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Sample records for microsomal protein synthesis

  1. Microsomal protein synthesis inhibition: an early manifestation of gentamicin nephrotoxicity

    SciTech Connect

    Bennett, W.M.; Mela-Riker, L.M.; Houghton, D.C.; Gilbert, D.N.; Buss, W.C.

    1988-08-01

    Aminoglycoside antibiotics achieve bacterial killing by binding to bacterial ribosomes and inhibiting protein synthesis. To examine whether similar mechanisms could be present in renal tubular cells prior to the onset of overt proximal tubular necrosis due to these drugs, we isolated microsomes from Fischer rats given 20 mg/kg gentamicin every 12 h subcutaneously for 2 days and from vehicle-injected controls. Concomitant studies of renal structure, function, and mitochondrial respiration were carried out. (3H)leucine incorporation into renal microsomes of treated animals was reduced by 21.9% (P less than 0.01), whereas brain and liver microsomes from the same animals were unaffected. Gentamicin concentration in the renal microsomal preparation was 56 micrograms/ml, a value 7- to 10-fold above concentrations necessary to inhibit bacterial growth. Conventional renal function studies were normal (blood urea, serum creatinine, creatinine clearance). Treated animals showed only a mild reduction of inulin clearance, 0.71 compared with 0.93 ml.min-1.100 g-1 in controls (P less than 0.05), and an increase in urinary excretion of N-acetylglucosaminidase of 20 compared with 14.8 units/l (P less than 0.05). Renal slice transport of p-aminohippuric acid, tetraethylammonium, and the fractional excretion of sodium were well preserved. There was no evidence, as seen by light microscopy, of proximal tubular necrosis. Mitochondrial cytochrome concentrations were normal and respiratory activities only slightly reduced. Processes similar to those responsible for bacterial killing could be involved in experimental gentamicin nephrotoxicity before overt cellular necrosis.

  2. Multiple functions of microsomal triglyceride transfer protein

    PubMed Central

    2012-01-01

    Microsomal triglyceride transfer protein (MTP) was first identified as a major cellular protein capable of transferring neutral lipids between membrane vesicles. Its role as an essential chaperone for the biosynthesis of apolipoprotein B (apoB)-containing triglyceride-rich lipoproteins was established after the realization that abetalipoproteinemia patients carry mutations in the MTTP gene resulting in the loss of its lipid transfer activity. Now it is known that it also plays a role in the biosynthesis of CD1, glycolipid presenting molecules, as well as in the regulation of cholesterol ester biosynthesis. In this review, we will provide a historical perspective about the identification, purification and characterization of MTP, describe methods used to measure its lipid transfer activity, and discuss tissue expression and function. Finally, we will review the role MTP plays in the assembly of apoB-lipoprotein, the regulation of cholesterol ester synthesis, biosynthesis of CD1 proteins and propagation of hepatitis C virus. We will also provide a brief overview about the clinical potentials of MTP inhibition. PMID:22353470

  3. Protein Targets of Reactive Electrophiles in Human Liver Microsomes

    PubMed Central

    Shin, Nah-Young; Liu, Qinfeng; Stamer, Sheryl L.; Liebler, Daniel C.

    2008-01-01

    Liver microsomes are widely used to study xenobiotic metabolism in vitro and covalent binding to microsomal proteins serves as a surrogate marker for toxicity mediated by reactive metabolites. We have applied liquid chromatography-tandem mass spectrometry (LC-MS-MS) to identify protein targets of the biotin-tagged model electrophiles 1-biotinamido-4-(4′-[maleimidoethylcyclohexane]-carboxamido)butane (BMCC) and N-iodoacetyl-N-biotinylhexylenediamine (IAB) in human liver microsomes. The biotin-tagged peptides resulting from in-gel tryptic digestion were enriched by biotin-avidin chromatography and LC-MS-MS was used to identify 376 microsomal cysteine thiol targets of BMCC and IAB in 263 proteins. Protein adduction was selective and reproducible and only 90 specific cysteine sites in 70 proteins (approximately 25% of the total) were adducted by both electrophiles. Differences in adduction selectivity correlated with different biological effects of the compounds, as IAB, but not BMCC induced ER stress in HEK293 cells. Targeted LC-MS-MS analysis of microsomal glutathione-S-transferase cysteine 50, a target of both IAB and BMCC, detected time-dependent adduction by the reactive acetaminophen metabolite N-acetyl-p-benzoquinoneimine during microsomal incubations. The results indicate that electrophiles selectively adduct microsomal proteins, but display differing target selectivities that correlate with differences in toxicity. Analysis of selected microsomal protein adduction reactions thus could provide a more specific indication of potential toxicity than bulk covalent binding of radiