astrophysics Data System (ADS)

Singh, Jaideep; Her, Cheenou; Krishnan, V. V.

2018-02-01

The anomerization of carbohydrates is an essential process that determines the relative stabilization of stereoisomers in an aqueous solution. In a typical real-time enzyme kinetics experiment, the substrate (sucrose) is converted to glucose and fructose by the enzyme invertase. The product (α-D-glucose) starts to convert to β-D-glucose immediately by hydrolysis. Though the anomerization process is independent of the enzyme catalysis, the progress curve describing the production of β-D-glucose from α-D-glucose is directly affected by the kinetics of consecutive reactions. When α-D-glucose is continually converted to β-D-glucose, by the enzymatic action, the time course of both α- and β-D-glucose is influenced by the enzyme kinetics. Thus, a reversible first-order rate equation is not adequate to model the reaction mechanism, leading to erroneous results on the rates of formation of the glucose anomers. In this manuscript, we incorporate an approximate method to address consecutive general reactions involving enzyme kinetics and first-order reaction processes. The utility of the approach is demonstrated in the real-time NMR measurement of the anomerization process of α-D-glucose (enzymatically produced from sucrose) to β-D-glucose, as a function of invertase enzyme concentration. Variable temperature experiments were used to estimate the thermodynamic parameters of the anomerization process and are consistent with literature values.

Pathway for biodegradation of p-nitrophenol in a Moraxella sp

DOE Office of Scientific and Technical Information (OSTI.GOV)

Spain, J.C.; Gibson, D.T.

1991-03-01

A Moraxella strain grew on p-nitrophenol with stoichiometric release of nitrite. During induction of the enzymes for growth on p-nitrophenol, traces of hydroquinone accumulated in the medium. In the presence of 2,2{prime}-dipyidyl, p-nitrophenol, was converted stoichiometrically to hydroquinone. Particulate enzymes catalyzed the conversion of p-nitrophenol to hydroquinone in the presence of NADPH and oxygen. Soluble enzymes catalyzed the conversion of hydroquinone to {gamma}-hydroxymuconic semialdehyde, which was identified by high-performance liquid chromatography (HPLC)-mass spectroscopy. Upon addition of catalytic amounts of NAD{sup +}, {gamma}-hydroxymuconic semialdehyde was converted to {beta}-ketoadipic acid. In the presence of pyruvate and lactic dehydrogenase, substrate amounts of NADmore » were required and {gamma}-hydroxymuconic semialdehyde was converted to maleylacetic acid, which was identified by HPLC-mass spectroscopy. Similar results were obtained when the reaction was carried out in the presence of potassium ferricyanide. Extracts prepared from p-nitrophenol-grown cells also contained an enzyme that catalyzed the oxidation of 1,2,4-benzenetriol to maleylacetic acid. The enzyme responsible for the oxidation of 1,2,4-benzenetriol was separated from the enzyme responsible for hydroquinone oxidation by DEAE-cellulose chromatography. The results indicate that the pathway for biodegradation of p-nitrophenol involves the initial removal of the nitro group as nitrite and formation of hydroquinone.« less

A structural basis for the pH-dependent xanthophyll cycle in Arabidopsis thaliana.

PubMed

Arnoux, Pascal; Morosinotto, Tomas; Saga, Giorgia; Bassi, Roberto; Pignol, David

2009-07-01

Plants adjust their photosynthetic activity to changing light conditions. A central regulation of photosynthesis depends on the xanthophyll cycle, in which the carotenoid violaxanthin is converted into zeaxanthin in strong light, thus activating the dissipation of the excess absorbed energy as heat and the scavenging of reactive oxygen species. Violaxanthin deepoxidase (VDE), the enzyme responsible for zeaxanthin synthesis, is activated by the acidification of the thylakoid lumen when photosynthetic electron transport exceeds the capacity of assimilatory reactions: at neutral pH, VDE is a soluble and inactive enzyme, whereas at acidic pH, it attaches to the thylakoid membrane where it binds its violaxanthin substrate. VDE also uses ascorbate as a cosubstrate with a pH-dependent Km that may reflect a preference for ascorbic acid. We determined the structures of the central lipocalin domain of VDE (VDEcd) at acidic and neutral pH. At neutral pH, VDEcd is monomeric with its active site occluded within a lipocalin barrel. Upon acidification, the barrel opens up and the enzyme appears as a dimer. A channel linking the two active sites of the dimer can harbor the entire carotenoid substrate and thus may permit the parallel deepoxidation of the two violaxanthin beta-ionone rings, making VDE an elegant example of the adaptation of an asymmetric enzyme to its symmetric substrate.

A Structural Basis for the pH-Dependent Xanthophyll Cycle in Arabidopsis thaliana[C][W

PubMed Central

Arnoux, Pascal; Morosinotto, Tomas; Saga, Giorgia; Bassi, Roberto; Pignol, David

2009-01-01

Plants adjust their photosynthetic activity to changing light conditions. A central regulation of photosynthesis depends on the xanthophyll cycle, in which the carotenoid violaxanthin is converted into zeaxanthin in strong light, thus activating the dissipation of the excess absorbed energy as heat and the scavenging of reactive oxygen species. Violaxanthin deepoxidase (VDE), the enzyme responsible for zeaxanthin synthesis, is activated by the acidification of the thylakoid lumen when photosynthetic electron transport exceeds the capacity of assimilatory reactions: at neutral pH, VDE is a soluble and inactive enzyme, whereas at acidic pH, it attaches to the thylakoid membrane where it binds its violaxanthin substrate. VDE also uses ascorbate as a cosubstrate with a pH-dependent Km that may reflect a preference for ascorbic acid. We determined the structures of the central lipocalin domain of VDE (VDEcd) at acidic and neutral pH. At neutral pH, VDEcd is monomeric with its active site occluded within a lipocalin barrel. Upon acidification, the barrel opens up and the enzyme appears as a dimer. A channel linking the two active sites of the dimer can harbor the entire carotenoid substrate and thus may permit the parallel deepoxidation of the two violaxanthin β-ionone rings, making VDE an elegant example of the adaptation of an asymmetric enzyme to its symmetric substrate. PMID:19638474

Endothelin-converting enzyme 2 differentially regulates opioid receptor activity

PubMed Central

Gupta, A; Fujita, W; Gomes, I; Bobeck, E; Devi, L A

2015-01-01

BACKGROUND AND PURPOSE Opioid receptor function is modulated by post-activation events such as receptor endocytosis, recycling and/or degradation. While it is generally understood that the peptide ligand gets co-endocytosed with the receptor, relatively few studies have investigated the role of the endocytosed peptide and peptide processing enzymes in regulating receptor function. In this study, we focused on endothelin-converting enzyme 2 (ECE2), a member of the neprilysin family of metallopeptidases that exhibits an acidic pH optimum, localizes to an intracellular compartment and selectively processes neuropeptides including opioid peptides in vitro, and examined its role in modulating μ receptor recycling and resensitization. EXPERIMENTAL APPROACH The effect of ECE2 inhibition on hydrolysis of the endocytosed peptide was examined using thin-layer chromatography and on μ opioid receptor trafficking using either elisa or microscopy. The effect of ECE2 inhibition on receptor signalling was measured using a cAMP assay and, in vivo, on antinociception induced by intrathecally administered opioids by the tail-flick assay. KEY RESULTS The highly selective ECE2 inhibitor, S136492, significantly impaired μ receptor recycling and signalling by only those ligands that are ECE2 substrates and this was seen both in heterologous cells and in cells endogenously co-expressing μ receptors with ECE2. We also found that ECE2 inhibition attenuated antinociception mediated only by opioid peptides that are ECE2 substrates. CONCLUSIONS AND IMPLICATIONS These results suggest that ECE2, by selectively processing endogenous opioid peptides in the endocytic compartment, plays a role in modulating opioid receptor activity. LINKED ARTICLES This article is part of a themed section on Opioids: New Pathways to Functional Selectivity. To view the other articles in this section visit http://dx.doi.org/10.1111/bph.2015.172.issue-2 PMID:24990314

Increasing thermal stability and catalytic activity of glutamate decarboxylase in E. coli: An in silico study.

PubMed

Tavakoli, Yasaman; Esmaeili, Abolghasem; Saber, Hossein

2016-10-01

Glutamate decarboxylase (GAD) is an enzyme that converts l-glutamate to gamma amino butyric acid (GABA) that is a widely used drug to treat mental disorders like Alzheimer's disease. In this study for the first time point mutation was performed virtually in the active site of the E. coli GAD in order to increase thermal stability and catalytic activity of the enzyme. Energy minimization and addition of water box were performed using GROMACS 5.4.6 package. PoPMuSiC 2.1 web server was used to predict potential spots for point mutation and Modeller software was used to perform point mutation on three dimensional model. Molegro virtual docker software was used for cavity detection and stimulated docking study. Results indicate that performing mutation separately at positions 164, 302, 304, 393, 396, 398 and 410 increase binding affinity to substrate. The enzyme is predicted to be more thermo- stable in all 7 mutants based on ΔΔG value. Copyright © 2016 Elsevier Ltd. All rights reserved.

Role of serum angiotensin converting enzyme in sarcoidosis.

PubMed

Khan, A H; Ghani, F; Khan, A; Khan, M A; Khurshid, M

1998-05-01

This study was conducted to determine the role of Serum Angiotensin Converting Enzyme (SACE) as a marker in the differential diagnosis of pulmonary diseases and prognosis of sarcoidosis. A retrospective analysis of 113 medical records of patients at The Aga Khan University Hospital, with laboratory investigation for SACE was performed. Among 113 patients, 51 cases were found to have sarcoidosis, 44 of them had SACE levels greater than 52 IU/L (mean ACE 104.44). SACE levels were also found elevated in other clinical conditions like tuberculosis (mean 58.64 IU/L), but the enzyme level were less (p 0.04) than those found in sarcoidosis (mean (92.97 IU/L). SACE activity was found to be considerably lower in other chronic lung diseases such as, fibrosing alveolitis (mean 43.98 IU/L), interstitial lung disease (mean 42.11 IU/L) and chronic obstructive lung disease (mean 40.85 IU/L). Twenty patients of sarcoidosis, who received steroid treatment subsequently showed a decline in the SACE levels. SACE is a useful marker in differential diagnosis as 37.2% cases of sarcoidosis compared to only 9.09% of tuberculosis had SACE levels greater than 100 IU/L. In addition, our data also suggest that serum ACE is useful for the diagnosis as well as monitoring prognosis in sarcoidosis.

Carbonyl reduction in the biosynthesis of a male sex pheromone secreted by the grape borer Xylotrechus pyrrhoderus.

PubMed

Iwabuchi, Kikuo; Arakawa, Maki; Kiyota, Ryutaro; Hoshino, Keita; Ando, Tetsu

2014-10-01

Males of the cerambycid beetle Xylotrechus pyrrhoderus release a mixture of (S)-2-hydroxy-3-octanone [(S)-1] and (2S,3S)-2,3-octanediol [(2S,3S)-2] as a sex pheromone that attracts conspecific females. The chemical structures of these pheromone components include a common motif and are assumed to be biosynthetically related. Here, we show that deuterated (S)-1, applied on the cuticle of a pronotal pheromone gland, was converted into (2S,3S)-2, that included deuterium atoms, but a reverse conversion did not take place. These results reveal a carbonyl reductase to be active in the pheromone gland, and that the ketol is a biosynthetic precursor of the diol. Males did not produce (R)-1; however, deuterated (R)-1 was converted into (2R,3R)-2, indicating an attack of the enzyme from the opposite side of the hydroxyl group at the 2-position. Furthermore, to understand the substrate specificity of the enzyme, racemates of 2-hydroxy-3-hexanone and 2-hydroxy-3-decanone were synthesized and applied to the gland. Their conversion into the corresponding diols suggests that the enzyme reduces the carbonyl group at the 3-position, regardless of the chain length.

Central nervous system considerations in the use of beta-blockers, angiotensin-converting enzyme inhibitors, and thiazide diuretics in managing essential hypertension.

PubMed

Gengo, F M; Gabos, C

1988-07-01

The most common mild side effects occurring with use of beta-blockers, thiazide diuretics, and angiotensin-converting enzyme inhibitors for blood pressure control are central nervous system symptoms, specifically lethargy, sedation, and fatigue. These symptoms affect 5% to 10% of patients taking these drugs. The mechanism by which beta-blockers may induce central nervous system effects is uncertain. Relative lipophilicity as a factor affecting penetrance of the blood-brain barrier has not proved to be a reliable predictor of whether the drug will cause such disturbances. Comparisons of atenolol (hydrophilic) and metoprolol (lipophilic) have shown no differences between these drugs with respect to side effects of the central nervous system. The incidence of central nervous system effects with angiotensin-converting enzyme inhibitors is similar to that for most beta-blockers. The precise role of the angiotensin-converting enzyme in the central nervous system is not well defined. Most thiazide diuretics are not associated with major complications of the central nervous system, although electrolyte imbalance may occasionally lead to complaints of neurologic symptoms. Because the incidence of central nervous system effects with these three classes of drugs is so low, concern for the side effects of the central nervous system is not a prime consideration in the choice of an initial antihypertensive agent.

Hydrolysates of sheep cheese whey as a source of bioactive peptides with antioxidant and angiotensin-converting enzyme inhibitory activities.

PubMed

Corrêa, Ana Paula Folmer; Daroit, Daniel Joner; Fontoura, Roberta; Meira, Stela Maris Meister; Segalin, Jeferson; Brandelli, Adriano

2014-11-01

Enzymatic proteolysis may be employed to release bioactive peptides, which have been investigated for potential benefits from both technological and human health perspectives. In this study, sheep cheese whey (SCW) was hydrolyzed with a protease preparation from Bacillus sp. P7, and the hydrolysates were evaluated for antioxidant and angiotensin I-converting enzyme (ACE)-inhibitory activities. Soluble protein and free amino acids increased during hydrolysis of SCW for up to 4h. Antioxidant activity of hydrolysates, evaluated by the 2,2'azino-bis-(3-ethylbenzothiazoline)-6-sulfonic acid radical scavenging method, increased 3.2-fold from 0 h (15.9%) to 6h of hydrolysis (51.3%). Maximum Fe(2+) chelation was reached in 3h hydrolysates, and the reducing power peaked at 1h of hydrolysis, representing 6.2 and 2.1-fold increase, respectively, when compared to that of non-hydrolyzed SCW. ACE inhibition by SCW (12%) was improved through hydrolysis, reaching maximal values (55% inhibition) in 4h, although 42% inhibition was already observed after 1h hydrolysis. The peptide LAFNPTQLEGQCHV, derived from β-lactoglobulin, was identified from 4-h hydrolysates. Such a biotechnological approach might be an interesting strategy for SCW processing, potentially contributing to the management and valorization of this abundant dairy byproduct. Copyright © 2014 Elsevier Inc. All rights reserved.

Development of predictive pharmacophore model for in silico screening, and 3D QSAR CoMFA and CoMSIA studies for lead optimization, for designing of potent tumor necrosis factor alpha converting enzyme inhibitors

NASA Astrophysics Data System (ADS)

Murumkar, Prashant Revan; Zambre, Vishal Prakash; Yadav, Mange Ram

2010-02-01

A chemical feature-based pharmacophore model was developed for Tumor Necrosis Factor-α converting enzyme (TACE) inhibitors. A five point pharmacophore model having two hydrogen bond acceptors (A), one hydrogen bond donor (D) and two aromatic rings (R) with discrete geometries as pharmacophoric features was developed. The pharmacophore model so generated was then utilized for in silico screening of a database. The pharmacophore model so developed was validated by using four compounds having proven TACE inhibitory activity which were grafted into the database. These compounds mapped well onto the five listed pharmacophoric features. This validated pharmacophore model was also used for alignment of molecules in CoMFA and CoMSIA analysis. The contour maps of the CoMFA/CoMSIA models were utilized to provide structural insight for activity improvement of potential novel TACE inhibitors. The pharmacophore model so developed could be used for in silico screening of any commercial/in house database for identification of TACE inhibiting lead compounds, and the leads so identified could be optimized using the developed CoMSIA model. The present work highlights the tremendous potential of the two mutually complementary ligand-based drug designing techniques (i.e. pharmacophore mapping and 3D-QSAR analysis) using TACE inhibitors as prototype biologically active molecules.

Characterization of ACE and ACE2 Expression within Different Organs of the NOD Mouse

PubMed Central

Roca-Ho, Heleia; Riera, Marta; Palau, Vanesa; Pascual, Julio; Soler, Maria Jose

2017-01-01

Renin angiotensin system (RAS) is known to play a key role in several diseases such as diabetes, and renal and cardiovascular pathologies. Its blockade has been demonstrated to delay chronic kidney disease progression and cardiovascular damage in diabetic patients. In this sense, since local RAS has been described, the aim of this study is to characterize angiotensin converting enzyme (ACE) and ACE2 activities, as well as protein expression, in several tissues of the non-obese diabetic (NOD) mice model. After 21 or 40 days of diabetes onset, mouse serums and tissues were analyzed for ACE and ACE2 enzyme activities and protein expression. ACE and ACE2 enzyme activities were detected in different tissues. Their expressions vary depending on the studied tissue. Thus, whereas ACE activity was highly expressed in lungs, ACE2 activity was highly expressed in pancreas among the studied tissues. Interestingly, we also observed that diabetes up-regulates ACE mainly in serum, lung, heart, and liver, and ACE2 mainly in serum, liver, and pancreas. In conclusion, we found a marked serum and pulmonary alteration in ACE activity of diabetic mice, suggesting a common regulation. The increase of ACE2 activity within the circulation in diabetic mice may be ascribed to a compensatory mechanism of RAS. PMID:28273875

Characterization of ACE and ACE2 Expression within Different Organs of the NOD Mouse.

PubMed

Roca-Ho, Heleia; Riera, Marta; Palau, Vanesa; Pascual, Julio; Soler, Maria Jose

2017-03-05

Renin angiotensin system (RAS) is known to play a key role in several diseases such as diabetes, and renal and cardiovascular pathologies. Its blockade has been demonstrated to delay chronic kidney disease progression and cardiovascular damage in diabetic patients. In this sense, since local RAS has been described, the aim of this study is to characterize angiotensin converting enzyme (ACE) and ACE2 activities, as well as protein expression, in several tissues of the non-obese diabetic (NOD) mice model. After 21 or 40 days of diabetes onset, mouse serums and tissues were analyzed for ACE and ACE2 enzyme activities and protein expression. ACE and ACE2 enzyme activities were detected in different tissues. Their expressions vary depending on the studied tissue. Thus, whereas ACE activity was highly expressed in lungs, ACE2 activity was highly expressed in pancreas among the studied tissues. Interestingly, we also observed that diabetes up-regulates ACE mainly in serum, lung, heart, and liver, and ACE2 mainly in serum, liver, and pancreas. In conclusion, we found a marked serum and pulmonary alteration in ACE activity of diabetic mice, suggesting a common regulation. The increase of ACE2 activity within the circulation in diabetic mice may be ascribed to a compensatory mechanism of RAS.

Gestational Protein Restriction Increases Angiotensin II Production in Rat Lung1

PubMed Central

Gao, Haijun; Yallampalli, Uma; Yallampalli, Chandra

2013-01-01

ABSTRACT Gestational protein restriction (PR) alters the renin-angiotensin system in uterine arteries and placentas and elevates plasma levels of angiotensin II in pregnant rats. To date, how PR increases maternal plasma levels of angiotensin II remains unknown. In this study, we hypothesize that the expression and/or the activity of angiotensin I converting enzyme (peptidyl-dipeptidase A) 1 (ACE) in lungs, but not kidneys and blood, largely contribute to elevated plasma angiotensin II levels in pregnant rats subject to gestational PR. Time-scheduled pregnant Sprague-Dawley rats were fed a normal or low-protein diet from Day 3 of pregnancy until euthanized at Day 19 or 22. Expressions of Ace and Ace2 (angiotens in I converting enzyme [peptidyl-dipeptidase A] 2) in lungs and kidneys from pregnant rats by quantitative real-time PCR and Western blotting, and the activities of these proteins in lungs, kidneys, and plasma, were measured. The mRNA levels of Ace and Ace2 in lungs were elevated by PR at both Days 19 and 22 of pregnancy. The abundance of ACE protein in lungs was increased, but ACE2 protein was decreased, by PR. The activities of ACE, but not ACE2, in lungs were increased by PR. PR did not change expressions of Ace and Ace2, the activities of both ACE and ACE2 in kidneys, and the abundance and activity of plasma ACE. These findings suggest that maternal lungs contribute to the elevated plasma levels of angiotensin II by increasing both the expression and the activity of ACE in response to gestational PR. PMID:23365412

Angiotensin I-Converting Enzyme Inhibitor Activity on Egg Albumen Fermentation

PubMed Central

Nahariah, N.; Legowo, A. M.; Abustam, E.; Hintono, A.

2015-01-01

Lactobacillus plantarum is used for fermentation of fish products, meat and milk. However, the utilization of these bacteria in egg processing has not been done. This study was designed to evaluate the potential of fermented egg albumen as a functional food that is rich in angiotensin I-converting enzyme inhibitors activity (ACE-inhibitor activity) and is antihypertensive. A completely randomized design was used in this study with six durations of fermentation (6, 12, 18, 24, 30, and 36 h) as treatments. Six hundred eggs obtained from the same chicken farm were used in the experiment as sources of egg albumen. Bacteria L. plantarum FNCC 0027 used in the fermentation was isolated from cow’s milk. The parameters measured were the total bacteria, dissolved protein, pH, total acid and the activity of ACE-inhibitors. The results showed that there were significant effects of fermentation time on the parameters tested. Total bacteria increased significantly during fermentation for 6, 12, 18, and 24 h and then decreased with the increasing time of fermentation to 30 and 36 h. Soluble protein increased significantly during fermentation to 18 h and then subsequently decreased during of fermentation to 24, 30, and 36 h. The pH value decreased markedly during fermentation. The activities of ACE-inhibitor in fermented egg albumen increased during fermentation to 18 h and then decreased with the increasing of the duration of fermentation to 24, 30, and 36 h. The egg albumen which was fermented for 18 h resulted in a functional food that was rich in ACE-inhibitor activity. PMID:25715689

[Risk for hyperkalemia during long-term treatment with angiotensin-converting enzyme inhibitors in insulin-dependent type 2 diabetics in relation to the glomerular filtration rate].

PubMed

Raml, A; Schmekal, B; Grafinger, P; Biesenbach, G

2001-11-23

The risk for hyperkalaemia during therapy with angiotensin-converting enzyme inhibitors is especially increased in the elderly diabetic because of a decrease in glomerular filtration rate (GFR), as well as the occurrence of hyporeninaemic hypoaldosteronism. We evaluated the risk for hyperkalaemia under long-term angiotensin-converting enyzme inhibition in 86 insulin-dependent type 2 diabetic patients in relation to their GFR. We compared the influence of a 3 to 6 months long treatment with angiotensin-converting enzyme inhibitors on the serum potassium levels, the creatinine clearance and the urinary albumin excretion in insulin-dependent type 2 diabetic patients with an initial creatinine clearance < 50 ml/min/1.73m(2) (n = 15, age 66 +/- 6 years) and >/= 50 ml/min/1.73m(2) respectively (n = 71, age 61 +/- 10 years). In addition, we also investigated the influence on the metabolic control and the blood pressure values in both groups of patients. In the patients with creatinine clearance >/= 50 ml/min/1,73m(2) the mean potassium level increased from 4.3 +/- 0.2 to 4.6 +/- 0.4 mmol/l (P < 0,01), while the incidence of a potassium level > 5 mmol/l was 17 %. In the group with a creatinine clearance < 50 ml/min/1.73m(2) the potassium level rose from 4.5 +/- 0.2 to 5.0 +/- 0.4 mmol/l (P < 0.01). The incidence of potassium levels > 5 mmol/l was 66 % (P < 0,01). In both patient groups the creatinine clearances did not change significantly during angiotensin-converting enzyme inhibition, and the urinary albumin excretion as well as the HbA(1c) values and blood pressure showed only a tendency towards a decrease. Long-term treatment with angiotensin-converting enzyme inhibitors in insulin-dependent type 2 diabetic patients leads to a significant increase in serum potassium. The incidence of hyperkalaemia with potassium levels > 5 mmol/l is significantly higher in the patients with initial creatinine clearance < 50 ml/min/1.73m(2). Severe hyperkalaemia with potassium levels > 6 mmol/l was not observed.

Convergent evidences from human and animal studies implicate angiotensin I-converting enzyme activity in cognitive performance in schizophrenia.

PubMed

Gadelha, A; Vendramini, A M; Yonamine, C M; Nering, M; Berberian, A; Suiama, M A; Oliveira, V; Lima-Landman, M T; Breen, G; Bressan, R A; Abílio, V; Hayashi, M A F

2015-12-08

In schizophrenia (SCZ), higher angiotensin I-converting enzyme (ACE) levels have been reported in patient's blood and cerebrospinal fluid (CSF). Hereby, we propose to explore whether the ACE activity levels are associated to cognitive performance in SCZ. Seventy-two patients with SCZ or schizoaffective disorder diagnosis, and 69 healthy controls (HCs) underwent a cognitive battery with parallel collection of peripheral blood samples to measure ACE activity. Significant higher ACE activity levels were confirmed in the plasma of SCZ patients compared with HCs (Student's t=-5.216; P<0.001). ACE activity significantly correlated to Hopkins delayed recall measures (r=-0.247; P=0.004) and Hopkins total (r=-0.214; P=0.012). Subjects grouped as high ACE activity (above average) had worse performance compared with low ACE activity level group for Hopkins delayed recall measure, even after correction for clinical condition, age, gender and years of education (P=0.029). The adjusted R squared for this final model was 0.343. This result was evident only comparing extreme groups for ACE activity, when splitting the sample in three groups with similar number of subjects. To clarify this finding, we performed an evaluation of the cognitive performance of transgenic mice with three copies of ACE gene in novel object recognition (NOR) test, which showed that such animals presented impairment in NOR (P<0.05) compared with two copies of wild-type animals. The results observed in SCZ patients and animal model suggest both the association of ACE to cognitive deficits in SCZ. This finding may support the evaluation of novel treatment protocols and/or of innovative drugs for specific intervention of cognitive deficits in SCZ envisioning concomitant ACE activity and behavior evaluations.

Biochemical Characterization of the Lactobacillus reuteri Glycoside Hydrolase Family 70 GTFB Type of 4,6-α-Glucanotransferase Enzymes That Synthesize Soluble Dietary Starch Fibers.

PubMed

Bai, Yuxiang; van der Kaaij, Rachel Maria; Leemhuis, Hans; Pijning, Tjaard; van Leeuwen, Sander Sebastiaan; Jin, Zhengyu; Dijkhuizen, Lubbert

2015-10-01

4,6-α-Glucanotransferase (4,6-α-GTase) enzymes, such as GTFB and GTFW of Lactobacillus reuteri strains, constitute a new reaction specificity in glycoside hydrolase family 70 (GH70) and are novel enzymes that convert starch or starch hydrolysates into isomalto/maltopolysaccharides (IMMPs). These IMMPs still have linear chains with some α1→4 linkages but mostly (relatively long) linear chains with α1→6 linkages and are soluble dietary starch fibers. 4,6-α-GTase enzymes and their products have significant potential for industrial applications. Here we report that an N-terminal truncation (amino acids 1 to 733) strongly enhances the soluble expression level of fully active GTFB-ΔN (approximately 75-fold compared to full-length wild type GTFB) in Escherichia coli. In addition, quantitative assays based on amylose V as the substrate are described; these assays allow accurate determination of both hydrolysis (minor) activity (glucose release, reducing power) and total activity (iodine staining) and calculation of the transferase (major) activity of these 4,6-α-GTase enzymes. The data show that GTFB-ΔN is clearly less hydrolytic than GTFW, which is also supported by nuclear magnetic resonance (NMR) analysis of their final products. From these assays, the biochemical properties of GTFB-ΔN were characterized in detail, including determination of kinetic parameters and acceptor substrate specificity. The GTFB enzyme displayed high conversion yields at relatively high substrate concentrations, a promising feature for industrial application. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

Generation of proton-motive force by an archaeal terminal quinol oxidase from Sulfolobus acidocaldarius.

PubMed

Gleissner, M; Elferink, M G; Driessen, A J; Konings, W N; Anemüller, S; Schäfer, G

1994-09-15

The terminal quinol oxidase of the cytochrome aa3 type was isolated from the extreme thermoacidophilic archaeon Sulfolobus acidocaldarius. In micellar solution, the enzyme oxidized various quinols and exerted the highest activity with the physiological substrate caldariella quinol. The enzyme was functionally reconstituted into monolayer liposomes composed of archaeal tetraether lipids also derived from S. acidocaldarius. With the electron donor system ascorbate and N,N,N',N'-tetramethyl-p-phenylenediamine, the reconstituted enzyme was more active in the archaeal lipids as compared to lipids derived from Escherichia coli at temperatures above 50 degrees C. Due to the low proton permeability of the tetraether lipids, it was possible to generate a steady-state transmembrane electrical potential (delta psi, interior negative), and transmembrane pH gradient (delta pH, interior alkaline) at temperatures up to 70 degrees C. The successful functional reconstitution of the cytochrome aa3-type quinol oxidase from Sulfolobus identifies it as the key energy converter in the respiratory system of this hyperthermophilic archaeon.

Nicotinamide riboside kinase structures reveal new pathways to NAD+.

PubMed

Tempel, Wolfram; Rabeh, Wael M; Bogan, Katrina L; Belenky, Peter; Wojcik, Marzena; Seidle, Heather F; Nedyalkova, Lyudmila; Yang, Tianle; Sauve, Anthony A; Park, Hee-Won; Brenner, Charles

2007-10-02

The eukaryotic nicotinamide riboside kinase (Nrk) pathway, which is induced in response to nerve damage and promotes replicative life span in yeast, converts nicotinamide riboside to nicotinamide adenine dinucleotide (NAD+) by phosphorylation and adenylylation. Crystal structures of human Nrk1 bound to nucleoside and nucleotide substrates and products revealed an enzyme structurally similar to Rossmann fold metabolite kinases and allowed the identification of active site residues, which were shown to be essential for human Nrk1 and Nrk2 activity in vivo. Although the structures account for the 500-fold discrimination between nicotinamide riboside and pyrimidine nucleosides, no enzyme feature was identified to recognize the distinctive carboxamide group of nicotinamide riboside. Indeed, nicotinic acid riboside is a specific substrate of human Nrk enzymes and is utilized in yeast in a novel biosynthetic pathway that depends on Nrk and NAD+ synthetase. Additionally, nicotinic acid riboside is utilized in vivo by Urh1, Pnp1, and Preiss-Handler salvage. Thus, crystal structures of Nrk1 led to the identification of new pathways to NAD+.

Involvement of phenoloxidase in browning during grinding of Tenebrio molitor larvae

PubMed Central

Lakemond, Catriona M. M.; Fogliano, Vincenzo; Renzone, Giovanni; Scaloni, Andrea; Vincken, Jean-Paul

2017-01-01

Insects are investigated as alternative protein source to meet the increasing demand for proteins in the future. Enzymatic browning occurring during grinding of insect and subsequent extraction of proteins can influence the proteins’ properties, but it is unclear which enzymes are responsible for this phenomenon. This study was performed on larvae of three commonly used insect species, namely Tenebrio molitor, Alphitobius diaperinus and Hermetia illucens. Oxygen consumption measurements on protein extracts showed activity on L-tyrosine, L-3,4-di-hydroxy-phenylalanine (L-DOPA) and L-dopamine, indicating phenoloxidase as a key player in browning. Furthermore, no reaction on 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) was observed, ruling out an important contribution of laccase to browning. The browning reaction was most prominent at pH 6 for T. molitor and A. diaperinus, and 7 for H. illucens. As the enzyme activity of H. illucens was the lowest with the darkest color formation, this was likely caused by another factor. The activity of phenoloxidase was confirmed for T. molitor and A. diaperinus by activity measurements after fractionation by anion-exchange chromatography. Color measurements showed the presence of activity on both L-DOPA and L-tyrosine in the same fractions. Both substrates were converted into dopachrome after incubation with enzyme-enriched fractions. No DOPA-decarboxylase, tyrosine hydroxylase and peroxidase activities were observed. By using native PAGE with L-DOPA as staining-solution, active T. molitor protein bands were resolved and characterized, identifying a tyrosinase/phenoloxidase as the active enzyme species. All together, these data confirmed that tyrosinase is an important enzyme in causing enzymatic browning in T. molitor and likely in A. diaperinus. PMID:29244828

Involvement of phenoloxidase in browning during grinding of Tenebrio molitor larvae.

PubMed

Janssen, Renske H; Lakemond, Catriona M M; Fogliano, Vincenzo; Renzone, Giovanni; Scaloni, Andrea; Vincken, Jean-Paul

2017-01-01

Insects are investigated as alternative protein source to meet the increasing demand for proteins in the future. Enzymatic browning occurring during grinding of insect and subsequent extraction of proteins can influence the proteins' properties, but it is unclear which enzymes are responsible for this phenomenon. This study was performed on larvae of three commonly used insect species, namely Tenebrio molitor, Alphitobius diaperinus and Hermetia illucens. Oxygen consumption measurements on protein extracts showed activity on L-tyrosine, L-3,4-di-hydroxy-phenylalanine (L-DOPA) and L-dopamine, indicating phenoloxidase as a key player in browning. Furthermore, no reaction on 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) was observed, ruling out an important contribution of laccase to browning. The browning reaction was most prominent at pH 6 for T. molitor and A. diaperinus, and 7 for H. illucens. As the enzyme activity of H. illucens was the lowest with the darkest color formation, this was likely caused by another factor. The activity of phenoloxidase was confirmed for T. molitor and A. diaperinus by activity measurements after fractionation by anion-exchange chromatography. Color measurements showed the presence of activity on both L-DOPA and L-tyrosine in the same fractions. Both substrates were converted into dopachrome after incubation with enzyme-enriched fractions. No DOPA-decarboxylase, tyrosine hydroxylase and peroxidase activities were observed. By using native PAGE with L-DOPA as staining-solution, active T. molitor protein bands were resolved and characterized, identifying a tyrosinase/phenoloxidase as the active enzyme species. All together, these data confirmed that tyrosinase is an important enzyme in causing enzymatic browning in T. molitor and likely in A. diaperinus.

Casein Fermentate of Lactobacillus animalis DPC6134 Contains a Range of Novel Propeptide Angiotensin-Converting Enzyme Inhibitors▿

PubMed Central

Hayes, M.; Stanton, C.; Slattery, H.; O'Sullivan, O.; Hill, C.; Fitzgerald, G. F.; Ross, R. P.

2007-01-01

This work evaluated the angiotensin-converting-enzyme (ACE)-inhibitory activities of a bovine sodium caseinate fermentate generated using the proteolytic capabilities of the porcine small intestinal isolate Lactobacillus animalis DPC6134 (NCIMB deposit 41355). The crude 10-kDa L. animalis DPC6134 fermentate exhibited ACE-inhibitory activity of 85.51% (±15%) and had a 50% inhibitory concentration (IC50) of 0.8 mg protein/ml compared to captopril, which had an IC50 value of 0.005 mg/ml. Fractionation of the crude L. animalis DPC6134 fermentate by membrane filtration and reversed-phase high-performance liquid chromatography (HPLC) generated three bioactive fractions from a total of 72 fractions. Fractions 10, 19, and 43 displayed ACE-inhibitory activity percentages of 67.53 (±15), 83.71 (±19), and 42.36 (±11), respectively, where ACE inhibition was determined with 80 μl of the fractions with protein concentrations of 0.5 mg/ml. HPLC and mass spectrometry analysis identified 25 distinct peptide sequences derived from α-, β-, and κ-caseins. In silico predictions, based on the C-terminal tetrapeptide sequences, suggested that peptide NIPPLTQTPVVVPPFIQ, corresponding to β-casein f(73-89); peptide IGSENSEKTTMP, corresponding to αs1-casein f(201212); peptide SQSKVLPVPQ, corresponding to β-casein f(166-175); peptide MPFPKYPVEP, corresponding to β-casein f(124133); and peptide EPVLGPVRGPFP, corresponding to β-casein f(210-221), contained ACE-inhibitory activities. These peptides were chosen for chemical synthesis to confirm the ACE-inhibitory activity of the fractions. Chemically synthesized peptides displayed IC50 values in the range of 92 μM to 790 μM. Additionally, a simulated gastrointestinal digestion confirmed that the ACE-inhibitory 10-kDa L. animalis DPC6134 fermentation was resistant to a cocktail of digestive enzymes found in the gastrointestinal tract. PMID:17483275

Enzymatic characterization of a class II lysyl-tRNA synthetase, LysS, from Myxococcus xanthus.

PubMed

Oka, Manami; Takegawa, Kaoru; Kimura, Yoshio

2015-08-01

Lysyl-tRNA synthetases efficiently produce diadenosine tetraphosphate (Ap4A) from lysyl-AMP with ATP in the absence of tRNA. We characterized recombinant class II lysyl-tRNA synthetase (LysS) from Myxococcus xanthus and found that it is monomeric and requires Mn(2+) for the synthesis of Ap4A. Surprisingly, Zn(2+) inhibited enzyme activity in the presence of Mn(2+). When incubated with ATP, Mn(2+), lysine, and inorganic pyrophosphatase, LysS first produced Ap4A and ADP, then converted Ap4A to diadenosine triphosphate (Ap3A), and finally converted Ap3A to ADP, the end product of the reaction. Recombinant LysS retained Ap4A synthase activity without lysine addition. Additionally, when incubated with Ap4A (minus pyrophosphatase), LysS converted Ap4A mainly ATP and AMP, or ADP in the presence or absence of lysine, respectively. These results demonstrate that M. xanthus LysS has different enzymatic properties from class II lysyl-tRNA synthetases previously reported. Copyright © 2015 Elsevier Inc. All rights reserved.

A Haloalkane Dehalogenase from a Marine Microbial Consortium Possessing Exceptionally Broad Substrate Specificity.

PubMed

Buryska, Tomas; Babkova, Petra; Vavra, Ondrej; Damborsky, Jiri; Prokop, Zbynek

2018-01-15

The haloalkane dehalogenase enzyme DmmA was identified by marine metagenomic screening. Determination of its crystal structure revealed an unusually large active site compared to those of previously characterized haloalkane dehalogenases. Here we present a biochemical characterization of this interesting enzyme with emphasis on its structure-function relationships. DmmA exhibited an exceptionally broad substrate specificity and degraded several halogenated environmental pollutants that are resistant to other members of this enzyme family. In addition to having this unique substrate specificity, the enzyme was highly tolerant to organic cosolvents such as dimethyl sulfoxide, methanol, and acetone. Its broad substrate specificity, high overexpression yield (200 mg of protein per liter of cultivation medium; 50% of total protein), good tolerance to organic cosolvents, and a broad pH range make DmmA an attractive biocatalyst for various biotechnological applications. IMPORTANCE We present a thorough biochemical characterization of the haloalkane dehalogenase DmmA from a marine metagenome. This enzyme with an unusually large active site shows remarkably broad substrate specificity, high overexpression, significant tolerance to organic cosolvents, and activity under a broad range of pH conditions. DmmA is an attractive catalyst for sustainable biotechnology applications, e.g., biocatalysis, biosensing, and biodegradation of halogenated pollutants. We also report its ability to convert multiple halogenated compounds to corresponding polyalcohols. Copyright © 2018 American Society for Microbiology.

Spectral and catalytic properties of aryl-alcohol oxidase, a fungal flavoenzyme acting on polyunsaturated alcohols

PubMed Central

2005-01-01

Spectral and catalytic properties of the flavoenzyme AAO (aryl-alcohol oxidase) from Pleurotus eryngii were investigated using recombinant enzyme. Unlike most flavoprotein oxidases, AAO does not thermodynamically stabilize a flavin semiquinone radical and forms no sulphite adduct. AAO catalyses the oxidative dehydrogenation of a wide range of unsaturated primary alcohols with hydrogen peroxide production. This differentiates the enzyme from VAO (vanillyl-alcohol oxidase), which is specific for phenolic compounds. Moreover, AAO is optimally active in the pH range of 5–6, whereas VAO has an optimum at pH 10. Kinetic studies showed that AAO is most active with p-anisyl alcohol and 2,4-hexadien-1-ol. AAO converts m- and p-chlorinated benzyl alcohols at a similar rate as it does benzyl alcohol, but introduction of a p-methoxy substituent in benzyl alcohol increases the reaction rate approx. 5-fold. AAO also exhibits low activity on aromatic aldehydes. 19F NMR analysis showed that fluorinated benzaldehydes are converted into the corresponding benzoic acids. Inhibition studies revealed that the AAO active site can bind a wide range of aromatic ligands, chavicol (4-allylphenol) and p-anisic (4-methoxybenzoic) acid being the best competitive inhibitors. Uncompetitive inhibition was observed with 4-methoxybenzylamine. The properties described above render AAO a unique oxidase. The possible mechanism of AAO binding and oxidation of substrates is discussed in the light of the results of the inhibition and kinetic studies. PMID:15813702

Short communication: Potential of Fresco-style cheese whey as a source of protein fractions with antioxidant and angiotensin-I-converting enzyme inhibitory activities.

PubMed

Tarango-Hernández, S; Alarcón-Rojo, A D; Robles-Sánchez, M; Gutiérrez-Méndez, N; Rodríguez-Figueroa, J C

2015-11-01

Recently, traditional Mexican Fresco-style cheese production has been increasing, and the volume of cheese whey generated represents a problem. In this study, we investigated the chemical composition of Fresco-style cheese wheys and their potential as a source of protein fractions with antioxidant and angiotensin-I-converting enzyme (ACE)-inhibitory activities. Three samples from Fresco, Panela, and Ranchero cheeses whey were physicochemically characterized. Water-soluble extracts were fractionated to obtain whey fractions with different molecular weights: 10-5, 5-3, 3-1 and <1 kDa. The results indicated differences in the lactose, protein, ash, and dry matter contents (% wt/wt) in the different Fresco-style cheese wheys. All whey fractions had antioxidant and ACE-inhibitory activities. The 10-5 kDa whey fraction of Ranchero cheese had the highest Trolox equivalent antioxidant capacity (0.62 ± 0.00 mM), and the 3-1 kDa Panela and Fresco cheese whey fractions showed the highest ACE-inhibitory activity (0.57 ± 0.02 and 0.59 ± 0.04 μg/mL 50%-inhibitory concentration values, respectively). These results suggest that Fresco-style cheese wheys may be a source of protein fractions with bioactivity, and thus could be useful ingredients in the manufacture of functional foods with increased nutritional value. Copyright © 2015 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.

Angiotensin I-Converting Enzyme (ACE) Inhibitory Activity, Antioxidant Properties, Phenolic Content and Amino Acid Profiles of Fucus spiralis L. Protein Hydrolysate Fractions

PubMed Central

Paiva, Lisete; Neto, Ana Isabel; Baptista, José

2017-01-01

Food protein-derived hydrolysates with multi-bioactivities such as antihypertensive and antioxidant properties have recently received special attention since both activities can play significant roles in preventing cardiovascular diseases. This study reports, for the first time, the angiotensin I-converting enzyme (ACE)-inhibition and antioxidant properties of ultrafiltrate fractions (UF) with different molecular weight ranges (<1, 1–3 and ≥3 kDa) obtained from Fucus spiralis protein hydrolysate (FSPH) digested with cellulase–bromelain. The amino acids profile, recovery yield, protein, peptide and total phenolic contents of these FSPH-UF, and the in vitro digestibility of F. spiralis crude protein were also investigated. FSPH-UF ≥3 kDa presented remarkably higher ACE-inhibition, yield, peptide and polyphenolic (phlorotannins) contents. Antioxidant analysis showed that FSPH-UF <1 kDa and ≥3 kDa exhibited significantly higher scavenging of 2,2-diphenyl-1-picrylhydrazyl radical and ferrous ion-chelating (FIC) activity. FSPH-UF ≥3 kDa had also notably higher ferric reducing antioxidant power (FRAP). Strong correlations were observed between ACE-inhibition and antioxidant activities (FIC and FRAP). The results suggest that ACE-inhibition and antioxidant properties of FSPH-UF may be due to the bioactive peptides and polyphenols released during the enzymatic hydrolysis. In conclusion, this study shows the potential use of defined size FSPH-UF for the prevention/treatment of hypertension and/or oxidative stress-related diseases. PMID:29027934

Substance P increases sympathetic activity during combined angiotensin-converting enzyme and dipeptidyl peptidase-4 inhibition.

PubMed

Devin, Jessica K; Pretorius, Mias; Nian, Hui; Yu, Chang; Billings, Frederic T; Brown, Nancy J

2014-05-01

Dipeptidyl peptidase-4 inhibitors prevent the degradation of incretin hormones and reduce postprandial hyperglycemia in patients with type 2 diabetes mellitus. Dipeptidyl peptidase-4 degrades other peptides with a penultimate proline or alanine, including bradykinin and substance P, which are also substrates of angiotensin-converting enzyme (ACE). During ACE inhibition, substance P is inactivated primarily by dipeptidyl peptidase-4, whereas bradykinin is first inactivated by aminopeptidase P. This study tested the hypothesis that dipeptidyl peptidase-4 inhibition potentiates vasodilator and fibrinolytic responses to substance P when ACE is inhibited. Twelve healthy subjects participated in this randomized, double-blinded, placebo-controlled crossover study. On each study day, subjects received sitagliptin 200 mg by mouth or placebo. Substance P and bradykinin were infused via brachial artery before and during intra-arterial enalaprilat. Sitagliptin and enalaprilat each reduced forearm vascular resistance and increased forearm blood flow without affecting mean arterial pressure, but there was no interactive effect of the inhibitors. Enalaprilat increased bradykinin-stimulated vasodilation and tissue plasminogen activator release; sitagliptin did not affect these responses to bradykinin. The vasodilator response to substance P was unaffected by sitagliptin and enalaprilat; however, substance P increased heart rate and vascular release of norepinephrine during combined ACE and dipeptidyl peptidase-4 inhibition. In women, sitagliptin diminished tissue plasminogen activator release in response to substance P both alone and during enalaprilat. Substance P increases sympathetic activity during combined ACE and dipeptidyl peptidase-4 inhibition. - URL: http://www.clinicaltrials.gov. Unique identifier: NCT01413542.

Comparison of the effects of candesartan cilexetil and enalapril maleate on right ventricular myocardial remodeling in dogs with experimentally induced pulmonary stenosis.

PubMed

Yamane, Tsuyoshi; Fujii, Yoko; Orito, Kensuke; Osamura, Kaori; Kanai, Takao; Wakao, Yoshito

2008-12-01

To compare the effects of candesartan cilexetil and enalapril maleate on right ventricular myocardial remodeling in dogs with experimentally induced pulmonary stenosis. 24 Beagles. 18 dogs underwent pulmonary arterial banding (PAB) to induce right ventricular pressure overload, and 6 healthy dogs underwent sham operations (thoracotomy only [sham-operated group]). Dogs that underwent PAB were allocated to receive 1 of 3 treatments (6 dogs/group): candesartan (1 mg/kg, PO, q 24 h [PABC group]), enalapril (0.5 mg/kg, PO, q 24 h [PABE group]), or no treatment (PABNT group). Administration of treatments was commenced the day prior to surgery; control dogs received no cardiac medications. Sixty days after surgery, right ventricular wall thickness was assessed echocardiographically and plasma renin activity, angiotensin-converting enzyme activity, and angiotensin I and II concentrations were assessed; all dogs were euthanatized, and collagenous fiber area, cardiomyocyte diameter, and tissue angiotensin-converting enzyme and chymase-like activities in the right ventricle were evaluated. After 60 days of treatment, right ventricular wall thickness, cardiomyocyte diameter, and collagenous fiber area in the PABNT and PABE groups were significantly increased, compared with values in the PABC and sham-operated groups. Chymase-like activity was markedly greater in the PABE group than in other groups. Results indicated that treatment with candesartan but not enalapril effectively prevented myocardial remodeling in dogs with experimentally induced subacute right ventricular pressure overload.

Heat treatment of curdlan enhances the enzymatic production of biologically active β-(1,3)-glucan oligosaccharides.

PubMed

Kumagai, Yuya; Okuyama, Masayuki; Kimura, Atsuo

2016-08-01

Biologically active β-(1,3)-glucan oligosaccharides were prepared from curdlan using GH64 enzyme (KfGH64). KfGH64 showed low activity toward native curdlan; thereby pretreatment conditions of curdlan were evaluated. KfGH64 showed the highest activity toward curdlan with heat treatment. The most efficient pretreatment (90°C for 0.5h) converted approximately 60% of curdlan into soluble saccharides under the optimized enzyme reaction conditions (pH 5.5, 37°C, 100rpm mixing speed, 24h, and 10μg of KfGH64/1g of curdlan). The resulting products were predominantly laminaripentaose and a small amount of β-(1,3)-glucans with an average degree of polymerization (DP) of 13 and 130. The products did not contain small oligosaccharides (DP<5), indicating that the hydrolysis of heat-treated curdlan by KfGH64 is a suitable method for the production of biologically active β-(1,3)-glucan oligosaccharides. Copyright © 2016 Elsevier Ltd. All rights reserved.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Strittmatter, S.M.; Snyder, S.H.

We demonstrate that (3H)captopril selectively labels angiotensin converting enzyme (EC 3.14.15.1) (ACE) and employ this technique to probe enzyme-inhibitor interactions. (3H)Captopril binding sites copurify with ACE activity from rat lung or rat brain. At each stage of the purification the Vmax/Bmax ratio, or kcat is 17,000 min-1 with hippuryl-L-histidyl-L-leucine as substrate. The specificity of (3H)captopril binding is apparent in the similar pharmacologic profile of inhibition in crude and pure enzyme preparations. Furthermore, binding sites and enzyme activity comigrate in gel filtration and sucrose gradient sedimentation experiments. Equilibrium analysis of (3H)captopril binding to purified ACE reveals a Bmax of 6 nmol/mgmore » of protein (KD = 2 nM), demonstrating the presence of one inhibitor binding site per polypeptide chain. The kinetics of (3H)captopril binding are characterized by monophasic association and dissociation rate constants of 0.026 nM-1 min-1 and 0.034 min-1, respectively. The affinity of ACE for both (3H) captopril and enalaprilat is greater at 37 degrees than at 0 degree, demonstrating that these interactions are entropically driven, perhaps by an isomerization of the enzyme molecule. The ionic requirements for (3H)captopril binding and substrate catalysis differ. Chloride and bromide ion, but not fluoride, are about 100-fold more potent stimulators of binding than catalysis. When the active site Zn2+ ion is replaced by Co2+, catalysis was stimulated 2-fold, whereas binding activity was decreased by 70%.« less

Angiotensin-converting enzyme inhibitor prevents oxidative stress, inflammation, and fibrosis in carbon tetrachloride-treated rat liver.

PubMed

Reza, Hasan Mahmud; Tabassum, Nabila; Sagor, Md Abu Taher; Chowdhury, Mohammed Riaz Hasan; Rahman, Mahbubur; Jain, Preeti; Alam, Md Ashraful

2016-01-01

Hepatic fibrosis is a common feature of chronic liver injury, and the involvement of angiotensin II in such process has been studied earlier. We hypothesized that anti-angiotensin II agents may be effective in preventing hepatic fibrosis. In this study, Long Evans female rats were used and divided into four groups such as Group-I, Control; Group-II, Control + ramipril; Group-III, CCl4; and Group-IV, CCl4 + ramipril. Group II and IV are treated with ramipril for 14 d. At the end of treatment, the livers were removed, and the level of hepatic marker enzymes (aspartate aminotransferase, Alanine aminotransferase, and alkaline phosphatase), nitric oxide, advanced protein oxidation product , catalase activity, and lipid peroxidation were determined. The degree of fibrosis was evaluated through histopathological staining with Sirius red and trichrome milligan staining. Carbon-tetrachloride (CCl4) administration in rats developed hepatic dysfunction and raised the hepatic marker enzymes activities significantly. CCl4 administration in rats also produced oxidative stress, inflammation, and fibrosis in liver. Furthermore, angiotensinogen-inhibitor ramipril normalized the hepatic enzymes activities and improved the antioxidant enzyme catalase activity. Moreover, ramipril treatment ameliorated lipid peroxidation and hepatic inflammation in CCl4-treated rats. Ramipril treatment also significantly reduced hepatic fibrosis in CCl4-administered rats. In conclusion, our investigation suggests that the antifibrotic effect of ramipril may be attributed to inhibition of angiotensin-II mediated oxidative stress and inflammation in liver CCl4-administered rats.

Molecular and catalytic properties of 2,4'-dihydroxyacetophenone dioxygenase from Burkholderia sp. AZ11.

PubMed

Enya, Mayu; Aoyagi, Keiko; Hishikawa, Yoshihiro; Yoshimura, Azusa; Mitsukura, Koichi; Maruyama, Kiyofumi

2012-01-01

The gene dad encoding 2,4'-dihydroxyacetophenone (DHAP) dioxygenase was cloned from Burkholderia sp. AZ11. The initiation codon GTG was converted to ATG for high-level expression of the enzyme in Escherichia coli. The enzyme was moderately thermostable, and the recombinant enzyme was briefly purified. The enzyme (M(r)=90 kDa) was a homotetramer with a subunit M(r) of 23 kDa. It contained 1.69 mol of non-heme iron, and had a dark gray color. On anaerobic incubation of it with DHAP, the absorption at around 400 nm increased due to the formation of an enzyme-DHAP complex. Multiple sequence alignment suggested that His77, His79, His115, and Glu96 in the cupin fold were possible metal ligands. The apparent K(m) for DHAP and the apparent V(max) were estimated to be 1.60 µM and 6.28 µmol/min/mg respectively. 2-Hydroxyacetophenone was a poor substrate. CuCl(2) and HgCl(2) strongly inhibited the enzyme, while FeSO(4) weakly activated it.

Nitrosomonas europaea cytochrome P460 is a direct link between nitrification and nitrous oxide emission.

PubMed

Caranto, Jonathan D; Vilbert, Avery C; Lancaster, Kyle M

2016-12-20

Ammonia oxidizing bacteria (AOB) are major contributors to the emission of nitrous oxide (N 2 O). It has been proposed that N 2 O is produced by reduction of NO. Here, we report that the enzyme cytochrome (cyt) P460 from the AOB Nitrosomonas europaea converts hydroxylamine (NH 2 OH) quantitatively to N 2 O under anaerobic conditions. Previous literature reported that this enzyme oxidizes NH 2 OH to nitrite ([Formula: see text]) under aerobic conditions. Although we observe [Formula: see text] formation under aerobic conditions, its concentration is not stoichiometric with the NH 2 OH concentration. By contrast, under anaerobic conditions, the enzyme uses 4 oxidizing equivalents (eq) to convert 2 eq of NH 2 OH to N 2 O. Enzyme kinetics coupled to UV/visible absorption and electron paramagnetic resonance (EPR) spectroscopies support a mechanism in which an Fe III -NH 2 OH adduct of cyt P460 is oxidized to an {FeNO} 6 unit. This species subsequently undergoes nucleophilic attack by a second equivalent of NH 2 OH, forming the N-N bond of N 2 O during a bimolecular, rate-determining step. We propose that [Formula: see text] results when nitric oxide (NO) dissociates from the {FeNO} 6 intermediate and reacts with dioxygen. Thus, [Formula: see text] is not a direct product of cyt P460 activity. We hypothesize that the cyt P460 oxidation of NH 2 OH contributes to NO and N 2 O emissions from nitrifying microorganisms.

Cognitive enhancing effect of angiotensin-converting enzyme inhibitors and angiotensin receptor blockers on learning and memory

PubMed Central

Nade, V. S.; Kawale, L. A.; Valte, K. D.; Shendye, N. V.

2015-01-01

Objective: The present study was designed to investigate cognitive enhancing property of angiotensin-converting enzymes inhibitors (ACEI) and angiotensin receptor blockers (ARBs) in rats. Materials and Methods: The elevated plus maze (EPM), passive avoidance test (PAT), and water maze test (WMT) were used to assess cognitive enhancing activity in young and aged rats. Ramipril (10 mg/kg, p.o.), perindopril (10 mg/kg, i.p), losartan (20 mg/kg, i.p), and valsartan (20 mg/kg, p.o) were administered to assess their effect on learning and memory. Scopolamine (1 mg/kg, i.p) was used to impair cognitive function. Piracetam (200 mg/kg, i.p) was used as reference drug. Results: All the treatments significantly attenuated amnesia induced by aging and scopolamine. In EPM, aged and scopolamine-treated rats showed an increase in transfer latency (TL) whereas, ACEI and ARBs showed a significant decrease in TL. Treatment with ACEI and ARBs significantly increased step down latencies and decreased latency to reach the platform in target quadrant in young, aged and scopolamine-treated animals in PAT and WMT, respectively. The treatments inhibited acetylcholinesterase (AChE) enzyme in the brain. Similarly, all the treatments attenuated scopolamine-induced lipid peroxidation and normalize antioxidant enzymes. Conclusion: The results suggest that the cognitive enhancing effect of ACEI and ARBs may be due to inhibition of AChE or by regulation of antioxidant system or increase in formation of angiotensin IV. PMID:26069362

The influence of carbon nanotubes on enzyme activity and structure: investigation of different immobilization procedures through enzyme kinetics and circular dichroism studies.

PubMed

Cang-Rong, Jason Teng; Pastorin, Giorgia

2009-06-24

In the last decade, many environmental organizations have devoted their efforts to identifying renewable biosystems, which could provide sustainable fuels and thus enhance energy security. Amidst the myriad of possibilities, some biofuels make use of different types of waste biomasses, and enzymes are often employed to hydrolyze these biomasses and produce sugars that will be subsequently converted into ethanol. In this project, we aimed to bridge nanotechnology and biofuel production: here we report on the activity and structure of the enzyme amyloglucosidase (AMG), physically adsorbed or covalently immobilized onto single-walled carbon nanotubes (SWNTs) and multi-walled carbon nanotubes (MWNTs). In fact, carbon nanotubes (CNTs) present several properties that render them ideal support systems, without the diffusion limitations displayed by porous material and with the advantage of being further functionalizable at their surface. Chemical ligation was achieved both on oxidized nanotubes (via carbodiimide chemistry), as well as on amino-functionalized nanotubes (via periodate-oxidized AMG). Results showed that AMG retained a certain percentage of its specific activity for all enzyme-carbon nanotubes complexes prepared, with the physically adsorbed samples displaying better catalytic efficiency than the covalently immobilized samples. Analysis of the enzyme's structure through circular dichroism (CD) spectroscopy revealed significant structural changes in all samples, the degree of change being consistent with the activity profiles. This study proves that AMG interacts differently with carbon nanotubes depending on the method employed. Due to the higher activity reported by the enzyme physically adsorbed onto CNTs, these samples demonstrated a vast potential for further development. At the same time, the possibility of inducing magnetic properties into CNTs offers the opportunity to easily separate them from the original solution. Hence, substances to which they have been attached can be separated from a reaction medium, or directed by an external magnetic field to achieve efficient biofuel production. This paves the way for future design of efficient CNT-enzyme nanostructure bioreactors.

The influence of carbon nanotubes on enzyme activity and structure: investigation of different immobilization procedures through enzyme kinetics and circular dichroism studies

NASA Astrophysics Data System (ADS)

Cang-Rong, Jason Teng; Pastorin, Giorgia

2009-06-01

In the last decade, many environmental organizations have devoted their efforts to identifying renewable biosystems, which could provide sustainable fuels and thus enhance energy security. Amidst the myriad of possibilities, some biofuels make use of different types of waste biomasses, and enzymes are often employed to hydrolyze these biomasses and produce sugars that will be subsequently converted into ethanol. In this project, we aimed to bridge nanotechnology and biofuel production: here we report on the activity and structure of the enzyme amyloglucosidase (AMG), physically adsorbed or covalently immobilized onto single-walled carbon nanotubes (SWNTs) and multi-walled carbon nanotubes (MWNTs). In fact, carbon nanotubes (CNTs) present several properties that render them ideal support systems, without the diffusion limitations displayed by porous material and with the advantage of being further functionalizable at their surface. Chemical ligation was achieved both on oxidized nanotubes (via carbodiimide chemistry), as well as on amino-functionalized nanotubes (via periodate-oxidized AMG). Results showed that AMG retained a certain percentage of its specific activity for all enzyme-carbon nanotubes complexes prepared, with the physically adsorbed samples displaying better catalytic efficiency than the covalently immobilized samples. Analysis of the enzyme's structure through circular dichroism (CD) spectroscopy revealed significant structural changes in all samples, the degree of change being consistent with the activity profiles. This study proves that AMG interacts differently with carbon nanotubes depending on the method employed. Due to the higher activity reported by the enzyme physically adsorbed onto CNTs, these samples demonstrated a vast potential for further development. At the same time, the possibility of inducing magnetic properties into CNTs offers the opportunity to easily separate them from the original solution. Hence, substances to which they have been attached can be separated from a reaction medium, or directed by an external magnetic field to achieve efficient biofuel production. This paves the way for future design of efficient CNT-enzyme nanostructure bioreactors.

Neurohormonal axis in patients with pulmonary arterial hypertension: friend or foe?

PubMed

de Man, Frances S; Handoko, M Louis; Guignabert, Christophe; Bogaard, Harm J; Vonk-Noordegraaf, Anton

2013-01-01

Despite its description some 25 years ago, neurohormonal activation has long been neglected as an important factor in the pathophysiology of pulmonary arterial hypertension (PAH). Neurohormonal activation was interpreted as a necessary compensatory response to maintain cardiac contractility and systemic blood pressure. Therefore, inhibitors of neurohormonal activity (like β-blockers or angiotensin-converting enzyme inhibitors) are considered contraindicated in current PAH management guidelines. However, recent data revealed that sympathetic overstimulation is strongly related to mortality, and blockade of neurohormonal activity in experimental PAH improved survival and cardiac function. These novel insights shed new light on the role of neurohormonal activity in PAH.

Association between angiotensin II receptor gene polymorphism and serum angiotensin converting enzyme (SACE) activity in patients with sarcoidosis

PubMed Central

Takemoto, Y.; Sakatani, M.; Takami, S.; Tachibana, T.; Higaki, J.; Ogihara, T.; Miki, T.; Katsuya, T.; Tsuchiyama, T.; Yoshida, A.; Yu, H.; Tanio, Y.; Ueda, E.

1998-01-01

BACKGROUND—Serum angiotensin converting enzyme (SACE) is considered to reflect disease activity in sarcoidosis. SACE activity is increased in many patients with active sarcoid lesions. The mechanism for the increased SACE activity in this disease has not been clarified. ACE insertion/deletion (I/D) gene polymorphism has been reported to have an association with SACE levels in sarcoidosis, but no evidence of an association between angiotensin II receptor gene polymorphism and SACE in this disease has been found. A study of the association of angiotensin II receptor gene polymorphisms with sarcoidosis was therefore undertaken.
METHODS—ACE (I/D), angiotensin II type 1 receptor (AGTR1), and angiotensin II type 2 receptor (AGTR2 ) gene polymorphisms were investigated by polymerase chain reaction (PCR) and SACE levels were measured in three groups of patients: those with sarcoidosis or tuberculosis and normal controls.
RESULTS—There was no difference in allele frequency of AGTR1 and AGTR2 polymorphism among the three groups. Neither AGTR1 nor AGTR2 polymorphisms were associated with sarcoidosis. SACE activity was higher in patients with sarcoidosis with the AGTR1 A/C genotype than in others. However, this tendency was not detected in patients with tuberculosis.
CONCLUSIONS—The AGTR1 allele C is associated with high activity of SACE in patients with sarcoidosis. It is another predisposing factor for high levels of SACE in patients with sarcoidosis and is considered to be an independent factor from the ACE D allele for high levels of SACE in sarcoidosis. This fact could be one of the explanations for the increased SACE activity in sarcoidosis.

 PMID:9713444

Optimized angiotensin-converting enzyme activity assay for the accurate diagnosis of sarcoidosis.

PubMed

Csongrádi, Alexandra; Enyedi, Attila; Takács, István; Végh, Tamás; Mányiné, Ivetta S; Pólik, Zsófia; Altorjay, István Tibor; Balla, József; Balla, György; Édes, István; Kappelmayer, János; Tóth, Attila; Papp, Zoltán; Fagyas, Miklós

2018-06-27

Serum angiotensin-converting enzyme (ACE) activity determination can aid the early diagnosis of sarcoidosis. We aimed to optimize a fluorescent kinetic assay for ACE activity by screening the confounding effects of endogenous ACE inhibitors and interfering factors. Genotype-dependent and genotype-independent reference values of ACE activity were established, and their diagnostic accuracies were validated in a clinical study. Internally quenched fluorescent substrate, Abz-FRK(Dnp)P-OH was used for ACE-activity measurements. A total of 201 healthy individuals and 59 presumably sarcoidotic patients were enrolled into this study. ACE activity and insertion/deletion (I/D) genotype of the ACE gene were determined. Here we report that serum samples should be diluted at least 35-fold to eliminate the endogenous inhibitor effect of albumin. No significant interferences were detected: up to a triglyceride concentration of 16 mM, a hemoglobin concentration of 0.71 g/L and a bilirubin concentration of 150 μM. Genotype-dependent reference intervals were considered as 3.76-11.25 U/L, 5.22-11.59 U/L, 7.19-14.84 U/L for II, ID and DD genotypes, respectively. I/D genotype-independent reference interval was established as 4.85-13.79 U/L. An ACE activity value was considered positive for sarcoidosis when it exceeded the upper limit of the reference interval. The optimized assay with genotype-dependent reference ranges resulted in 42.5% sensitivity, 100% specificity, 100% positive predictive value and 32.4% negative predictive value in the clinical study, whereas the genotype-independent reference range proved to have inferior diagnostic efficiency. An optimized fluorescent kinetic assay of serum ACE activity combined with ACE I/D genotype determination is an alternative to invasive biopsy for confirming the diagnosis of sarcoidosis in a significant percentage of patients.

Wheat Bread with Pumpkin (Cucurbita maxima L.) Pulp
as a Functional Food Product

PubMed Central

Gawlik-Dziki, Urszula; Dziki, Dariusz; Jakubczyk, Anna; Karaś, Monika; Różyło, Krzysztof

2014-01-01

Summary In this study, a new application of pumpkin pulp in bread production is shown. The aim of this work is to determine the influence of the addition of fresh pumpkin pulp directly into wheat flour on physical, sensorial and biological properties of bread. The bioaccessibility of active compounds was also studied. An increase in the addition of pumpkin pulp from 5 to 20% (converted to dry matter) caused a decrease of bread volume and increase of crumb hardness and cohesiveness. The sensory characteristics of the bread showed that a partial replacement of wheat flour with up to 10% of pumpkin pulp gave satisfactory results. The taste, aroma and overall acceptability of control bread and bread containing 5 or 10% of pulp had the highest degree of liking. The addition of higher levels of pumpkin pulp caused an unpleasant aroma and taste. Pumpkin pulp is a good material to complement the bread with potentially bioaccessible phenolics (including flavonoids) and, especially, with peptides. The highest antioxidant activity was observed, in most cases, of the samples with added 10 and 15% of pumpkin pulp. The addition of the pulp significantly enriched the bread with potentially bioaccessible angiotensin-converting enzyme (ACE) inhibitors. The highest activity was determined in the bread with 15 and 20% pumpkin pulp. ACE inhibitors from the tested bread were highly bioaccessible in vitro. Pumpkin pulp seems to be a valuable source of active compounds to complement the wheat bread. Adding the pulp directly to the wheat flour gives satisfactory baking results and reduces the cost of production. Additionally, pumpkin pulp is sometimes treated as waste material after the acquisition of seeds, thus using it as bread supplement also has environmental and economic benefits. Key words: pumpkin, bread, texture, antioxidants, bioaccessibility in vitro, angiotensin-converting enzyme (ACE) inhibition PMID:27904316

The enzymic preparation of (14)C-kaurene.

PubMed

Graebe, J E

1969-06-01

Endosperm from immature seeds of Cucurbita pepo L. converts 2-(14)C-DL-mevalonate to (14)C-(-)-kaurene with a yield of nearly 40% of the active isomer. Kaurene is the main product and the only diterpene hydrocarbon which is formed from mevalonate in the system and is therefore easily obtained radiochemically pure. The product was identified by thin-layer chromatography and recrystallization with authentic (-)-kaurene to constant specific radioactivity.

Angiotensin I-Converting Enzyme Inhibitor Derived from Cross-Linked Oyster Protein

PubMed Central

Xie, Cheng-Liang; Kim, Jin-Soo; Ha, Jong-Myung; Choung, Se-Young

2014-01-01

Following cross-linking by microbial transglutaminase, modified oyster proteins were hydrolyzed to improve inhibitory activity against angiotensin-converting enzyme (ACE) inhibitory activity with the use of a single protease, or a combination of six proteases. The oyster hydrolysate with the lowest 50% ACE inhibitory concentration (IC50) of 0.40 mg/mL was obtained by two-step hydrolysis of the cross-linked oyster protein using Protamex and Neutrase. Five ACE inhibitory peptides were purified from the oyster hydrolysate using a multistep chromatographic procedure comprised of ion-exchange, size exclusion, and reversed-phase liquid chromatography. Their sequences were identified as TAY, VK, KY, FYN, and YA, using automated Edman degradation and mass spectrometry. These peptides were synthesized, and their IC50 values were measured to be 16.7, 29.0, 51.5, 68.2, and 93.9 μM, respectively. Toxicity of the peptides on the HepG2 cell line was not detected. The oyster hydrolysate also significantly decreased the systolic blood pressure of spontaneously hypertensive rats (SHR). The antihypertensive effect of the oyster hydrolysate on SHR was rapid and long-lasting, compared to commercially obtained sardine hydrolysate. These results suggest that the oyster hydrolysate could be a source of effective nutraceuticals against hypertension. PMID:25140307

Combination of six enzymes of a marine Novosphingobium converts the stereoisomers of β-O-4 lignin model dimers into the respective monomers

PubMed Central

Ohta, Yukari; Nishi, Shinro; Hasegawa, Ryoichi; Hatada, Yuji

2015-01-01

Lignin, an aromatic polymer of phenylpropane units joined predominantly by β-O-4 linkages, is the second most abundant biomass component on Earth. Despite the continuous discharge of terrestrially produced lignin into marine environments, few studies have examined lignin degradation by marine microorganisms. Here, we screened marine isolates for β-O-4 cleavage activity and determined the genes responsible for this enzymatic activity in one positive isolate. Novosphingobium sp. strain MBES04 converted all four stereoisomers of guaiacylglycerol-β-guaiacyl ether (GGGE), a structural mimic of lignin, to guaiacylhydroxypropanone as an end metabolite in three steps involving six enzymes, including a newly identified Nu-class glutathione-S-transferase (GST). In silico searches of the strain MBES04 genome revealed that four GGGE-metabolizing GST genes were arranged in a cluster. Transcriptome analysis demonstrated that the lignin model compounds GGGE and (2-methoxyphenoxy)hydroxypropiovanillone (MPHPV) enhanced the expression of genes in involved in energy metabolism, including aromatic-monomer assimilation, and evoked defense responses typically expressed upon exposure to toxic compounds. The findings from this study provide insight into previously unidentified bacterial enzymatic systems and the physiological acclimation of microbes associated with the biological transformation of lignin-containing materials in marine environments. PMID:26477321

The N Domain of Human Angiotensin-I-converting Enzyme

PubMed Central

Anthony, Colin S.; Corradi, Hazel R.; Schwager, Sylva L. U.; Redelinghuys, Pierre; Georgiadis, Dimitris; Dive, Vincent; Acharya, K. Ravi; Sturrock, Edward D.

2010-01-01

Angiotensin-I-converting enzyme (ACE) plays a critical role in the regulation of blood pressure through its central role in the renin-angiotensin and kallikrein-kinin systems. ACE contains two domains, the N and C domains, both of which are heavily glycosylated. Structural studies of ACE have been fraught with severe difficulties because of surface glycosylation of the protein. In order to investigate the role of glycosylation in the N domain and to create suitable forms for crystallization, we have investigated the importance of the 10 potential N-linked glycan sites using enzymatic deglycosylation, limited proteolysis, and mass spectrometry. A number of glycosylation mutants were generated via site-directed mutagenesis, expressed in CHO cells, and analyzed for enzymatic activity and thermal stability. At least eight of 10 of the potential glycan sites are glycosylated; three C-terminal sites were sufficient for expression of active N domain, whereas two N-terminal sites are important for its thermal stability. The minimally glycosylated Ndom389 construct was highly suitable for crystallization studies. The structure in the presence of an N domain-selective phosphinic inhibitor RXP407 was determined to 2.0 Å resolution. The Ndom389 structure revealed a hinge region that may contribute to the breathing motion proposed for substrate binding. PMID:20826823

Activating Intrinsic Carbohydrate-Active Enzymes of the Smut Fungus Ustilago maydis for the Degradation of Plant Cell Wall Components

PubMed Central

Geiser, Elena; Reindl, Michèle; Blank, Lars M.; Feldbrügge, Michael

2016-01-01

ABSTRACT The microbial conversion of plant biomass to valuable products in a consolidated bioprocess could greatly increase the ecologic and economic impact of a biorefinery. Current strategies for hydrolyzing plant material mostly rely on the external application of carbohydrate-active enzymes (CAZymes). Alternatively, production organisms can be engineered to secrete CAZymes to reduce the reliance on externally added enzymes. Plant-pathogenic fungi have a vast repertoire of hydrolytic enzymes to sustain their lifestyle, but expression of the corresponding genes is usually highly regulated and restricted to the pathogenic phase. Here, we present a new strategy in using the biotrophic smut fungus Ustilago maydis for the degradation of plant cell wall components by activating its intrinsic enzyme potential during axenic growth. This fungal model organism is fully equipped with hydrolytic enzymes, and moreover, it naturally produces value-added substances, such as organic acids and biosurfactants. To achieve the deregulated expression of hydrolytic enzymes during the industrially relevant yeast-like growth in axenic culture, the native promoters of the respective genes were replaced by constitutively active synthetic promoters. This led to an enhanced conversion of xylan, cellobiose, and carboxymethyl cellulose to fermentable sugars. Moreover, a combination of strains with activated endoglucanase and β-glucanase increased the release of glucose from carboxymethyl cellulose and regenerated amorphous cellulose, suggesting that mixed cultivations could be a means for degrading more complex substrates in the future. In summary, this proof of principle demonstrates the potential applicability of activating the expression of native CAZymes from phytopathogens in a biocatalytic process. IMPORTANCE This study describes basic experiments that aim at the degradation of plant cell wall components by the smut fungus Ustilago maydis. As a plant pathogen, this fungus contains a set of lignocellulose-degrading enzymes that may be suited for biomass degradation. However, its hydrolytic enzymes are specifically expressed only during plant infection. Here, we provide the proof of principle that these intrinsic enzymes can be synthetically activated during the industrially relevant yeast-like growth. The fungus is known to naturally synthesize valuable compounds, such as itaconate or glycolipids. Therefore, it could be suited for use in a consolidated bioprocess in which more complex and natural substrates are simultaneously converted to fermentable sugars and to value-added compounds in the future. PMID:27316952

The enzyme lecithin-cholesterol acyltransferase esterifies cerebrosterol and limits the toxic effect of this oxysterol on SH-SY5Y cells.

PubMed

La Marca, Valeria; Spagnuolo, Maria Stefania; Cigliano, Luisa; Marasco, Daniela; Abrescia, Paolo

2014-07-01

Cholesterol is mostly removed from the CNS by its conversion to cerebrosterol (24(S)-hydroxycholesterol, 24(S)OH-C), which is transported to the circulation for bile formation in liver. A neurotoxic role of this oxysterol was previously demonstrated in cell culture. Here, we provide evidence that the enzyme lecithin-cholesterol acyltransferase, long known to esterify cholesterol, also produces monoesters of 24(S)OH-C. Proteoliposomes containing apolipoprotein A-I or apolipoprotein E were used to stimulate the enzyme activity and entrap the formed esters. Proteoliposomes with apolipoprotein A-I were found to be more active than those with apolipoprotein E in stimulating the production of oxysteryl esters. Cholesterol and 24(S)OH-C were found to compete for enzyme activity. High levels of haptoglobin, as those circulating during the acute inflammatory phase, inhibited 24(S)OH-C esterification. When highly neurotoxic 24(S)OH-C was treated with enzyme and proteoliposomes before incubation with differentiated SH-SY5Y cells, the neuron survival improved. The esters of 24(S)OH-C, embedded into proteoliposomes by the enzyme and isolated from unesterified 24(S)OH-C by gel filtration chromatography, did not enter the neurons in culture. These results suggest that the enzyme, in the presence of the apolipoproteins, converts 24(S)OH-C into esters restricted to the extracellular environment, thus preventing or limiting oxysterol-induced neurotoxic injuries to neurons in culture. 24-hydroxycholesterol (24(S)OH-C) is neurotoxic. The enzyme lecithin-cholesterol acyltransferase (LCAT) synthesizes monoesters of 24(S)OH-C in reaction mixtures with proteoliposomes containing phospholipids and apolipoprotein A-I or apolipoprotein E. The esters, also produced by incubation of cerebrospinal fluid only with tritiated 24(S)OH-C, are embedded into lipoproteins that do not enter neurons in culture. The enzyme activity limits the toxicity of 24-hydroxycholesterol in neuron culture. © 2014 International Society for Neurochemistry.

Real-time investigation of human topoisomerase I reaction kinetics using an optical sensor: a fast method for drug screening and determination of active enzyme concentrations

NASA Astrophysics Data System (ADS)

Kristoffersen, Emil L.; Jørgensen, Line A.; Franch, Oskar; Etzerodt, Michael; Frøhlich, Rikke; Bjergbæk, Lotte; Stougaard, Magnus; Ho, Yi-Ping; Knudsen, Birgitta R.

2015-05-01

Human DNA topoisomerase I (hTopI) is a nuclear enzyme that catalyzes relaxation of super helical tension that arises in the genome during essential DNA metabolic processes. This is accomplished through a common reaction mechanism shared among the type IB topoisomerase enzymes, including eukaryotic and poxvirus topoisomerase I. The mechanism of hTopI is specifically targeted in cancer treatment using camptothecin derivatives. These drugs convert the hTopI activity into a cellular poison, and hence the cytotoxic effects of camptothecin derivatives correlate with the hTopI activity. Therefore, fast and reliable techniques for high throughput measurements of hTopI activity are of high clinical interest. Here we demonstrate potential applications of a fluorophore-quencher based DNA sensor designed for measurement of hTopI cleavage-ligation activities, which are the catalytic steps affected by camptothecin. The kinetic analysis of the hTopI reaction with the DNA sensor exhibits a characteristic burst profile. This is the result of a two-step ping-pong reaction mechanism, where a fast first reaction, the one creating the signal, is followed by a slower second reaction necessary for completion of the catalytic cycle. Hence, the burst profile holds information about two reactions in the enzymatic mechanism. Moreover, it allows the amount of active enzyme in the reaction to be determined. The presented results pave the way for future high throughput drug screening and the potential of measuring active hTopI concentrations in clinical samples for individualized treatment.Human DNA topoisomerase I (hTopI) is a nuclear enzyme that catalyzes relaxation of super helical tension that arises in the genome during essential DNA metabolic processes. This is accomplished through a common reaction mechanism shared among the type IB topoisomerase enzymes, including eukaryotic and poxvirus topoisomerase I. The mechanism of hTopI is specifically targeted in cancer treatment using camptothecin derivatives. These drugs convert the hTopI activity into a cellular poison, and hence the cytotoxic effects of camptothecin derivatives correlate with the hTopI activity. Therefore, fast and reliable techniques for high throughput measurements of hTopI activity are of high clinical interest. Here we demonstrate potential applications of a fluorophore-quencher based DNA sensor designed for measurement of hTopI cleavage-ligation activities, which are the catalytic steps affected by camptothecin. The kinetic analysis of the hTopI reaction with the DNA sensor exhibits a characteristic burst profile. This is the result of a two-step ping-pong reaction mechanism, where a fast first reaction, the one creating the signal, is followed by a slower second reaction necessary for completion of the catalytic cycle. Hence, the burst profile holds information about two reactions in the enzymatic mechanism. Moreover, it allows the amount of active enzyme in the reaction to be determined. The presented results pave the way for future high throughput drug screening and the potential of measuring active hTopI concentrations in clinical samples for individualized treatment. Electronic supplementary information (ESI) available. See DOI: 10.1039/c5nr01474c

The allosteric activator ATP induces a substrate-dependent alteration of the quaternary structure of a mutant aspartate transcarbamoylase impaired in active site closure.

PubMed Central

Baker, D. P.; Fetler, L.; Vachette, P.; Kantrowitz, E. R.

1996-01-01

Aspartate transcarbamoylase from Escherichia coli shows homotropic cooperativity for aspartate as well as heterotropic regulation by nucleotides. Structurally, it consists of two trimeric catalytic subunits and three dimeric regulatory subunits, each chain being comprised of two domains. Glu-50 and Ser-171 are involved in stabilizing the closed conformation of the catalytic chain. Replacement of Glu-50 or Ser-171 by Ala in the holoenzyme has been shown previously to result in marked decreases in the maximal observed specific activity, homotropic cooperativity, and affinity for aspartate (Dembowski NJ, Newton CJ, Kantrowitz ER, 1990, Biochemistry 29:3716-3723; Newton CJ, Kantrowitz ER, 1990, Biochemistry 29:1444-1451). We have constructed a double mutant enzyme combining both mutations. The resulting Glu-50/ser-171-->Ala enzyme is 9-fold less active than the Ser-171-->Ala enzyme, 69-fold less active than the Glu-50-->Ala enzyme, and shows 1.3-fold and 1.6-fold increases in the [S]0.5Asp as compared to the Ser-171-->Ala and Glu-50-->Ala enzymes, respectively. However, the double mutant enzyme exhibits some enhancement of homotropic cooperativity with respect to aspartate, relative to the single mutant enzymes. At subsaturating concentrations of aspartate, the Glu-50/Ser-171 -->Ala enzyme is activated less by ATP than either the Glu-50-->Ala or Ser-171-->Ala enzyme, whereas CTP inhibition is intermediate between that of the two single mutants. As opposed to the wild-type enzyme, the Glu-50/Ser-171 -->Ala enzyme is activated by ATP and inhibited by CTP at saturating concentrations of aspartate. Structural analysis of the Ser-171-->Ala and Glu-50/Ser-171-->Ala enzymes by solution X-ray scattering indicates that both mutants exist in the same T quaternary structure as the wild-type enzyme in the absence of ligands, and in the same R quaternary structure in the presence of saturating N-(phosphonoacetyl)-L-aspartate. However, saturating concentrations of carbamoyl phosphate and succinate are unable to convert a significant fraction of either mutant enzyme population to the R quaternary structure, as has been observed previously for the Glu-50-->Ala enzyme. The curves for both the Ser-171-->Ala and Glu-50/Ser-171-->Ala enzymes obtained in the presence of substoichiometric amounts of PALA are linear combinations of the two extreme T and R states. The structural consequences of nucleotide binding to these two enzymes were also investigated. Most surprisingly, the direction and amplitude of the effect of ATP upon the double mutant enzyme were shown to vary depending upon the substrate analogue used. PMID:8931146

In vivo synergistic activity of a CAZyme cassette from Acidothermus cellulolyticus significantly improves the cellulolytic activity of the C. bescii exoproteome

DOE PAGES

Kim, Sun -Ki; Chung, Daehwan; Himmel, Michael E.; ...

2017-06-26

The use of microbial cells to convert plant biomass directly to fuels and chemicals is referred to as consolidated bioprocessing (CBP). Members of the bacterial genus, Caldicellulosiruptor (Gram-positive, anaerobic hyperthermophiles) are capable of deconstructing plant biomass without enzymatic or chemical pretreatment. This is accomplished by the production and secretion of free, multi-domain enzymes that outperform commercial enzyme cocktails on some substrates. Here, we show that the exoproteome of C. bescii may be enhanced by the heterologous expression of enzymes from Acidothermus cellulolyticus that act synergistically to improve sugar release from complex substrates; as well as improve cell growth. In thismore » work, co-expression of the A. cellulolyticus Acel_0615 ..beta..-glucanase (GH6 and GH12) and E1 endoglucanase (GH5) enzymes resulted in an increase in the activity of the exoproteome on Avicel; as well as an increase in growth of C. bescii on Avicel compared to the parental strain or the strain expressing the ..beta..-glucanase alone. As a result, our ability to engineer the composition and effectiveness of the exoproteome of these bacteria provides insight into the natural mechanism of plant cell wall deconstruction, as well as future directions for improving CBP.« less

In vivo synergistic activity of a CAZyme cassette from Acidothermus cellulolyticus significantly improves the cellulolytic activity of the C. bescii exoproteome

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kim, Sun -Ki; Chung, Daehwan; Himmel, Michael E.

The use of microbial cells to convert plant biomass directly to fuels and chemicals is referred to as consolidated bioprocessing (CBP). Members of the bacterial genus, Caldicellulosiruptor (Gram-positive, anaerobic hyperthermophiles) are capable of deconstructing plant biomass without enzymatic or chemical pretreatment. This is accomplished by the production and secretion of free, multi-domain enzymes that outperform commercial enzyme cocktails on some substrates. Here, we show that the exoproteome of C. bescii may be enhanced by the heterologous expression of enzymes from Acidothermus cellulolyticus that act synergistically to improve sugar release from complex substrates; as well as improve cell growth. In thismore » work, co-expression of the A. cellulolyticus Acel_0615 ..beta..-glucanase (GH6 and GH12) and E1 endoglucanase (GH5) enzymes resulted in an increase in the activity of the exoproteome on Avicel; as well as an increase in growth of C. bescii on Avicel compared to the parental strain or the strain expressing the ..beta..-glucanase alone. As a result, our ability to engineer the composition and effectiveness of the exoproteome of these bacteria provides insight into the natural mechanism of plant cell wall deconstruction, as well as future directions for improving CBP.« less

Characterization of the 4,6-α-glucanotransferase GTFB enzyme of Lactobacillus reuteri 121 isolated from inclusion bodies.

PubMed

Bai, Yuxiang; van der Kaaij, Rachel Maria; Woortman, Albert Jan Jacob; Jin, Zhengyu; Dijkhuizen, Lubbert

2015-06-09

The GTFB enzyme of the probiotic bacterium Lactobacillus reuteri 121 is a 4,6-α-glucanotransferase of glycoside hydrolase family 70 (GH70; http://www.cazy.org ). Contrary to the glucansucrases in GH70, GTFB is unable to use sucrose as substrate, but instead converts malto-oligosaccharides and starch into isomalto-/malto- polymers that may find application as prebiotics and dietary fibers. The GTFB enzyme expresses well in Escherichia coli BL21 Star (DE3), but mostly accumulates in inclusion bodies (IBs) which generally contain wrongly folded protein and inactive enzyme. Denaturation followed by refolding, as well as ncIB preparation were used for isolation of active GTFB protein from inclusion bodies. Soluble, refolded and ncIB GTFB were compared using activity assays, secondary structure analysis by FT-IR, and product analyses by NMR, HPAEC and SEC. Expression of GTFB in E. coli yielded > 100 mg/l relatively pure and active but mostly insoluble GTFB protein in IBs, regardless of the expression conditions used. Following denaturing, refolding of GTFB protein was most efficient in double distilled H2O. Also, GTFB ncIBs were active, with approx. 10 % of hydrolysis activity compared to the soluble protein. When expressed as units of activity obtained per liter E. coli culture, the total amount of ncIB GTFB expressed possessed around 180 % hydrolysis activity and 100 % transferase activity compared to the amount of soluble GTFB enzyme obtained from one liter culture. The product profiles obtained for the three GTFB enzyme preparations were similar when analyzed by HPAEC and NMR. SEC investigation also showed that these 3 enzyme preparations yielded products with similar size distributions. FT-IR analysis revealed extended β-sheet formation in ncIB GTFB providing an explanation at the molecular level for reduced GTFB activity in ncIBs. The thermostability of ncIB GTFB was relatively high compared to the soluble and refolded GTFB. In view of their relatively high yield, activity and high thermostability, both refolded and ncIB GTFB derived from IBs in E. coli may find industrial application in the synthesis of modified starches.

21 CFR 184.1388 - Lactase enzyme preparation from Kluyveromyces lactis.

Code of Federal Regulations, 2014 CFR

2014-04-01

...) This enzyme preparation is derived from the nonpathogenic, nontoxicogenic yeast Kluyveromyces lactis... 683), which converts lactose to glucose and galactose. It is prepared from yeast that has been grown...

Manganese(IV) Oxide Production by Acremonium sp. Strain KR21-2 and Extracellular Mn(II) Oxidase Activity

PubMed Central

Miyata, Naoyuki; Tani, Yukinori; Maruo, Kanako; Tsuno, Hiroshi; Sakata, Masahiro; Iwahori, Keisuke

2006-01-01

Ascomycetes that can deposit Mn(III, IV) oxides are widespread in aquatic and soil environments, yet the mechanism(s) involved in Mn oxide deposition remains unclear. A Mn(II)-oxidizing ascomycete, Acremonium sp. strain KR21-2, produced a Mn oxide phase with filamentous nanostructures. X-ray absorption near-edge structure (XANES) spectroscopy showed that the Mn phase was primarily Mn(IV). We purified to homogeneity a laccase-like enzyme with Mn(II) oxidase activity from cultures of strain KR21-2. The purified enzyme oxidized Mn(II) to yield suspended Mn particles; XANES spectra indicated that Mn(II) had been converted to Mn(IV). The pH optimum for Mn(II) oxidation was 7.0, and the apparent half-saturation constant was 0.20 mM. The enzyme oxidized ABTS [2,2′-azinobis(3-ethylbenzothiazoline-6-sulfonic acid)] (pH optimum, 5.5; Km, 1.2 mM) and contained two copper atoms per molecule. Moreover, the N-terminal amino acid sequence (residues 3 to 25) was 61% identical with the corresponding sequence of an Acremonium polyphenol oxidase and 57% identical with that of a Myrothecium bilirubin oxidase. These results provide the first evidence that a fungal multicopper oxidase can convert Mn(II) to Mn(IV) oxide. The present study reinforces the notion of the contribution of multicopper oxidase to microbially mediated precipitation of Mn oxides and suggests that Acremonium sp. strain KR21-2 is a good model for understanding the oxidation of Mn in diverse ascomycetes. PMID:17021194

Introduction to the Glutamate-Glutamine Cycle.

PubMed

Sonnewald, Ursula; Schousboe, Arne

2016-01-01

The term 'glutamate-glutamine cycle' was coined several decades ago based on the observation that using certain 14 C-labeled precursors for studies of brain metabolism the specific radioactivity of glutamine generated from glutamate was higher than that of glutamate, its immediate precursor. This is metabolically impossible unless it is assumed that at least two distinct pools of these amino acids exist. This combined with the finding that the enzyme synthesizing glutamine from glutamate was expressed in astrocytes but not in neurons formed the basis of the notion that a cycle must exist in which glutamate released from neurons is transported into astrocytes, converted to glutamine which is subsequently returned to neurons and converted to glutamate by an enzyme the activity of which is much higher in neurons than in astrocytes. Originally this cycle was supposed to function in a stoichiometric fashion but more recent research has seriously questioned this.This volume of Advances in Neurobiology is intended to provide a detailed discussion of recent developments in research aimed at delineating the functional roles of the cycle taking into account that in order for this system to work there must be a tight coupling between metabolism of glutamate in astrocytes, transfer of glutamine to neurons and de novo synthesis of glutamine in astrocytes. To understand this, knowledge about the activity and regulation of the enzymes and transporters involved in these processes is required and as can be seen from the table of contents these issues will be dealt with in detail in the individual chapters of the book.

ANGIOTENSIN CONVERTING ENZYME INHIBITION AND NOVEL CARDIOVASCULAR RISK BIOMARKERS

PubMed Central

Cesari, Matteo; Kritchevsky, Stephen B.; Atkinson, Hal H.; Penninx, Brenda W.; Di Bari, Mauro; Tracy, Russell P.; Pahor, Marco

2015-01-01

Background Beneficial effects of angiotensin converting enzyme (ACE) inhibitors seem to be mediated by mechanisms that are partly independent of blood pressure lowering. The present study evaluates effects of an ACE-inhibitor (i.e. fosinopril) intervention on novel cardiovascular risk factors. Methods Data are from the Trial of Angiotensin Converting Enzyme Inhibition and Novel Cardiovascular Risk Factors (TRAIN), a double-blind, crossover, randomized, placebo-controlled trial enrolling subjects aged ≥55 years and older with high cardiovascular disease risk profile. Biomarkers of hemostasis (i.e. plasminogen activator inhibitor-1 [PAI-1], D-dimer), inflammation (i.e. C-reactive protein [CRP], interleukin-6 [IL-6]), and endothelial function (i.e. endothelin-1, vascular cell adhesion molecule-1 [VCAM-1]) were measured at the baseline, at the mid-term, and at end of follow-up (after one year) clinic visits. Paired t-test analyses (after Sidak’s adjustment, p value<0.009) were performed to compare biomarkers modifications after fosinopril/placebo interventions. Results Mean age of the sample (n=290, women 43.4%) was 66.0 years old. No significant differences were reported for CRP, IL-6, PAI-1, VCAM-1, and endothelin-1 levels in the comparisons between fosinopril and placebo interventions. D-Dimer was the only biomarker showing a significant difference between fosinopril intervention (median 0.32 [interquartile range, IQR 0.22–0.52] µg/mL) and placebo (median 0.29 [IQR 0.20–0.47] µg/mL, p=0.007) when analyses were restricted to participants with higher compliance to treatment and receiving the maximum ACE-inhibitor dosage. Conclusions ACE-inhibition does not significantly modify major biomarkers of inflammation, hemostasis, and endothelial function. Further studies should confirm the possible effect of ACE-inhibitors on the fibrinolysis pathway. PMID:19185642

Isolation of 2-deoxy-scyllo-inosose (DOI)-assimilating yeasts and cloning and characterization of the DOI reductase gene of Cryptococcus podzolicus ND1.

PubMed

Ara, Satoshi; Yamazaki, Harutake; Takaku, Hiroaki

2018-04-01

2-Deoxy-scyllo-inosose (DOI) is the first intermediate in the 2-deoxystreptamine-containing aminoglycoside antibiotic biosynthesis pathway and has a six-membered carbocycle structure. DOI is a valuable material because it is easily converted to aromatic compounds and carbasugar derivatives. In this study, we isolated yeast strains capable of assimilating DOI as a carbon source. One of the strains, Cryptococcus podzolicus ND1, mainly converted DOI to scyllo-quercitol and (-)-vibo-quercitol, which is a valuable compound used as an antihypoglycemia agent and as a heat storage material. An NADH-dependent DOI reductase coding gene, DOIR, from C. podzolicus ND1 was cloned and successfully overexpressed in Escherichia coli. The purified protein catalyzed the irreversible reduction of DOI with NADH and converted DOI into (-)-vibo-quercitol. The enzyme had an optimal pH of 8.5 and optimal temperature of 35°C, respectively. The k cat of this enzyme was 9.98 s -1 , and the K m values for DOI and NADH were 4.38 and 0.24 mM, respectively. The enzyme showed a strong preference for NADH and showed no activity with NADPH. Multiple-alignment analysis of DOI reductase revealed that it belongs to the GFO_IDH_MocA protein family and is an inositol dehydrogenase homolog in other fungi, such as Cryptococcus gattii, and bacteria, such as Bacillus subtilis. This is the first identification of a DOI-assimilating yeast and a gene involved in DOI metabolism in fungi. Copyright © 2017 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

Association of angiotensin-converting enzyme (ACE) gene polymorphism with elevated serum ACE activity and major depression in an Iranian population.

PubMed

Firouzabadi, Negar; Shafiei, Massoumeh; Bahramali, Ehsan; Ebrahimi, Soltan Ahmed; Bakhshandeh, Hooman; Tajik, Nader

2012-12-30

Genetic factors contribute substantially to the likelihood of developing major depressive disorder (MDD). The importance of renin-angiotensin system (RAS) elements in cognition and behaviour and their involvement in aetiology and treatment of depression imply that RAS gene polymorphism(s) associated with RAS overactivity might also be associated with depression. In the present study, genotype and allele frequencies of six common polymorphisms of genes encoding for RAS components were determined in DNAs extracted from venous blood of 191 depressed and 104 healthy individuals using polymerase chain reaction (PCR) and polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) and serum angiotensin-converting enzyme (ACE) activity was assayed using a high-performance liquid chromatography (HPLC) method. The results showed, for the first time, that GG genotype of ACE A2350G was significantly associated with MDD among Iranian participants (P=0.001; odds ratio (OR)=6.2; 95% confidence interval (CI)=2.1-18.3). Significant higher serum ACE activity (P=0.0001) as well as higher diastolic blood pressure (P=0.036) were observed in depressed patients compared to the healthy control group. Depressed patients carrying GG genotype of the A2350G polymorphism had a significantly higher serum ACE activity (P=0.02) than individuals with either AA or AG genotype. In conclusion, this study supports the hypothesis of RAS overactivity in depression in that the genotype associated with higher serum ACE activity in an Iranian population was also associated with MDD. Copyright © 2012 Elsevier Ireland Ltd. All rights reserved.

A Novel Angiotensin I-Converting Enzyme Mutation (S333W) Impairs N-Domain Enzymatic Cleavage of the Anti-Fibrotic Peptide, AcSDKP

PubMed Central

Danilov, Sergei M.; Wade, Michael S.; Schwager, Sylva L.; Douglas, Ross G.; Nesterovitch, Andrew B.; Popova, Isolda A.; Hogarth, Kyle D.; Bhardwaj, Nakul; Schwartz, David E.; Sturrock, Edward D.; Garcia, Joe G. N.

2014-01-01

Background Angiotensin I-converting enzyme (ACE) has two functional N- and C-domain active centers that display differences in the metabolism of biologically-active peptides including the hemoregulatory tetrapeptide, Ac-SDKP, hydrolysed preferentially by the N domain active center. Elevated Ac-SDKP concentrations are associated with reduced tissue fibrosis. Results We identified a patient of African descent exhibiting unusual blood ACE kinetics with reduced relative hydrolysis of two synthetic ACE substrates (ZPHL/HHL ratio) suggestive of the ACE N domain center inactivation. Inhibition of blood ACE activity by anti-catalytic mAbs and ACE inhibitors and conformational fingerprint of blood ACE suggested overall conformational changes in the ACE molecule and sequencing identified Ser333Trp substitution in the N domain of ACE. In silico analysis demonstrated S333W localized in the S1 pocket of the active site of the N domain with the bulky Trp adversely affecting binding of ACE substrates due to steric hindrance. Expression of mutant ACE (S333W) in CHO cells confirmed altered kinetic properties of mutant ACE and conformational changes in the N domain. Further, the S333W mutant displayed decreased ability (5-fold) to cleave the physiological substrate AcSDKP compared to wild-type ACE. Conclusions and Significance A novel Ser333Trp ACE mutation results in dramatic changes in ACE kinetic properties and lowered clearance of Ac-SDKP. Individuals with this mutation (likely with significantly increased levels of the hemoregulatory tetrapeptide in blood and tissues), may confer protection against fibrosis. PMID:24505347

Serum and tissue angiotensin-converting enzyme in patients with alopecia areata.

PubMed

Fahim, Shabnam; Montazer, Fatemeh; Tohidinik, Hamid Reza; Naraghi, Zahra Safaei; Abedini, Robabeh; Nasimi, Maryam; Ghandi, Narges

2018-03-27

Alopecia areata is an immune-dependent disorder characterized by the interaction of T-lymphocytes with follicular antigens. Recent studies have shown the existence of a local renin-angiotensin system in the skin, where angiotensin-converting enzyme (ACE) plays a role in autoimmunity and inflammation. The objective of this study was to evaluate serum and tissue ACE activity in patients with alopecia areata. This case-control study was conducted on patients with alopecia areata and healthy controls. Serum and tissue ACE activity were assessed and compared between the two groups. Twenty-five alopecia areata patients (60% male, mean age 32.1 ± 9.9 years) and 24 controls (50% male, mean age 37.4 ± 8.8 years) were included. Mean serum ACE activity was 52.1 ± 9 U/L in cases and 55.3 ± 14.7 U/L in controls (P = 0.37). Tissue ACE activity was significantly lower in cases in all parts of the skin i.e. epidermis (P = 0.016), follicular epithelium (P = 0.004), and endothelium (P = 0.037). Among cases, serum ACE activity was significantly higher in patients with more severe disease (P = 0.030), nonpatchy alopecia areata (alopecia universalis; ophiasis, patchy and ophiasis, diffuse) (P = 0.029), and with nail involvement (P = 0.027). The sample size was too small to draw definite conclusions. Further, most of the patients had only mild or moderate alopecia areata. Unlike in some other inflammatory diseases, the tissue level of ACE seems to be significantly lower in alopecia areata compared to normal controls. Serum ACE was significantly higher in patients with more severe disease.

Depletion of angiotensin-converting enzyme 2 reduces brain serotonin and impairs the running-induced neurogenic response.

PubMed

Klempin, Friederike; Mosienko, Valentina; Matthes, Susann; Villela, Daniel C; Todiras, Mihail; Penninger, Josef M; Bader, Michael; Santos, Robson A S; Alenina, Natalia

2018-04-20

Physical exercise induces cell proliferation in the adult hippocampus in rodents. Serotonin (5-HT) and angiotensin (Ang) II are important mediators of the pro-mitotic effect of physical activity. Here, we examine precursor cells in the adult brain of mice lacking angiotensin-converting enzyme (ACE) 2, and explore the effect of an acute running stimulus on neurogenesis. ACE2 metabolizes Ang II to Ang-(1-7) and is essential for the intestinal uptake of tryptophan (Trp), the 5-HT precursor. In ACE2-deficient mice, we observed a decrease in brain 5-HT levels and no increase in the number of BrdU-positive cells following exercise. Targeting the Ang II/AT1 axis by blocking the receptor, or experimentally increasing Trp/5-HT levels in the brain of ACE2-deficient mice, did not rescue the running-induced effect. Furthermore, mice lacking the Ang-(1-7) receptor, Mas, presented a normal neurogenic response to exercise. Our results identify ACE2 as a novel factor required for exercise-dependent modulation of adult neurogenesis and essential for 5-HT metabolism.

The angiotensin-converting-enzyme insertion/deletion polymorphism is not related to venous thrombosis.

PubMed

Köppel, Herwig; Renner, Wilfried; Gugl, Alexander; Cichocki, Lisa; Gasser, Robert; Wascher, Thomas C; Pilger, Ernst

2004-01-01

The insertion/deletion (I/D) polymorphism of the gene for angiotensin-converting-enzyme (ACE) is associated with ACE plasma levels and activity. Conflicting results have been reported about the relevance of this polymorphism for venous thrombosis. The aim of the present study was to analyze the role of this polymorphism for deep venous thrombosis. The study was designed as a case-control study, including 330 patients with documented deep venous thrombosis and 354 controls. ACE genotype was determined by size-analysis of polymerase chain reaction products. Results showed that, ACE genotype frequencies were similar between patients (II: 24.8%; ID: 43.3%; DD: 31.8%) and controls (II: 22.9%; ID: 50.6%; DD: 26.6%, P = 0.15). The adjusted odds ratio of carriers of the DD geno-type for venous thrombosis was 1.24 (95% confidence interval 0.90-1.80). The polymorphism was furthermore not associated with age at first thromboembolic event or the occurrence of pulmonary embolism. From these results, we can conclude that the ACE I/D polymorphism is not a significant risk factor for deep venous thrombosis.

Metabolism of captopril carboxyl ester derivatives for percutaneous absorption.

PubMed

Gullick, Darren R; Ingram, Matthew J; Pugh, W John; Cox, Paul A; Gard, Paul; Smart, John D; Moss, Gary P

2009-02-01

To determine the metabolism of captopril n-carboxyl derivatives and how this may impact on their use as transdermal prodrugs. The pharmacological activity of the ester derivatives was also characterised in order to compare the angiotensin converting enzyme inhibitory potency of the derivatives compared with the parent drug, captopril. The metabolism rates of the ester derivatives were determined in vitro (using porcine liver esterase and porcine ear skin) and in silico (using molecular modelling to investigate the potential to predict metabolism). Relatively slow pseudo first-order metabolism of the prodrugs was observed, with the ethyl ester displaying the highest rate of metabolism. A strong relationship was established between in-vitro methods, while in-silico methods support the use of in-vitro methods and highlight the potential of in-silico techniques to predict metabolism. All the prodrugs behaved as angiotensin converting enzyme inhibitors, with the methyl ester displaying optimum inhibition. In-vitro porcine liver esterase metabolism rates inform in-vitro skin rates well, and in-silico interaction energies relate well to both. Thus, in-silico methods may be developed that include interaction energies to predict metabolism rates.

Enzymes Required for Maltodextrin Catabolism in Enterococcus faecalis Exhibit Novel Activities

PubMed Central

Joyet, Philippe; Mokhtari, Abdelhamid; Riboulet-Bisson, Eliette; Blancato, Víctor S.; Espariz, Martin; Magni, Christian; Sauvageot, Nicolas

2017-01-01

ABSTRACT Maltose and maltodextrins are formed during the degradation of starch or glycogen. Maltodextrins are composed of a mixture of maltooligosaccharides formed by α-1,4- but also some α-1,6-linked glucosyl residues. The α-1,6-linked glucosyl residues are derived from branching points in the polysaccharides. In Enterococcus faecalis, maltotriose is mainly transported and phosphorylated by a phosphoenolpyruvate:carbohydrate phosphotransferase system. The formed maltotriose-6″-phosphate is intracellularly dephosphorylated by a specific phosphatase, MapP. In contrast, maltotetraose and longer maltooligosaccharides up to maltoheptaose are taken up without phosphorylation via the ATP binding cassette transporter MdxEFG-MsmX. We show that the maltose-producing maltodextrin hydrolase MmdH (GenBank accession no. EFT41964) in strain JH2-2 catalyzes the first catabolic step of α-1,4-linked maltooligosaccharides. The purified enzyme converts even-numbered α-1,4-linked maltooligosaccharides (maltotetraose, etc.) into maltose and odd-numbered (maltotriose, etc.) into maltose and glucose. Inactivation of mmdH therefore prevents the growth of E. faecalis on maltooligosaccharides ranging from maltotriose to maltoheptaose. Surprisingly, MmdH also functions as a maltogenic α-1,6-glucosidase, because it converts the maltotriose isomer isopanose into maltose and glucose. In addition, E. faecalis contains a glucose-producing α-1,6-specific maltodextrin hydrolase (GenBank accession no. EFT41963, renamed GmdH). This enzyme converts panose, another maltotriose isomer, into glucose and maltose. A gmdH mutant had therefore lost the capacity to grow on panose. The genes mmdH and gmdH are organized in an operon together with GenBank accession no. EFT41962 (renamed mmgT). Purified MmgT transfers glucosyl residues from one α-1,4-linked maltooligosaccharide molecule to another. For example, it catalyzes the disproportionation of maltotriose by transferring a glucosyl residue to another maltotriose molecule, thereby forming maltotetraose and maltose together with a small amount of maltopentaose. IMPORTANCE The utilization of maltodextrins by Enterococcus faecalis has been shown to increase the virulence of this nosocomial pathogen. However, little is known about how this organism catabolizes maltodextrins. We identified two enzymes involved in the metabolism of various α-1,4- and α-1,6-linked maltooligosaccharides. We found that one of them functions as a maltose-producing α-glucosidase with relaxed linkage specificity (α-1,4 and α-1,6) and exo- and endoglucosidase activities. A third enzyme, which resembles amylomaltase, exclusively transfers glucosyl residues from one maltooligosaccharide molecule to another. Similar enzymes are present in numerous other Firmicutes, such as streptococci and lactobacilli, suggesting that these organisms follow the same maltose degradation pathway as E. faecalis. PMID:28455338

Characterization of thermostable cellulase produced by Bacillus strains isolated from solid waste of carrageenan

NASA Astrophysics Data System (ADS)

Listyaningrum, N. P.; Sutrisno, A.; Wardani, A. K.

2018-03-01

Cellulase-producing bacteria was isolated from solid waste of carrageenan and identified as Bacillus licheniformis C55 by 16S rRNA sequencing. The optimum condition for cellulase production was obtained at pH and temperature of 8.0 and 50°C, respectively in a medium containing glucose as carbon source and 1.0% carboxymethyl cellulose (CMC) to stimulate the cellulase production. Most remarkably, the enzyme retained its relative activity over 50% after incubation at 50°C for 90 minutes. Substrate specificity suggested that the enzyme is an endoglucanase. The molecular mass of Bacillus licheniformis C55 crude cellulase was found about 18 kDa by SDS-PAGE analysis. This thermostable enzyme would facilitate development of more efficient and cost-effective forms of the process to convert lignocellulosic biomass into high-value products.

Top1 May Do More Than Relax DNA | Center for Cancer Research

Cancer.gov

Topoisomerase 1 (Top1) is an enzyme with a well known role in relaxing DNA supercoils by making reversible nicks in DNA. The ribonuclease (RNase) H class of enzymes is equally well known for removing ribonucleotides from hybrid duplex DNA when they are misincorporated during DNA replication. Recently, Shar-yin Huang, Ph.D., and Yves Pommier, M.D., Ph.D., in CCR’s Laboratory of Molecular Pharmacology teamed up with Sue Jinks-Robertson of Duke University’s Department of Molecular Genetics and Microbiology and Thomas Kunkel of the NIEHS, NIH to show that in yeast, Top1 can act like the RNase H class enzymes and convert misincorporated single ribonucleotides into irreversible single-strand breaks, an activity that produces deletion mutations.  They reported this discovery in Science.

Enzymes: A Workshop for Secondary School Students.

ERIC Educational Resources Information Center

Bering, C. Larry

1994-01-01

Describes the weekend science workshop on enzymes and includes several projects that involve students directly, parts of which can be incorporated into a traditional chemistry, biology, or physical science course at the secondary level. Subjects include catalysts and catalytic converters in cars, enzymes as consumer products and in industrial…

Proteomic analysis reveals large amounts of decomposition enzymes and major metabolic pathways involved in algicidal process of Trametes versicolor F21a.

PubMed

Gao, Xueyan; Wang, Congyan; Dai, Wei; Ren, Shenrong; Tao, Fang; He, Xingbing; Han, Guomin; Wang, Wei

2017-06-20

A recent algicidal mode indicates that fungal mycelia can wrap and eliminate almost all co-cultivated algal cells within a short time span. However, the underlying molecular mechanism is rarely understood. We applied proteomic analysis to investigate the algicidal process of Trametes versicolor F21a and identified 3,754 fungal proteins. Of these, 30 fungal enzymes with endo- or exoglycosidase activities such as β-1,3-glucanase, α-galactosidase, α-glucosidase, alginate lyase and chondroitin lyase were significantly up-regulated. These proteins belong to Glycoside Hydrolases, Auxiliary Activities, Carbohydrate Esterases and Polysaccharide Lyases, suggesting that these enzymes may degrade lipopolysaccharides, peptidoglycans and alginic acid of algal cells. Additionally, peptidase, exonuclease, manganese peroxidase and cytochrome c peroxidase, which decompose proteins and DNA or convert other small molecules of algal cells, could be other major decomposition enzymes. Gene Ontology and KEGG pathway enrichment analysis demonstrated that pyruvate metabolism and tricarboxylic acid cycle pathways play a critical role in response to adverse environment via increasing energy production to synthesize lytic enzymes or uptake molecules. Carbon metabolism, selenocompound metabolism, sulfur assimilation and metabolism, as well as several amino acid biosynthesis pathways could play vital roles in the synthesis of nutrients required by fungal mycelia.

Characterization of angiotensin-converting enzyme inhibitory activity of fermented milk produced by Lactobacillus helveticus.

PubMed

Chen, Yongfu; Li, Changkun; Xue, Jiangang; Kwok, Lai-yu; Yang, Jie; Zhang, Heping; Menghe, Bilige

2015-08-01

Hypertension affects up to 30% of the adult population in most countries. It is a known risk factor for cardiovascular diseases, including coronary heart disease, peripheral artery disease, and stroke. Owing to the increased health awareness of consumers, the application of angiotensin-converting enzyme (ACE)-inhibitory peptides produced by Lactobacillushelveticus to prevent or control high blood pressure has drawn wide attention. A total of 59 L. helveticus strains were isolated from traditional fermented dairy products and the ACE-inhibitory activity of the fermented milks produced with the isolated microorganisms was assayed. The ACE-inhibitory activity of 38 L. helveticus strains was more than 50%, and 3 strains (IMAU80872, IMAU80852, and IMAU80851) expressing the highest ACE-inhibitory activity were selected for further studies. Particularly, the gastrointestinal protease tolerance and thermostability of the ACE-inhibitory activity in the fermented milks were assessed. Based on these 2 criteria, IMAU80872 was found to be superior over the other 2 strains. Furthermore, IMAU80872 exhibited a high in vitro ACE-inhibitory activity at the following fermentation conditions: fermentation temperature at 40°C, inoculation concentration of 1×10(6) cfu/mL, and fermentation for 18h. Finally, by using ultra-performance liquid chromatography coupled with electrospray ionization quadrupole time-of-flight tandem mass spectrometry analysis, we observed changes of the metabolome along the milk fermentation process of IMAU80872. Furthermore, 6 peptides were identified, which might have ACE-inhibitory activity. In conclusion, we identified a novel ACE-inhibitory L. helveticus strain suitable for the production of fermented milk or other functional dairy products. Copyright © 2015 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.

Expanding the Enzyme Universe: Accessing Non-Natural Reactions by Mechanism-Guided Directed Evolution

PubMed Central

Renata, Hans; Wang, Z. Jane

2015-01-01

High selectivities and exquisite control over reaction outcomes entice chemists to use biocatalysts in organic synthesis. However, many useful reactions are not accessible because they are not in nature’s known repertoire. We will use this review to outline an evolutionary approach to engineering enzymes to catalyze reactions not found in nature. We begin with examples of how nature has discovered new catalytic functions and how such evolutionary progressions have been recapitulated in the laboratory starting from extant enzymes. We then examine non-native enzyme activities that have been discovered and exploited for chemical synthesis, emphasizing reactions that do not have natural counterparts. The new functions have mechanistic parallels to the native reaction mechanisms that often manifest as catalytic promiscuity and the ability to convert from one function to the other with minimal mutation. We present examples of how non-natural activities have been improved by directed evolution, mimicking the process used by nature to create new catalysts. Examples of new enzyme functions include epoxide opening reactions with non-natural nucleophiles catalyzed by a laboratory-evolved halohydrin dehalogenase, cyclopropanation and other carbene transfer reactions catalyzed by cytochrome P450 variants, and non-natural modes of cyclization by a modified terpene synthase. Lastly, we describe discoveries of non-native catalytic functions that may provide future opportunities for expanding the enzyme universe. PMID:25649694

Unusual 4-hydroxybenzaldehyde synthase activity from tissue cultures of the vanilla orchid Vanilla planifolia.

PubMed

Podstolski, Andrzej; Havkin-Frenkel, Daphna; Malinowski, Jacek; Blount, Jack W; Kourteva, Galina; Dixon, Richard A

2002-11-01

Tissue cultures of the vanilla orchid, Vanilla planifolia, produce the flavor compound vanillin (4-hydroxy-3-methoxybenzaldehyde) and vanillin precursors such as 4-hydroxybenzaldehyde. A constitutively expressed enzyme activity catalyzing chain shortening of a hydroxycinnamic acid, believed to be the first reaction specific for formation of vanilla flavor compounds, was identified in these cultures. The enzyme converts 4-coumaric acid non-oxidatively to 4-hydroxybenzaldehyde in the presence of a thiol reagent but with no co-factor requirement. Several forms of this 4-hydroxybenzaldehyde synthase (4HBS) were resolved and partially purified by a combination of hydrophobic interaction, ion exchange and gel filtration chromatography. These forms appear to be interconvertible. The unusual properties of the 4HBS, and its appearance in different protein fractions, raise questions as to its physiological role in vanillin biosynthesis in vivo.

Combinatorial Enzyme Design Probes Allostery and Cooperativity in the Trypsin Fold

DOE Office of Scientific and Technical Information (OSTI.GOV)

Page, Michael J.; Di Cera, Enrico; St. Louis-MED)

2010-06-14

Converting one enzyme into another is challenging due to the uneven distribution of important amino acids for function in both protein sequence and structure. We report a strategy for protein engineering allowing an organized mixing and matching of genetic material that leverages lower throughput with increased quality of screens. Our approach successfully tested the contribution of each surface-exposed loop in the trypsin fold alone and the cooperativity of their combinations towards building the substrate selectivity and Na{sup +}-dependent allosteric activation of the protease domain of human coagulation factor Xa into a bacterial trypsin. As the created proteases lack additional proteinmore » domains and protein co-factor activation mechanism requisite for the complexity of blood coagulation, they are stepping-stones towards further understanding and engineering of artificial clotting factors.« less

The effect of combination treatment with aliskiren and blockers of the renin-angiotensin system on hyperkalaemia and acute kidney injury: systematic review and meta-analysis

PubMed Central

Gilbert, Cameron; Wald, Ron; Bell, Chaim; Perl, Jeff; Juurlink, David; Beyene, Joseph; Shah, Prakesh S

2012-01-01

Objective To examine the safety of using aliskiren combined with agents used to block the renin-angiotensin system. Design Systematic review and meta-analysis of randomised controlled trials. Data sources Medline, Embase, the Cochrane Library, and two trial registries, published up to 7 May 2011. Study selection Published and unpublished randomised controlled trials that compared combined treatment using aliskiren and angiotensin converting enzyme inhibitors or angiotensin receptor blockers with monotherapy using these agents for at least four weeks and that provided numerical data on the adverse event outcomes of hyperkalaemia and acute kidney injury. A random effects model was used to calculate pooled risk ratios and 95% confidence intervals for these outcomes. Results 10 randomised controlled studies (4814 participants) were included in the analysis. Combination therapy with aliskiren and angiotensin converting enzyme inhibitors or angiotensin receptor blockers significantly increased the risk of hyperkalaemia compared with monotherapy using angiotensin converting enzymes or angiotensin receptor blockers (relative risk 1.58, 95% confidence interval 1.24 to 2.02) or aliskiren alone (1.67, 1.01 to 2.79). The risk of acute kidney injury did not differ significantly between the combined therapy and monotherapy groups (1.14, 0.68 to 1.89). Conclusion Use of aliskerin in combination with angiotensin converting enzyme inhibitors or angiotensin receptor blockers is associated with an increased risk for hyperkalaemia. The combined use of these agents warrants careful monitoring of serum potassium levels. PMID:22232539

EFFECT OF SYSTEMIC BETA-BLOCKERS, ACE INHIBITORS, AND ANGIOTENSIN RECEPTOR BLOCKERS ON DEVELOPMENT OF CHOROIDAL NEOVASCULARIZATION IN PATIENTS WITH AGE-RELATED MACULAR DEGENERATION.

PubMed

Thomas, Akshay S; Redd, Travis; Hwang, Thomas

2015-10-01

Recent studies have suggested that the use of systemic beta-blockers, angiotensin-converting enzyme inhibitors, and angiotensin receptor blockers can induce regression of choroidal neovascularization in rodent models. The purpose of this study is to evaluate if these agents have a protective effect against the development of choroidal neovascularization in patients with age-related macular degeneration. In this single-center retrospective case-control study, the charts of 250 patients with neovascular age-related macular degeneration were compared with those of 250 controls with dry age-related macular degeneration. Charts were reviewed for current and past use of beta-blockers, angiotensin-converting enzyme inhibitors, and angiotensin receptor blockers. Frequency tables were generated, and associations were examined using chi-square tests, t-tests, and multivariate logistic regression. There was no statistically significant difference between rates of beta-blocker use (P = 0.57), angiotensin-converting enzyme inhibitors use (P = 0.20), or angiotensin receptor blockers use (P = 0.61) between the 2 groups. Additionally, there was no statistically significant difference between rates of use of combinations of the above drugs between the two groups. Although there is growing evidence that beta-blockers, angiotensin-converting enzyme inhibitors, and angiotensin receptor blockers can induce regression of choroidal neovascularization in rodent models, these medications do not seem to confer a protective effect against the development of choroidal neovascularization in patients with age-related macular degeneration.

Non-specific activities of the major herbicide-resistance gene BAR.

PubMed

Christ, Bastien; Hochstrasser, Ramon; Guyer, Luzia; Francisco, Rita; Aubry, Sylvain; Hörtensteiner, Stefan; Weng, Jing-Ke

2017-12-01

Bialaphos resistance (BAR) and phosphinothricin acetyltransferase (PAT) genes, which convey resistance to the broad-spectrum herbicide phosphinothricin (also known as glufosinate) via N-acetylation, have been globally used in basic plant research and genetically engineered crops 1-4 . Although early in vitro enzyme assays showed that recombinant BAR and PAT exhibit substrate preference toward phosphinothricin over the 20 proteinogenic amino acids 1 , indirect effects of BAR-containing transgenes in planta, including modified amino acid levels, have been seen but without the identification of their direct causes 5,6 . Combining metabolomics, plant genetics and biochemical approaches, we show that transgenic BAR indeed converts two plant endogenous amino acids, aminoadipate and tryptophan, to their respective N-acetylated products in several plant species. We report the crystal structures of BAR, and further delineate structural basis for its substrate selectivity and catalytic mechanism. Through structure-guided protein engineering, we generated several BAR variants that display significantly reduced non-specific activities compared with its wild-type counterpart in vivo. The transgenic expression of enzymes can result in unintended off-target metabolism arising from enzyme promiscuity. Understanding such phenomena at the mechanistic level can facilitate the design of maximally insulated systems featuring heterologously expressed enzymes.

Efficient Conversion of Phenylpyruvic Acid to Phenyllactic Acid by Using Whole Cells of Bacillus coagulans SDM

PubMed Central

Zheng, Zhaojuan; Ma, Cuiqing; Gao, Chao; Li, Fengsong; Qin, Jiayang; Zhang, Haiwei; Wang, Kai; Xu, Ping

2011-01-01

Background Phenyllactic acid (PLA), a novel antimicrobial compound with broad and effective antimicrobial activity against both bacteria and fungi, can be produced by many microorganisms, especially lactic acid bacteria. However, the concentration and productivity of PLA have been low in previous studies. The enzymes responsible for conversion of phenylpyruvic acid (PPA) into PLA are equivocal. Methodology/Principal Findings A novel thermophilic strain, Bacillus coagulans SDM, was isolated for production of PLA. When the solubility and dissolution rate of PPA were enhanced at a high temperature, whole cells of B. coagulans SDM could effectively convert PPA into PLA at a high concentration (37.3 g l−1) and high productivity (2.3 g l−1 h−1) under optimal conditions. Enzyme activity staining and kinetic studies identified NAD-dependent lactate dehydrogenases as the key enzymes that reduced PPA to PLA. Conclusions/Significance Taking advantage of the thermophilic character of B. coagulans SDM, a high yield and productivity of PLA were obtained. The enzymes involved in PLA production were identified and characterized, which makes possible the rational design and construction of microorganisms suitable for PLA production with metabolic engineering. PMID:21533054

Photometric Characterization of the Reductive Amination Scope of the Imine Reductases from Streptomyces tsukubaensis and Streptomyces ipomoeae.

PubMed

Matzel, Philipp; Krautschick, Lukas; Höhne, Matthias

2017-10-18

Imine reductases (IREDs) have emerged as promising enzymes for the asymmetric synthesis of secondary and tertiary amines starting from carbonyl substrates. Screening the substrate specificity of the reductive amination reaction is usually performed by time-consuming GC analytics. We found two highly active IREDs in our enzyme collection, IR-20 from Streptomyces tsukubaensis and IR-Sip from Streptomyces ipomoeae, that allowed a comprehensive substrate screening with a photometric NADPH assay. We screened 39 carbonyl substrates combined with 17 amines as nucleophiles. Activity data from 663 combinations provided a clear picture about substrate specificity and capabilities in the reductive amination of these enzymes. Besides aliphatic aldehydes, the IREDs accepted various cyclic (C 4 -C 8 ) and acyclic ketones, preferentially with methylamine. IR-Sip also accepted a range of primary and secondary amines as nucleophiles. In biocatalytic reactions, IR-Sip converted (R)-3-methylcyclohexanone with dimethylamine or pyrrolidine with high diastereoselectivity (>94-96 % de). The nucleophile acceptor spectrum depended on the carbonyl substrate employed. The conversion of well-accepted substrates could also be detected if crude lysates were employed as the enzyme source. © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

STRUCTURAL AND FUNCTIONAL CONSEQUENCES OF CIRCULAR PERMUTATION ON THE ACTIVE SITE OF OLD YELLOW ENZYME.

PubMed

Daugherty, Ashley B; Horton, John R; Cheng, Xiaodong; Lutz, Stefan

2015-02-06

Circular permutation of the NADPH-dependent oxidoreductase Old Yellow Enzyme from Saccharomyces pastorianus (OYE1) can significantly enhance the enzyme's catalytic performance. Termini relocation into four regions of the protein (sectors I-IV) near the active site has proven effective in altering enzyme function. To better understand the structural consequences and rationalize the observed functional gains in these OYE1 variants, we selected representatives from sectors I-III for further characterization by biophysical methods and X-ray crystallography. These investigations not only show trends in enzyme stability and quaternary structure as a function of termini location, but also provide a possible explanation for the catalytic gains in our top-performing OYE variant (new N-terminus at residue 303; sector III). Crystallographic analysis indicates that termini relocation into sector III affects the loop β6 region (amino acid positions: 290-310) of OYE1 which forms a lid over the active site. Peptide backbone cleavage greatly enhances local flexibility, effectively converting the loop into a tether and consequently increasing the environmental exposure of the active site. Interestingly, such active site remodeling does not negatively impact the enzyme's activity and stereoselectivity, nor does it perturb the conformation of other key active site residues with the exception of Y375. These observations were confirmed in truncation experiments, deleting all residues of the loop β6 region in our OYE variant. Intrigued by the finding that circular permutation leaves most of the key catalytic residues unchanged, we also tested OYE permutants for possible additive or synergistic effects of amino acid substitutions. Distinct functional changes in these OYE variants were detected upon mutations at W116, known in native OYE1 to cause inversion of diastereo-selectivity for ( S )-carvone reduction. Our findings demonstrate the contribution of loop β6 toward determining the stereoselectivity of OYE1, an important insight for future OYE engineering efforts.

Systematic comparison of co-expression of multiple recombinant thermophilic enzymes in Escherichia coli BL21(DE3).

PubMed

Chen, Hui; Huang, Rui; Zhang, Y-H Percival

2017-06-01

The precise control of multiple heterologous enzyme expression levels in one Escherichia coli strain is important for cascade biocatalysis, metabolic engineering, synthetic biology, natural product synthesis, and studies of complexed proteins. We systematically investigated the co-expression of up to four thermophilic enzymes (i.e., α-glucan phosphorylase (αGP), phosphoglucomutase (PGM), glucose 6-phosphate dehydrogenase (G6PDH), and 6-phosphogluconate dehydrogenase (6PGDH)) in E. coli BL21(DE3) by adding T7 promoter or T7 terminator of each gene for multiple genes in tandem, changing gene alignment, and comparing one or two plasmid systems. It was found that the addition of T7 terminator after each gene was useful to decrease the influence of the upstream gene. The co-expression of the four enzymes in E. coli BL21(DE3) was demonstrated to generate two NADPH molecules from one glucose unit of maltodextrin, where NADPH was oxidized to convert xylose to xylitol. The best four-gene co-expression system was based on two plasmids (pET and pACYC) which harbored two genes. As a result, apparent enzymatic activities of the four enzymes were regulated to be at similar levels and the overall four-enzyme activity was the highest based on the formation of xylitol. This study provides useful information for the precise control of multi-enzyme-coordinated expression in E. coli BL21(DE3).

21 CFR 184.1387 - Lactase enzyme preparation from Candida pseudotropicalis.

Code of Federal Regulations, 2012 CFR

2012-04-01

..., nontoxicogenic yeast C. pseudotropicalis. It contains the enzyme lactase (β-D-galactoside galactohydrolase, EC 3.2.1.23), which converts lactose to glucose and galactose. It is prepared from yeast that has been...

21 CFR 184.1387 - Lactase enzyme preparation from Candida pseudotropicalis.

Code of Federal Regulations, 2013 CFR

2013-04-01

..., nontoxicogenic yeast C. pseudotropicalis. It contains the enzyme lactase (β-D-galactoside galactohydrolase, EC 3.2.1.23), which converts lactose to glucose and galactose. It is prepared from yeast that has been...

Genetics Home Reference: Crigler-Najjar syndrome

MedlinePlus

... from the body only after it undergoes a chemical reaction in the liver, which converts the toxic form ... the body. The bilirubin-UGT enzyme performs a chemical reaction called glucuronidation. During this reaction, the enzyme transfers ...

Biosynthesis of the enzymes of the cellulase system by T. Reesei QM 9414 in the presence of sophorose

NASA Astrophysics Data System (ADS)

Gritzali, M.

1982-12-01

As conventional, nonrenewable energy sources are rapidly depleted and it was necessary to search for alternative sources of energy. It was increasingly apparent that biomass and waste are alternatives well worth exploring. The sources of biomass and wastes that considered for conversion to useful products are quite diverse, but the most abundant constituent of almost every type is cellulose. Cellulose is cleanly converted to soluble fermentable sugars enzymatically, and cellulose enzymes were isolated from a number of microbial sources. It is generally agreed that the most effective system of enzymes for the conversion of cellulose to glucose is produced by species of the imperfect fungus Trichoderma. The mutant organism Trichoderma reesei QM 9414 is among the best producers of high levels of enzymes; these are extracellular and have carbonhydrate covalently bound to the peptide. Trichoderma produces three types of enzymes which, in a sequential and cooperative manner, convert cellulose to soluble oligosaccharides and glucose.

Antioxidant, antihypertensive, and antibacterial properties of endophytic Pestalotiopsis species from medicinal plants.

PubMed

Tejesvi, Mysore V; Kini, Kukkundoor R; Prakash, Harishchandra S; Subbiah, Ven; Shetty, Hunthrike S

2008-09-01

Pestalotiopsis species were most dominant endophytic species isolated from four medicinal plants including Terminalia arjuna, Terminalia chebula, Azadirachta indica, and Holarrhena antidysenterica. Thirty Pestalotiopsis species isolated from different parts of the medicinal plants were selected for the study. The antioxidant and antihypertensive properties of Pestalotiopsis isolates were determined by measuring 1,1-diphenyl-2-picrylhydrazyl inhibitory activity, lipid peroxidation, and angiotensin-converting enzyme inhibition activity. Pestalotiopsis isolates of T. arjuna origin exhibited maximum radical scavenging activity compared with the others. The IC50 values of Pestalotiopsis extracts for 1,1-diphenyl-2-picrylhydrazyl scavenging activity ranged from 14 to 27 microg/mL compared with 15 and 6 microg/mL for butylated hydroxytoluene and ascorbic acid, respectively. The DNA damage study was also done for three isolates, TC-315, TA-37, and TA-60; TA-37 gave 80% protection. The IC50 values of Pestalotiopsis extracts for lipid peroxidation ranged between 30 and 35.5 microg/mL, while for the positive control butylated hydroxytoluene, it was 26 microg/mL. Out of 32 fungal extracts screened for antihypertensive assay, five (TA-37, TA-60, TA-102, TA-103, and TC-320) showed >60% inhibition of angiotensin-converting enzyme. The IC50 values for five extracts ranged from 21 to 37 microg/mL and was 20 microg/mL for captopril used as a positive control. The antibacterial activity was measured by the microplate-based turbidity measurement method. Four Pestalotiopsis extracts (TA-04, TA-37, TA-60, and TA-102) showed >75% inhibition against five bacterial strains including Bacillus subtilis, Escherichia coli, Pseudomonas fluorescens, Xanthomonas axonopodis pv. malvacearum, and Staphylococcus aureus. The antioxidant, antibacterial, and antihypertensive activities demonstrated the potential of Pestalotiopsis extracts as therapeutic targets.

High-resolution crystal structures of Drosophila melanogaster angiotensin-converting enzyme in complex with novel inhibitors and antihypertensive drugs.

PubMed

Akif, Mohd; Georgiadis, Dimitris; Mahajan, Aman; Dive, Vincent; Sturrock, Edward D; Isaac, R Elwyn; Acharya, K Ravi

2010-07-16

Angiotensin I-converting enzyme (ACE), one of the central components of the renin-angiotensin system, is a key therapeutic target for the treatment of hypertension and cardiovascular disorders. Human somatic ACE (sACE) has two homologous domains (N and C). The N- and C-domain catalytic sites have different activities toward various substrates. Moreover, some of the undesirable side effects of the currently available and widely used ACE inhibitors may arise from their targeting both domains leading to defects in other pathways. In addition, structural studies have shown that although both these domains have much in common at the inhibitor binding site, there are significant differences and these are greater at the peptide binding sites than regions distal to the active site. As a model system, we have used an ACE homologue from Drosophila melanogaster (AnCE, a single domain protein with ACE activity) to study ACE inhibitor binding. In an extensive study, we present high-resolution structures for native AnCE and in complex with six known antihypertensive drugs, a novel C-domain sACE specific inhibitor, lisW-S, and two sACE domain-specific phosphinic peptidyl inhibitors, RXPA380 and RXP407 (i.e., nine structures). These structures show detailed binding features of the inhibitors and highlight subtle changes in the orientation of side chains at different binding pockets in the active site in comparison with the active site of N- and C-domains of sACE. This study provides information about the structure-activity relationships that could be utilized for designing new inhibitors with improved domain selectivity for sACE. 2010 Elsevier Ltd. All rights reserved.

Location and stoichiometry of the protease CspB and the cortex-lytic enzyme SleC in Clostridium perfringens spores.

PubMed

Banawas, Saeed; Korza, George; Paredes-Sabja, Daniel; Li, Yunfeng; Hao, Bing; Setlow, Peter; Sarker, Mahfuzur R

2015-09-01

The protease CspB and the cortex-lytic enzyme SleC are essential for peptoglycan cortex hydrolysis during germination of spores of the Clostridium perfringens food poisoning isolate SM101. In this study, Western blot analyses were used to demonstrate that CspB and SleC are present exclusively in the C. perfringens SM101 spore coat layer fraction and absent in the lysate from decoated spores and from the purified inner spore membrane. These results indicate why decoating treatments greatly reduce both germination and apparent viability of C. perfringens spores in the absence of an exogenous lytic enzyme. In addition, quantitative Western blot analyses showed that there are approximately 2000 and 130,000 molecules of CspB and pro-SleC, respectively, per C. perfringens SM101 spore, consistent with CspB's role in acting catalytically on pro-SleC to convert this zymogen to the active enzyme. Copyright © 2015 Elsevier Ltd. All rights reserved.

Role of glutamate-199 in the aging of cholinesterase. (Reannouncement with new availability information)

DOE Office of Scientific and Technical Information (OSTI.GOV)

Saxena, A.; Doctor, B.P.; Maxwell, D.M.

1993-11-30

Aging of organophosphate-conjugated acetylcholinesterase results from the loss of an alkoxy group with concomitant stabilization of the conjugate to spontaneous or nucleophile-induced deacylation. We have examined the kinetics of aging in a pinacolylmethylphosphonofluoridate (soman)-inhibited mutant enzyme in which the glutamate (E199) located at the amino-terminal to the active-site sense (S200) was converted to glutamine (Q). For wild type enzyme, the soman-acetylcholinesterase conjugate aged immediately, giving rise to a form of enzyme resistant to reactivation by oximes. In contrast, the E199Q mutant enzyme was largely resistant to aging and could be reactivated by oximes. Since the pH dependence for aging wasmore » not altered appreciably, the primary influence of the loss of charge appears to be on the intrinsic rate of aging. The negative charge on E199 likely imparts an inductive effect on the conjugated organophosphate to facilitate removal of the alkoxy group. Cholinesterases, Aging, Organophosphate.« less

Phe71 in Type III Trypanosomal Protein Arginine Methyltransferase 7 (TbPRMT7) Restricts the Enzyme to Monomethylation.

PubMed

Cáceres, Tamar B; Thakur, Abhishek; Price, Owen M; Ippolito, Nicole; Li, Jun; Qu, Jun; Acevedo, Orlando; Hevel, Joan M

2018-02-27

Protein arginine methyltransferase 7 (PRMT7) is unique within the PRMT family as it is the only isoform known to exclusively make monomethylarginine (MMA). Given its role in epigenetics, the mechanistic basis for the strict monomethylation activity is under investigation. It is thought that PRMT7 enzymes are unable to add a second methyl group because of steric hindrance in the active site that restricts them to monomethylation. To test this, we probed the active site of trypanosomal PRMT7 (TbPRMT7) using accelerated molecular dynamics, site-directed mutagenesis, kinetic, binding, and product analyses. Both the dynamics simulations and experimental results show that the mutation of Phe71 to Ile converts the enzyme from a type III methyltransferase into a mixed type I/II, that is, an enzyme that can now perform dimethylation. In contrast, the serine and alanine mutants of Phe71 preserve the type III behavior of the native enzyme. These results are inconsistent with a sterics-only model to explain product specificity. Instead, molecular dynamics simulations of these variants bound to peptides show hydrogen bonding between would-be substrates and Glu172 of TbPRMT7. Only in the case of the Phe71 to Ile mutation is this interaction between MMA and the enzyme maintained, and the geometry for optimal S N 2 methyl transfer is obtained. The results of these studies highlight the benefit of combined computational and experimental methods in providing a better understanding for how product specificity is dictated by PRMTs.

Potential utility of natural products as regulators of breast cancer-assoicated aromatase promoters

USDA-ARS?s Scientific Manuscript database

Aromatase, the key enzyme in estrogen biosynthesis, converts androstenedione to estrone and testosterone to estradiol. The enzyme is expressed in various tissues such as ovary, placenta, bone, brain, skin, and adipose tissue. Aromatase enzyme is encoded by a single gene CYP 19A1 and its expression i...

Martian Superoxide and Peroxide O2 Release (OR) Assay: A New Technology for Terrestrial and Planetary Applications

NASA Technical Reports Server (NTRS)

Georgiou, Christos D.; Zisimopoulos, Dimitrios; Panagiotidis, Konstantinos; Grintzalis, Kontantinos; Papapostolou, Ioannis; Quinn, Richard C.; McKay, Christopher P.; Sun, Henry J.

2015-01-01

This study presents an assay for the detection and quantification of soil metal superoxides and peroxides in regolith and soil. The O2 release (OR) assay is based on the enzymatic conversion of the hydrolysis products of metal oxides to O2, and their quantification by an O2 electrode based on the stoichiometry of the involved reactions: The intermediate product O2 from the hydrolysis of metal superoxides is converted by cytochrome c to O2, and also by superoxide dismutase (SOD) to 1/2 mol O2 and 1/2 mol H2O2, which is then converted by catalase (CAT) to 1/2 mol O2. The product H2O2 from the hydrolysis of metal peroxides and hydroperoxides is converted to 1/2 mol O2 by CAT. The assay-method was validated in a sealed sample chamber using a liquid-phase Clark-type O2 electrode with known concentrations of O2 and H2O2, and with commercial metal superoxide and peroxide mixed with Mars analogue Mojave and Atacama Desert soils. Carbonates and perchlorates, both present on Mars, do not interfere with the assay. The assay lower limit of detection, using luminescence quenching/optical sensing O2-electrodes, is 1 nmol O2 cm(exp. -3) or better. The activity of the assay enzymes SOD and cytochrome c was unaffected up to 6 Gy exposure by gamma-radiation, while CAT retained 100% and 40% of its activity at 3 and 6 Gy, respectively, demonstrating the suitability of these enzymes for planetary missions, e.g., in Mars or Europa.

Martian Superoxide and Peroxide O2 Release (OR) Assay: A New Technology for Terrestrial and Planetary Applications.

PubMed

Georgiou, Christos D; Zisimopoulos, Dimitrios; Panagiotidis, Konstantinos; Grintzalis, Konstantinos; Papapostolou, Ioannis; Quinn, Richard C; McKay, Christopher P; Sun, Henry J

2016-02-01

This study presents an assay for the detection and quantification of soil metal superoxides and peroxides in regolith and soil. The O2 release (OR) assay is based on the enzymatic conversion of the hydrolysis products of metal oxides to O2 and their quantification by an O2 electrode based on the stoichiometry of the involved reactions. The intermediate product O₂˙⁻ from the hydrolysis of metal superoxides is converted by cytochrome c to O2 and by superoxide dismutase (SOD) to ½ mol O2 and ½ mol H2O2, which is then converted by catalase (CAT) to ½ mol O2. The product H2O2 from the hydrolysis of metal peroxides and hydroperoxides is converted to ½ mol O2 by CAT. The assay method was validated in a sealed sample chamber by using a liquid-phase Clark-type O2 electrode with known concentrations of O₂˙⁻ and H2O2, and commercial metal superoxide and peroxide mixed with Mars analog Mojave and Atacama Desert soils. Carbonates and perchlorates, both present on Mars, do not interfere with the assay. The assay lower limit of detection, when using luminescence quenching/optical sensing O2-electrodes, is 1 nmol O2 cm(-3) or better. The activity of the assay enzymes SOD and cytochrome c was unaffected up to 6 Gy exposure by γ radiation, while CAT retained 100% and 40% of its activity at 3 and 6 Gy, respectively, which demonstrates the suitability of these enzymes for planetary missions, for example, on Mars or Europa.

The Thr-His Connection on the Distal Heme of Catalase-Related Hemoproteins: A Hallmark of Reaction with Fatty Acid Hydroperoxides.

PubMed

Mashhadi, Zahra; Newcomer, Marcia E; Brash, Alan R

2016-11-03

This review focuses on a group of heme peroxidases that retain the catalase fold in structure, yet show little or no reaction with hydrogen peroxide. Instead of having a role in oxidative defense, these enzymes are involved in secondary metabolite biosynthesis. The prototypical enzyme is catalase-related allene oxide synthase, an enzyme that converts a specific fatty acid hydroperoxide to the corresponding allene oxide (epoxide). Other catalase-related enzymes form allylic epoxides, aldehydes, or a bicyclobutane fatty acid. In all catalases (including these relatives), a His residue on the distal face of the heme is absolutely required for activity. Its immediate neighbor in sequence as well as in 3 D space is conserved as Val in true catalases and Thr in the fatty acid hydroperoxide-metabolizing enzymes. Thr-His on the distal face of the heme is critical in switching the substrate specificity from H 2 O 2 to fatty acid hydroperoxide. © 2016 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

Inhibition of metallopeptidases by flavonoids and related compounds.

PubMed

Bormann, H; Melzig, M F

2000-02-01

To elucidate possible mechanisms of activity in medicinal plants containing flavonoids, the inhibitory potency of twenty flavones, flavonols, flavanones, phenylacrylic acids and various hydroxylated phenylacetic acids on the activity of neutral endopeptidase (NEP; EC 3.4.24.11), angiotensin-converting enzyme (ACE; EC 3.4.15.1) and aminopeptidase N (APN; EC 3.4.11.2) was investigated in vitro. The screening generally resulted that inhibition of these enzymes requires free hydroxyl groups at the flavone molecule. Flavone and methoxylated compounds (sinensetin) were without effects. Flavonoids with free hydroxyl functions in position 3',4' and 5,7 inhibited the activity of NEP (quercetin, luteolin, fisetin), with myricetin (IC50 = 42 microM) as strongest inhibitor. Inhibition of ACE and APN did not depend on this class of compounds and substitution pattern. E.g. 3,4-dihydroxyphenylacetic acid and 4-methylcatechol (urinary metabolites of flavonoids) also inhibited both APN and ACE activity, but not NEP activity. The results demonstrate that some of the pharmacological activities of flavonoids might be related to the inhibition of metallopeptidases responsible for the splitting of regulatory neuropeptides.

Inhibition of angiotensin convertin enzyme (ACE) activity by the anthocyanins delphinidin- and cyanidin-3-O-sambubiosides from Hibiscus sabdariffa.

PubMed

Ojeda, Deyanira; Jiménez-Ferrer, Enrique; Zamilpa, Alejandro; Herrera-Arellano, Armando; Tortoriello, Jaime; Alvarez, Laura

2010-01-08

The beverages of Hibiscus sabdariffa calyces are widely used in Mexico as diuretic, for treating gastrointestinal disorders, liver diseases, fever, hypercholesterolemia and hypertension. Different works have demonstrated that Hibiscus sabdariffa extracts reduce blood pressure in humans, and recently, we demonstrated that this effect is due to angiotensin converting enzyme (ACE) inhibitor activity. The aim of the current study was to isolate and characterizer the constituents responsible of the ACE activity of the aqueous extract of Hibiscus sabdariffa. Bioassay-guided fractionation of the aqueous extract of dried calyces of Hibiscus sabdariffa using preparative reversed-phase HPLC, and the in vitro ACE Inhibition assay, as biological monitor model, were used for the isolation. The isolated compounds were characterized by spectroscopic methods. The anthocyanins delphinidin-3-O-sambubioside (1) and cyanidin-3-O-sambubioside (2) were isolated by bioassay-guided purification. These compounds showed IC(50) values (84.5 and 68.4 microg/mL, respectively), which are similar to those obtained by related flavonoid glycosides. Kinetic determinations suggested that these compounds inhibit the enzyme activity by competing with the substrate for the active site. The competitive ACE inhibitor activity of the anthocyanins 1 and 2 is reported for the first time. This activity is in good agreement with the folk medicinal use of Hibiscus sabdariffa calyces as antihypertensive. Copyright 2009 Elsevier Ireland Ltd. All rights reserved.

Stringency of the 2-His–1-Asp Active-Site Motif in Prolyl 4-Hydroxylase

PubMed Central

Gorres, Kelly L.; Pua, Khian Hong; Raines, Ronald T.

2009-01-01

The non-heme iron(II) dioxygenase family of enzymes contain a common 2-His–1-carboxylate iron-binding motif. These enzymes catalyze a wide variety of oxidative reactions, such as the hydroxylation of aliphatic C–H bonds. Prolyl 4-hydroxylase (P4H) is an α-ketoglutarate-dependent iron(II) dioxygenase that catalyzes the post-translational hydroxylation of proline residues in protocollagen strands, stabilizing the ensuing triple helix. Human P4H residues His412, Asp414, and His483 have been identified as an iron-coordinating 2-His–1-carboxylate motif. Enzymes that catalyze oxidative halogenation do so by a mechanism similar to that of P4H. These halogenases retain the active-site histidine residues, but the carboxylate ligand is replaced with a halide ion. We replaced Asp414 of P4H with alanine (to mimic the active site of a halogenase) and with glycine. These substitutions do not, however, convert P4H into a halogenase. Moreover, the hydroxylase activity of D414A P4H cannot be rescued with small molecules. In addition, rearranging the two His and one Asp residues in the active site eliminates hydroxylase activity. Our results demonstrate a high stringency for the iron-binding residues in the P4H active site. We conclude that P4H, which catalyzes an especially demanding chemical transformation, is recalcitrant to change. PMID:19890397

Structural Dissection of the Maltodextrin Disproportionation Cycle of the Arabidopsis Plastidial Disproportionating Enzyme 1 (DPE1)*

PubMed Central

O'Neill, Ellis C.; Stevenson, Clare E. M.; Tantanarat, Krit; Latousakis, Dimitrios; Donaldson, Matthew I.; Rejzek, Martin; Nepogodiev, Sergey A.; Limpaseni, Tipaporn; Field, Robert A.; Lawson, David M.

2015-01-01

The degradation of transitory starch in the chloroplast to provide fuel for the plant during the night requires a suite of enzymes that generate a series of short chain linear glucans. However, glucans of less than four glucose units are no longer substrates for these enzymes, whereas export from the plastid is only possible in the form of either maltose or glucose. In order to make use of maltotriose, which would otherwise accumulate, disproportionating enzyme 1 (DPE1; a 4-α-glucanotransferase) converts two molecules of maltotriose to a molecule of maltopentaose, which can now be acted on by the degradative enzymes, and one molecule of glucose that can be exported. We have determined the structure of the Arabidopsis plastidial DPE1 (AtDPE1), and, through ligand soaking experiments, we have trapped the enzyme in a variety of conformational states. AtDPE1 forms a homodimer with a deep, long, and open-ended active site canyon contained within each subunit. The canyon is divided into donor and acceptor sites with the catalytic residues at their junction; a number of loops around the active site adopt different conformations dependent on the occupancy of these sites. The “gate” is the most dynamic loop and appears to play a role in substrate capture, in particular in the binding of the acceptor molecule. Subtle changes in the configuration of the active site residues may prevent undesirable reactions or abortive hydrolysis of the covalently bound enzyme-substrate intermediate. Together, these observations allow us to delineate the complete AtDPE1 disproportionation cycle in structural terms. PMID:26504082

Primary Systemic Amyloidosis and High Levels of Angiotensin-Converting Enzyme: Two Case Reports

PubMed Central

Praena-Segovia, J.; Sanchez-Gastaldo, A.; Bernabeu-Wittel, M.; Ocete-Pérez, R.; Ávila-Polo, R.; Martino, M. L.

2013-01-01

Infiltrative heart diseases are caused by a heterogeneous group of disorders; amyloidosis and sarcoidosis are two frequent causes of myocardial infiltration, which differ in clinical and biological outcome and treatment issues. The presence of high levels of angiotensin-converting enzyme (ACE) in a patient with infiltrative heart disease may increase suspicion of sarcoidosis. Nevertheless, no mention about increased ACE levels in extracerebral primary systemic amyloidosis is available. We present two cases of primary systemic amyloidosis, which are cardiac involvement and elevated ACE levels. PMID:24826302

Bronchoalveolar lavage, serum angiotensin-converting enzyme, and /sup 67/Ga scanning in extrathoracic sarcoidosis

DOE Office of Scientific and Technical Information (OSTI.GOV)

Wallaert, B.; Ramon, P.; Fournier, E.

1982-11-01

Results of bronchoalveolar lavage (BAL), 67Ga scanning, and serum angiotensin-converting enzyme (SACE) assay are compared in the assessment of pulmonary involvement in ten cases of extrathoracic sarcoidosis. Standard clinical, radiologic, and pulmonary function tests detected no pulmonary changes in these patients, but BAL demonstrated an increased alveolar lymphocytosis in eight of ten cases. SACE levels were increased in two cases, and the thoracic gallium uptake was normal in all cases. BAL appears to be the best technique for diagnosing latent pulmonary involvement in extrathoracic sarcoidosis.

Enzyme Hydrolysates from Stichopus horrens as a New Source for Angiotensin-Converting Enzyme Inhibitory Peptides

PubMed Central

Forghani, Bita; Ebrahimpour, Afshin; Bakar, Jamilah; Abdul Hamid, Azizah; Hassan, Zaiton; Saari, Nazamid

2012-01-01

Stichopus horrens flesh was explored as a potential source for generating peptides with angiotensin-converting enzyme (ACE) inhibitory capacity using 6 proteases, namely alcalase, flavourzyme, trypsin, papain, bromelain, and protamex. Degree of hydrolysis (DH) and peptide profiling (SDS-PAGE) of Stichopus horrens hydrolysates (SHHs) was also assessed. Alcalase hydrolysate showed the highest DH value (39.8%) followed by flavourzyme hydrolysate (32.7%). Overall, alcalase hydrolysate exhibited the highest ACE inhibitory activity (IC50 value of 0.41 mg/mL) followed by flavourzyme hydrolysate (IC50 value of 2.24 mg/mL), trypsin hydrolysate (IC50 value of 2.28 mg/mL), papain hydrolysate (IC50 value of 2.48 mg/mL), bromelain hydrolysate (IC50 value of 4.21 mg/mL), and protamex hydrolysate (IC50 value of 6.38 mg/mL). The SDS-PAGE results showed that alcalase hydrolysate represented a unique pattern compared to others, which yielded potent ACE inhibitory peptides with molecular weight distribution lower than 20 kDa. The evaluation of the relationship between DH and IC50 values of alcalase and flavourzyme hydrolysates revealed that the trend between those parameters was related to the type of the protease used. We concluded that the tested SHHs would be used as a potential source of functional ACE inhibitory peptides for physiological benefits. PMID:22927875

Enzyme Hydrolysates from Stichopus horrens as a New Source for Angiotensin-Converting Enzyme Inhibitory Peptides.

PubMed

Forghani, Bita; Ebrahimpour, Afshin; Bakar, Jamilah; Abdul Hamid, Azizah; Hassan, Zaiton; Saari, Nazamid

2012-01-01

Stichopus horrens flesh was explored as a potential source for generating peptides with angiotensin-converting enzyme (ACE) inhibitory capacity using 6 proteases, namely alcalase, flavourzyme, trypsin, papain, bromelain, and protamex. Degree of hydrolysis (DH) and peptide profiling (SDS-PAGE) of Stichopus horrens hydrolysates (SHHs) was also assessed. Alcalase hydrolysate showed the highest DH value (39.8%) followed by flavourzyme hydrolysate (32.7%). Overall, alcalase hydrolysate exhibited the highest ACE inhibitory activity (IC(50) value of 0.41 mg/mL) followed by flavourzyme hydrolysate (IC(50) value of 2.24 mg/mL), trypsin hydrolysate (IC(50) value of 2.28 mg/mL), papain hydrolysate (IC(50) value of 2.48 mg/mL), bromelain hydrolysate (IC(50) value of 4.21 mg/mL), and protamex hydrolysate (IC(50) value of 6.38 mg/mL). The SDS-PAGE results showed that alcalase hydrolysate represented a unique pattern compared to others, which yielded potent ACE inhibitory peptides with molecular weight distribution lower than 20 kDa. The evaluation of the relationship between DH and IC(50) values of alcalase and flavourzyme hydrolysates revealed that the trend between those parameters was related to the type of the protease used. We concluded that the tested SHHs would be used as a potential source of functional ACE inhibitory peptides for physiological benefits.

Improvement on sugar cane bagasse hydrolysis using enzymatic mixture designed cocktail.

PubMed

Bussamra, Bianca Consorti; Freitas, Sindelia; Costa, Aline Carvalho da

2015-01-01

The aim of this work was to study cocktail supplementation for sugar cane bagasse hydrolysis, where the enzymes were provided from both commercial source and microorganism cultivation (Trichoderma reesei and genetically modified Escherichia coli), followed by purification. Experimental simplex lattice mixture design was performed to optimize the enzymatic proportion. The response was evaluated through hydrolysis microassays validated here. The optimized enzyme mixture, comprised of T. reesei fraction (80%), endoglucanase (10%) and β-glucosidase (10%), converted, theoretically, 72% of cellulose present in hydrothermally pretreated bagasse, whereas commercial Celluclast 1.5L converts 49.11%±0.49. Thus, a rational enzyme mixture designed by using synergism concept and statistical analysis was capable of improving biomass saccharification. Copyright © 2015 Elsevier Ltd. All rights reserved.

Investigating a Bio-Engineered Enzyme.

ERIC Educational Resources Information Center

Bullerwell, Lornie; And Others

1994-01-01

Describes science experiments with the enzyme lactose, which is available commercially as Lactaid and Dairy Ease. Experiments show how the rate of reaction of lactose converted to glucose and galactose is influenced by temperature, pH, and substrate concentration. (PR)

A re-evaluation of the final step of vanillin biosynthesis in the orchid Vanilla planifolia.

PubMed

Yang, Hailian; Barros-Rios, Jaime; Kourteva, Galina; Rao, Xiaolan; Chen, Fang; Shen, Hui; Liu, Chenggang; Podstolski, Andrzej; Belanger, Faith; Havkin-Frenkel, Daphna; Dixon, Richard A

2017-07-01

A recent publication describes an enzyme from the vanilla orchid Vanilla planifolia with the ability to convert ferulic acid directly to vanillin. The authors propose that this represents the final step in the biosynthesis of vanillin, which is then converted to its storage form, glucovanillin, by glycosylation. The existence of such a "vanillin synthase" could enable biotechnological production of vanillin from ferulic acid using a "natural" vanilla enzyme. The proposed vanillin synthase exhibits high identity to cysteine proteases, and is identical at the protein sequence level to a protein identified in 2003 as being associated with the conversion of 4-coumaric acid to 4-hydroxybenzaldehyde. We here demonstrate that the recombinant cysteine protease-like protein, whether expressed in an in vitro transcription-translation system, E. coli, yeast, or plants, is unable to convert ferulic acid to vanillin. Rather, the protein is a component of an enzyme complex that preferentially converts 4-coumaric acid to 4-hydroxybenzaldehyde, as demonstrated by the purification of this complex and peptide sequencing. Furthermore, RNA sequencing provides evidence that this protein is expressed in many tissues of V. planifolia irrespective of whether or not they produce vanillin. On the basis of our results, V. planifolia does not appear to contain a cysteine protease-like "vanillin synthase" that can, by itself, directly convert ferulic acid to vanillin. The pathway to vanillin in V. planifolia is yet to be conclusively determined. Copyright © 2017 Elsevier Ltd. All rights reserved.

Identification of natural inhibitors against angiotensin I converting enzyme for cardiac safety using induced fit docking and MM-GBSA studies

PubMed Central

Vijayakumar, Balakrishnan; Parasuraman, Subramani; Raveendran, Ramasamy; Velmurugan, Devadasan

2014-01-01

Background: Cleistanthins A and B are isolated compounds from the leaves of Cleistanthus collinus Roxb (Euphorbiaceae). This plant is poisonous in nature which causes cardiovascular abnormalities such as hypotension, nonspecific ST-T changes and QTc prolongation. The biological activity predictions spectra of the compounds show the presence of antihypertensive, diuretic and antitumor activities. Objective: Objective of the present study was to determine the in silico molecular interaction of cleistanthins A and B with Angiotensin I- Converting Enzyme (ACE-I) using Induced Fit Docking (IFD) protocols. Materials and Methods: All the molecular modeling calculations like IFD docking, binding free energy calculation and ADME/Tox were carried out using Glide software (Schrödinger LLC 2009, USA) in CentOS EL-5 workstation. Results: The IFD complexes showed favorable docking score, glide energy, glide emodel, hydrogen bond and hydrophobic interactions between the active site residues of ACE-I and the compounds. Binding free energy was calculated for the IFD complexes using Prime MM-GBSA method. The conformational changes induced by the inhibitor at the active site of ACE-I were observed based on changes of the back bone Cα atoms and side-chain chi (x) angles. The various physicochemical properties were calculated for these compounds. Both cleistanthins A and B showed better docking score, glide energy and glide emodel when compared to captopril inhibitor. Conclusion: These compounds have successively satisfied all the in silico parameters and seem to be potent inhibitors of ACE-I and potential candidates for hypertension. PMID:25298685

Location of the redox-active thiols of ribonucleotide reductase: sequences similarity between the Escherichia coli and Lactobacillus leichmannii enzymes

DOE Office of Scientific and Technical Information (OSTI.GOV)

Lin, A.N.I.; Ashley, G.W.; Stubbe, J.

1987-11-03

The redox-active thiols of Escherichia coli ribonucleoside diphosphate reductase and of Lactobacillus leichmannii ribonucleoside triphosphate reductase have been located by a procedure involving (1) prereduction of enzyme with dithiothreitol, (2) specific oxidation of the redox-active thiols by treatment with substrate in the absence of exogenous reductant, (3) alkylation of other thiols with iodoacetamide, and (4) reduction of the disulfides with dithiothreitol and alkylation with (1-/sup 14/C)iodoacetamide. The dithiothreitol-reduce E. coli B1 subunit is able to convert 3 equiv of CDP to dCDP and is labeled with 5.4 equiv of /sup 14/C. Sequencing of tryptic peptides shows that 2.8 equiv ofmore » /sup 14/C is on cysteines-752 and -757 at the C-terminus of B1, while 1.0-1.5 equiv of /sup 14/C is on cysteines-222 and -227. It thus appears that two sets of redox-active dithiols are involved in substrate reduction. The L. leichmannii reductase is able to convert 1.1 equiv of CTP to dCTP and is labeled with 2.1 equiv of /sup 14/C. Sequencing of tryptic peptides shows that 1.4 equiv of /sup 14/C is located on the two cysteines of C-E-G-G-A-C-P-I-K. This peptide shows remarkable and unexpected similarity to the thiol-containing region of the C-terminal peptide of E. coli B1, C-E-S-G-A-C-K-I.« less

Toward aldehyde and alkane production by removing aldehyde reductase activity in Escherichia coli.

PubMed

Rodriguez, Gabriel M; Atsumi, Shota

2014-09-01

Advances in synthetic biology and metabolic engineering have enabled the construction of novel biological routes to valuable chemicals using suitable microbial hosts. Aldehydes serve as chemical feedstocks in the synthesis of rubbers, plastics, and other larger molecules. Microbial production of alkanes is dependent on the formation of a fatty aldehyde intermediate which is converted to an alkane by an aldehyde deformylating oxygenase (ADO). However, microbial hosts such as Escherichia coli are plagued by many highly active endogenous aldehyde reductases (ALRs) that convert aldehydes to alcohols, which greatly complicates strain engineering for aldehyde and alkane production. It has been shown that the endogenous ALR activity outcompetes the ADO enzyme for fatty aldehyde substrate. The large degree of ALR redundancy coupled with an incomplete database of ALRs represents a significant obstacle in engineering E. coli for either aldehyde or alkane production. In this study, we identified 44 ALR candidates encoded in the E. coli genome using bioinformatics tools, and undertook a comprehensive screening by measuring the ability of these enzymes to produce isobutanol. From the pool of 44 candidates, we found five new ALRs using this screening method (YahK, DkgA, GldA, YbbO, and YghA). Combined deletions of all 13 known ALRs resulted in a 90-99% reduction in endogenous ALR activity for a wide range of aldehyde substrates (C2-C12). Elucidation of the ALRs found in E. coli could guide one in reducing competing alcohol formation during alkane or aldehyde production. Copyright © 2014 International Metabolic Engineering Society. Published by Elsevier Inc. All rights reserved.

UDP-4-Keto-6-Deoxyglucose, a Transient Antifungal Metabolite, Weakens the Fungal Cell Wall Partly by Inhibition of UDP-Galactopyranose Mutase

PubMed Central

Ma, Liang; Salas, Omar; Bowler, Kyle

2017-01-01

ABSTRACT Can accumulation of a normally transient metabolite affect fungal biology? UDP-4-keto-6-deoxyglucose (UDP-KDG) represents an intermediate stage in conversion of UDP-glucose to UDP-rhamnose. Normally, UDP-KDG is not detected in living cells, because it is quickly converted to UDP-rhamnose by the enzyme UDP-4-keto-6-deoxyglucose-3,5-epimerase/-4-reductase (ER). We previously found that deletion of the er gene in Botrytis cinerea resulted in accumulation of UDP-KDG to levels that were toxic to the fungus due to destabilization of the cell wall. Here we show that these negative effects are at least partly due to inhibition by UDP-KDG of the enzyme UDP-galactopyranose mutase (UGM), which reversibly converts UDP-galactopyranose (UDP-Galp) to UDP-galactofuranose (UDP-Galf). An enzymatic activity assay showed that UDP-KDG inhibits the B. cinerea UGM enzyme with a Ki of 221.9 µM. Deletion of the ugm gene resulted in strains with weakened cell walls and phenotypes that were similar to those of the er deletion strain, which accumulates UDP-KDG. Galf residue levels were completely abolished in the Δugm strain and reduced in the Δer strain, while overexpression of the ugm gene in the background of a Δer strain restored Galf levels and alleviated the phenotypes. Collectively, our results show that the antifungal activity of UDP-KDG is due to inhibition of UGM and possibly other nucleotide sugar-modifying enzymes and that the rhamnose metabolic pathway serves as a shunt that prevents accumulation of UDP-KDG to toxic levels. These findings, together with the fact that there is no Galf in mammals, support the possibility of developing UDP-KDG or its derivatives as antifungal drugs. PMID:29162710

Purification and characterization of an alkaline protease from the marine yeast Aureobasidium pullulans for bioactive peptide production from different sources.

PubMed

Ma, Chunling; Ni, Xiumei; Chi, Zhenming; Ma, Liyan; Gao, Lingmei

2007-01-01

The extracellular alkaline protease in the supernatant of cell culture of the marine yeast Aureobasidium pullulans 10 was purified to homogeneity with a 2.1-fold increase in specific protease activity as compared to that in the supernatant by ammonium sulfate fractionation, gel filtration chromatography (Sephadex G-75), and anion-exchange chromatography (DEAE Sepharose Fast Flow). According to the sodium dodecyl sulfate-polyacrylamide gel electrophoresis data, the molecular mass of the purified enzyme was estimated to be 32.0 kDa. The optimal pH and temperature of the purified enzyme were 9.0 and 45 degrees C, respectively. The enzyme was activated by Cu(2+) (at a concentration of 1.0 mM) and Mn(2+) and inhibited by Hg(2+), Fe(2+), Fe(3+), Zn(2+), and Co(2+). The enzyme was strongly inhibited by phenylmethylsulfonyl fluoride, but weakly inhibited by EDTA, 1-10-phenanthroline, and iodoacetic acid. The K(m) and V(max) values of the purified enzyme for casein were 0.25 mg/ml and 0.0286 micromol/min/mg of protein, respectively. After digestion of shrimp protein, spirulina (Arthospira platensis) protein, proteins of marine yeast strains N3C (Yarrowia lipolytica) and YA03a (Hanseniaspora uvarum), milk protein, and casein with the purified alkaline protease, angiotensin I converting enzyme (ACE) inhibitory activities of the resulting peptides reached 85.3%, 12.1%, 29.8%, 22.8%, 14.1%, and 15.5%, respectively, while the antioxidant activities of these were 52.1%. 54.6%, 25.1%, 35%, 12.5%, and 24.2%, respectively, indicating that ACE inhibitory activity of the resulting peptides from the shrimp protein and antioxidant activity of those produced from the spirulina protein were the highest, respectively. These results suggest that the bioactive peptides produced by digestion of the shrimp protein with the purified alkaline protease have potential applications in the food and pharmaceutical industries.

A fluorogenic near-infrared imaging agent for quantifying plasma and local tissue renin activity in vivo and ex vivo

PubMed Central

Zhang, Jun; Preda, Dorin V.; Vasquez, Kristine O.; Morin, Jeff; Delaney, Jeannine; Bao, Bagna; Percival, M. David; Xu, Daigen; McKay, Dan; Klimas, Michael; Bednar, Bohumil; Sur, Cyrille; Gao, David Z.; Madden, Karen; Yared, Wael; Rajopadhye, Milind

2012-01-01

The renin-angiotensin system (RAS) is well studied for its regulation of blood pressure and fluid homeostasis, as well as for increased activity associated with a variety of diseases and conditions, including cardiovascular disease, diabetes, and kidney disease. The enzyme renin cleaves angiotensinogen to form angiotensin I (ANG I), which is further cleaved by angiotensin-converting enzyme to produce ANG II. Although ANG II is the main effector molecule of the RAS, renin is the rate-limiting enzyme, thus playing a pivotal role in regulating RAS activity in hypertension and organ injury processes. Our objective was to develop a near-infrared fluorescent (NIRF) renin-imaging agent for noninvasive in vivo detection of renin activity as a measure of tissue RAS and in vitro plasma renin activity. We synthesized a renin-activatable agent, ReninSense 680 FAST (ReninSense), using a NIRF-quenched substrate derived from angiotensinogen that is cleaved specifically by purified mouse and rat renin enzymes to generate a fluorescent signal. This agent was assessed in vitro, in vivo, and ex vivo to detect and quantify increases in plasma and kidney renin activity in sodium-sensitive inbred C57BL/6 mice maintained on a low dietary sodium and diuretic regimen. Noninvasive in vivo fluorescence molecular tomographic imaging of the ReninSense signal in the kidney detected increased renin activity in the kidneys of hyperreninemic C57BL/6 mice. The agent also effectively detected renin activity in ex vivo kidneys, kidney tissue sections, and plasma samples. This approach could provide a new tool for assessing disorders linked to altered tissue and plasma renin activity and to monitor the efficacy of therapeutic treatments. PMID:22674025

The dogfish shark (Squalus acanthias) increases both hepatic and extrahepatic ornithine urea cycle enzyme activities for nitrogen conservation after feeding.

PubMed

Kajimura, Makiko; Walsh, Patrick J; Mommsen, Thomas P; Wood, Chris M

2006-01-01

Urea not only is utilized as a major osmolyte in marine elasmobranchs but also constitutes their main nitrogenous waste. This study investigated the effect of feeding, and thus elevated nitrogen intake, on nitrogen metabolism in the Pacific spiny dogfish Squalus acanthias. We determined the activities of ornithine urea cycle (O-UC) and related enzymes in liver and nonhepatic tissues. Carbamoyl phosphate synthetase III (the rate-limiting enzyme of the O-UC) activity in muscle is high compared with liver, and the activities in both tissues increased after feeding. The contribution of muscle to urea synthesis in the dogfish body appears to be much larger than that of liver when body mass is considered. Furthermore, enhanced activities of the O-UC and related enzymes (glutamine synthetase, ornithine transcarbamoylase, arginase) were seen after feeding in both liver and muscle and were accompanied by delayed increases in plasma urea, trimethylamine oxide, total free amino acids, alanine, and chloride concentrations, as well as in total osmolality. The O-UC and related enzymes also occurred in the intestine but showed little change after feeding. Feeding did not change the rate of urea excretion, indicating strong N retention after feeding. Ammonia excretion, which constituted only a small percentage of total N excretion, was raised in fed fish, while plasma ammonia did not change, suggesting that excess ammonia in plasma is quickly ushered into synthesis of urea or protein. In conclusion, we suggest that N conservation is a high priority in this elasmobranch and that feeding promotes ureogenesis and growth. Furthermore, exogenous nitrogen from food is converted into urea not only by the liver but also by the muscle and to a small extent by the intestine.

Angiotensin-Converting Enzyme I/D Polymorphism and Preeclampsia Risk: Evidence of Small-Study Bias

PubMed Central

Serrano, Norma C; Díaz, Luis A; Páez, Maria C; Mesa, Clara M; Cifuentes, Rodrigo; Monterrosa, Alvaro; González, Adriana; Smeeth, Liam; Hingorani, Aroon D; Casas, Juan P

2006-01-01

Background Inappropriate activation of the renin–angiotensin system may play a part in the development of preeclampsia. An insertion/deletion polymorphism within the angiotensin-I converting enzyme gene (ACE-I/D) has shown to be reliably associated with differences in angiotensin-converting enzyme (ACE) activity. However, previous studies of the ACE-I/D variant and preeclampsia have been individually underpowered to detect plausible genotypic risks. Methods and Findings A prospective case-control study was conducted in 1,711 unrelated young pregnant women (665 preeclamptic and 1,046 healthy pregnant controls) recruited from five Colombian cities. Maternal blood was obtained to genotype for the ACE-I/D polymorphism. Crude and adjusted odds ratio (OR) and 95% confidence interval (CI) using logistic regression models were obtained to evaluate the strength of the association between ACE-I/D variant and preeclampsia risk. A meta-analysis was then undertaken of all published studies to February 2006 evaluating the ACE-I/D variant in preeclampsia. An additive model (per-D-allele) revealed a null association between the ACE-I/D variant and preeclampsia risk (crude OR = 0.95 [95% CI, 0.81–1.10]) in the new case-control study. Similar results were obtained after adjusting for confounders (adjusted per-allele OR = 0.90 [95% CI, 0.77–1.06]) and using other genetic models of inheritance. A meta-analysis (2,596 cases and 3,828 controls from 22 studies) showed a per-allele OR of 1.26 (95% CI, 1.07–1.49). An analysis stratified by study size showed an attenuated OR toward the null as study size increased. Conclusions It is highly likely that the observed small nominal increase in risk of preeclampsia associated with the ACE D-allele is due to small-study bias, similar to that observed in cardiovascular disease. Reliable assessment of the origins of preeclampsia using a genetic approach may require the establishment of a collaborating consortium to generate a dataset of adequate size. PMID:17194198

Impact of scaffold rigidity on the design and evolution of an artificial Diels-Alderase

PubMed Central

Preiswerk, Nathalie; Beck, Tobias; Schulz, Jessica D.; Milovník, Peter; Mayer, Clemens; Siegel, Justin B.; Baker, David; Hilvert, Donald

2014-01-01

By combining targeted mutagenesis, computational refinement, and directed evolution, a modestly active, computationally designed Diels-Alderase was converted into the most proficient biocatalyst for [4+2] cycloadditions known. The high stereoselectivity and minimal product inhibition of the evolved enzyme enabled preparative scale synthesis of a single product diastereomer. X-ray crystallography of the enzyme–product complex shows that the molecular changes introduced over the course of optimization, including addition of a lid structure, gradually reshaped the pocket for more effective substrate preorganization and transition state stabilization. The good overall agreement between the experimental structure and the original design model with respect to the orientations of both the bound product and the catalytic side chains contrasts with other computationally designed enzymes. Because design accuracy appears to correlate with scaffold rigidity, improved control over backbone conformation will likely be the key to future efforts to design more efficient enzymes for diverse chemical reactions. PMID:24847076

Functional Foods and Nutraceuticals as Dietary Intervention in Chronic Diseases; Novel Perspectives for Health Promotion and Disease Prevention.

PubMed

Adefegha, Stephen Adeniyi

2017-12-27

Functional foods describe the importance of foods in promoting health and preventing diseases aside their primary role of providing the body with the required amount of essential nutrients such as proteins, carbohydrates, vitamins, fats, and oils needed for its healthy survival. This review explains the interaction of functional food bioactive compounds including polyphenols (phenolic acids [hydroxybenzoic acids and hydroxycinnamic acids], flavonoids [flavonols, flavones, flavanols, flavanones, isoflavones, proanthocyanidins], stilbenes, and lignans), terpenoids, carotenoids, alkaloids, omega-3 and polyunsaturated fatty acids, among others with critical enzymes (α- amylase, α- glucosidase, angiotensin-I converting enzyme [ACE], acetylcholinesterase [AChE], and arginase) linked to some degenerative diseases (type-2 diabetes, cardiovascular diseases [hypertension], neurodegenerative diseases [Alzheimer's disease] and erectile dysfunction). Different functional food bioactive compounds may synergistically/additively confer an overwhelming protection against these degenerative diseases by modulating/altering the activities of these critical enzymes of physiological importance.

A unique dual recognition hairpin probe mediated fluorescence amplification method for sensitive detection of uracil-DNA glycosylase and endonuclease IV activities.

PubMed

Wu, Yushu; Yan, Ping; Xu, Xiaowen; Jiang, Wei

2016-03-07

Uracil-DNA glycosylase (UDG) and endonuclease IV (Endo IV) play cooperative roles in uracil base-excision repair (UBER) and inactivity of either will interrupt the UBER to cause disease. Detection of UDG and Endo IV activities is crucial to evaluate the UBER process in fundamental research and diagnostic application. Here, a unique dual recognition hairpin probe mediated fluorescence amplification method was developed for sensitively and selectively detecting UDG and Endo IV activities. For detecting UDG activity, the uracil base in the probe was excised by the target enzyme to generate an apurinic/apyrimidinic (AP) site, achieving the UDG recognition. Then, the AP site was cleaved by a tool enzyme Endo IV, releasing a primer to trigger rolling circle amplification (RCA) reaction. Finally, the RCA reaction produced numerous repeated G-quadruplex sequences, which interacted with N-methyl-mesoporphyrin IX to generate an enhanced fluorescence signal. Alternatively, for detecting Endo IV activity, the uracil base in the probe was first converted into an AP site by a tool enzyme UDG. Next, the AP site was cleaved by the target enzyme, achieving the Endo IV recognition. The signal was then generated and amplified in the same way as those in the UDG activity assay. The detection limits were as low as 0.00017 U mL(-1) for UDG and 0.11 U mL(-1) for Endo IV, respectively. Moreover, UDG and Endo IV can be well distinguished from their analogs. This method is beneficial for properly evaluating the UBER process in function studies and disease prognoses.

Biogenesis of Selenium Nanoparticles Using Green Chemistry.

PubMed

Shoeibi, Sara; Mozdziak, Paul; Golkar-Narenji, Afsaneh

2017-11-09

Selenium binds some enzymes such as glutathione peroxidase and thioredoxin reductase, which may be activated in biological infections and oxidative stress. Chemical and physical methods for synthesizing nanoparticles, apart from being expensive, have their own particular risks. However, nanoparticle synthesis through green chemistry is a safe procedure that different biological sources such as bacteria, fungi, yeasts, algae and plants can be the catalyst bed for processing. Synthesis of selenium nanoparticles (SeNPs) by macro/microorganisms causes variation in morphology and shape of the particles is due to diversity of reduction enzymes in organisms. Reducing enzymes of microorganisms by changing the status of redox convert metal ions (Se 2- ) to SeNPs without charge (Se 0 ). Biological activity of SeNPs includes their protective role against DNA oxidation. Because of the biological and industrial properties, SeNPs have wide applications in the fields of medicine, microelectronic, agriculture and animal husbandry. SeNPs can show strong antimicrobial effects on the growth and proliferation of microorganisms in a dose-dependent manner. The objective of this review is to consider SeNPs applications to various organisms.

Environmentally benign process for the preparation of antimicrobial α-methylene-β-hydroxy-γ-butyrolactone (tulipalin B) from tulip biomass.

PubMed

Nomura, Taiji; Hayashi, Emiko; Kawakami, Shohei; Ogita, Shinjiro; Kato, Yasuo

2015-01-01

Tulipalin B (α-methylene-β-hydroxy-γ-butyrolactone, PaB) is an antimicrobial natural product occurring in tulip (Tulipa gesneriana). PaB is directly formed from the precursor glucose ester 6-tuliposide B (PosB) by endogenous Pos-converting enzyme (TCE). Despite the potential usefulness of antibacterial PaB in various industrial applications, lack of facile synthetic schemes hampers its practical use. Herein, we describe an environmentally benign and facile process for the preparation of PaB using tulip biomass materials based on one-step enzyme reaction catalyzed by TCE without the use of petroleum-derived solvents. By screening 115 tulip cultivars, we found three elite cultivars, which accumulated PosB almost exclusively in flower tissues. The flower extracts with aqueous ethanol were partially purified with activated charcoal and subjected to the enzyme reaction with reusable immobilized TCE prepared from bulb crude extracts. The reaction was completed in a few hours at room temperature, and PaB was purified with activated charcoal and ethanol in a batch-wise manner.

Cholecystokinin octa- and tetrapeptide degradation by synaptic membranes. II. Solubilization and separation of membrane-bound CCK-8 cleaving enzymes.

PubMed

Deschodt-Lanckman, M; Bui, N D; Koulischer, D; Paroutaud, P; Strosberg, A D

1983-01-01

Solubilization of rat synaptic membranes by Triton X-100, followed by DEAE-cellulose chromatography allowed the identification of different CCK-8 cleaving enzymes. The first one (in the order of elution) removed the N-terminal aspartic acid residue of CCK-8 and was active on L-aspartic acid beta naphtylamide, suggesting that a corresponded to an aminopeptidase A. Two aminopeptidases of broad specificity hydrolyzed sequentially all the peptide bonds of CCK-8 as far as the release of free tryptophan. The removal of the sulfated tyrosine residue of CCK-8 occurred at a slower rate than that of the unsulfated residue. Another peptidase converted CCK-8 into its C-terminal heptapeptide. This enzyme had a lower affinity for the sulfated octapeptide in comparison with the unsulfated form (app Km of respectively 180 and 40 muM). The CCK-7 generating proteases displayed a moderate regional variation in five rat brain areas, with the highest activity in olfactory bulbs membranes and the lowest in cerebellar membranes. This distribution followed (with a lower amplitude) that of the CCK receptors.

Possible involvement of G-proteins and cAMP in the induction of progesterone hydroxylating enzyme system in the vascular wilt fungus Fusarium oxysporum.

PubMed

Poli, Anna; Di Pietro, Antonio; Zigon, Dusan; Lenasi, Helena

2009-02-01

Fungi present the ability to hydroxylate steroids. In some filamentous fungi, progesterone induces an enzyme system which converts the compound into a less toxic hydroxylated product. We investigated the progesterone response in the vascular wilt pathogen Fusarium oxysporum, using mass spectrometry and high performance liquid chromatography (HPLC). Progesterone was mainly transformed into 15alpha-hydroxyprogesterone, which was found predominantly in the extracellular medium. The role of two conserved fungal signaling cascades in the induction of the progesterone-transforming enzyme system was studied, using knockout mutants lacking the mitogen-activated protein kinase Fmk1 or the heterotrimeric G-protein beta subunit Fgb1 functioning upstream of the cyclic adenosine monophosphate (cAMP) pathway. No steroid hydroxylation was induced in the Deltafgb1 strain, suggesting a role for the G-protein beta subunit in progesterone signaling. Exogenous cAMP restored the induction of progesterone-transforming activity in the Deltafgb1 strain, suggesting that steroid signaling in F. oxysporum is mediated by the cAMP-PKA pathway.

Aedes aegypti juvenile hormone acid methyl transferase, the ultimate enzyme in the biosynthetic pathway of juvenile hormone III, exhibits substrate control

USDA-ARS?s Scientific Manuscript database

We report on the cloning, sequencing, characterization, 3D modeling and docking of Aedes aegypti juvenile hormone acid methyl transferase (AeaJHAMT), the enzyme that converts juvenile hormone acid (JHA) into juvenile hormone (JH). Purified recombinant AeaJHAMT was extensively characterized for enzym...

Glyoxysomes in Megagamethophyte of Germinating Ponderosa Pine Seeds 12

PubMed Central

Ching, Te May

1970-01-01

Decoated ponderosa pine (Pinus ponderosa Laws) seeds contained 40% lipids, which were mainly stored in megagametophytic tissue and were utilized or converted to sugars via the glyoxylate cycle during germination. Mitochondria and glyoxysomes were isolated from the tissue by sucrose density gradient centrifugation at different stages of germination. It was found that isocitrate lyase, malate synthase, and catalase were mainly bound in glyoxysomes. Aconitase and fumarase were chiefly localized in mitochondria, whereas citrate synthase was common for both. Both organelles increased in quantity and specific activity of their respective marker enzymes with the advancement of germination. When the megagametophyte was exhausted at the end of germination, the quantity of these organelles and the activity of their marker enzymes decreased abruptly. At the stage of highest lipolysis, the isolated mitochondria and glyoxysomes were able to synthesize protein from labeled amino acids. Both organellar fractions contained RNA and DNA. Some degree of autonomy in glyoxysomes is indicated. Images PMID:16657489

Identification of an ACE-Inhibitory Peptide from Walnut Protein and Its Evaluation of the Inhibitory Mechanism.

PubMed

Wang, Cong; Tu, Maolin; Wu, Di; Chen, Hui; Chen, Cheng; Wang, Zhenyu; Jiang, Lianzhou

2018-04-11

In the present study, a novel angiotensin I-converting enzyme inhibitory (ACE inhibitory) peptide, EPNGLLLPQY, derived from walnut seed storage protein, fragment residues 80-89, was identified by ultra-high performance liquid chromatography electrospray ionization quadrupole time of flight mass spectrometry (UPLC-ESI-Q-TOF-MS/MS) from walnut protein hydrolysate. The IC 50 value of the peptide was 233.178 μM, which was determined by the high performance liquid chromatography method by measuring the amount of hippuric acid (HA) generated from the ACE decomposition substrate (hippuryl-l-histidyl-l-leucine (HHL) to assess the ACE activity. Enzyme inhibitory kinetics of the peptide against ACE were also conducted, by which the inhibitory mechanism of ACE-inhibitory peptide was confirmed. Moreover, molecular docking was simulated by Discovery Studio 2017 R2 software to provide the potential mechanisms underlying the ACE-inhibitory activity of EPNGLLLPQY.

Isolation and characterization of a cDNA clone coding for a glutathione S-transferase class delta enzyme from the biting midge Culicoides variipennis sonorensis Wirth and Jones.

PubMed

Abdallah, M A; Pollenz, R S; Droog, F N; Nunamaker, R A; Tabachnick, W J; Murphy, K E

2000-12-01

Culicoides variipennis sonorensis is the primary vector of bluetongue viruses in North America. Glutathione S-transferases (GSTs) are enzymes that catalyze nucleophilic substitutions, converting reactive lipophilic molecules into soluble conjugates. Increased GST activity is associated with development of insecticide resistance. Described here is the isolation of the first cDNA encoding a C. variipennis GST. The clone consists of 720 translated bases encoding a protein with a M(r) of approximately 24,800 composed of 219 amino acids. The deduced amino acid sequence is similar (64%-74%) to class Delta (previously named Theta) GSTs from the dipteran genera Musca, Drosophila, Lucilia and Anopheles. The cDNA was subcloned into pET-11b, expressed in Epicurian coli BL21 (DE3) and has a specific activity of approximately 28,000 units/mg for the substrate 1-chloro-2,4-dinitrobenzene.

Chitinase Chi1 from Myceliophthora thermophila C1, a Thermostable Enzyme for Chitin and Chitosan Depolymerization.

PubMed

Krolicka, Malgorzata; Hinz, Sandra W A; Koetsier, Martijn J; Joosten, Rob; Eggink, Gerrit; van den Broek, Lambertus A M; Boeriu, Carmen G

2018-02-21

A thermostable Chitinase Chi1 from Myceliophthora thermophila C1 was homologously produced and characterized. Chitinase Chi1 shows high thermostability at 40 °C (>140 h 90% activity), 50 °C (>168 h 90% activity), and 55 °C (half-life 48 h). Chitinase Chi1 has broad substrate specificity and converts chitin, chitosan, modified chitosan, and chitin oligosaccharides. The activity of Chitinase Chi1 is strongly affected by the degree of deacetylation (DDA), molecular weight (Mw), and side chain modification of chitosan. Chitinase Chi1 releases mainly (GlcNAc) 2 from insoluble chitin and chito-oligosaccharides with a polymerization degree (DP) ranging from 2 to 12 from chitosan, in a processive way. Chitinase Chi1 shows higher activity toward chitin oligosaccharides (GlcNAc) 4-6 than toward (GlcNAc) 3 and is inactive for (GlcNAc) 2 . During hydrolysis, oligosaccharides bind at subsites -2 to +2 in the enzyme's active site. Chitinase Chi1 can be used for chitin valorisation and for production of chitin- and chito-oligosaccharides at industrial scale.

Arginine production in the neonate

USDA-ARS?s Scientific Manuscript database

Endogenous arginine synthesis in adults is a complex multiorgan process, in which citrulline is synthesized in the gut, enters the general circulation, and is converted into arginine in the kidney, by what is known as the intestinal-renal axis. In neonates, the enzymes required to convert citrulline...

Arginine de novo and nitric oxide production in disease states

PubMed Central

Luiking, Yvette C.; Ten Have, Gabriella A. M.; Wolfe, Robert R.

2012-01-01

Arginine is derived from dietary protein intake, body protein breakdown, or endogenous de novo arginine production. The latter may be linked to the availability of citrulline, which is the immediate precursor of arginine and limiting factor for de novo arginine production. Arginine metabolism is highly compartmentalized due to the expression of the enzymes involved in arginine metabolism in various organs. A small fraction of arginine enters the NO synthase (NOS) pathway. Tetrahydrobiopterin (BH4) is an essential and rate-limiting cofactor for the production of NO. Depletion of BH4 in oxidative-stressed endothelial cells can result in so-called NOS3 “uncoupling,” resulting in production of superoxide instead of NO. Moreover, distribution of arginine between intracellular transporters and arginine-converting enzymes, as well as between the arginine-converting and arginine-synthesizing enzymes, determines the metabolic fate of arginine. Alternatively, NO can be derived from conversion of nitrite. Reduced arginine availability stemming from reduced de novo production and elevated arginase activity have been reported in various conditions of acute and chronic stress, which are often characterized by increased NOS2 and reduced NOS3 activity. Cardiovascular and pulmonary disorders such as atherosclerosis, diabetes, hypercholesterolemia, ischemic heart disease, and hypertension are characterized by NOS3 uncoupling. Therapeutic applications to influence (de novo) arginine and NO metabolism aim at increasing substrate availability or at influencing the metabolic fate of specific pathways related to NO bioavailability and prevention of NOS3 uncoupling. These include supplementation of arginine or citrulline, provision of NO donors including inhaled NO and nitrite (sources), NOS3 modulating agents, or the targeting of endogenous NOS inhibitors like asymmetric dimethylarginine. PMID:23011059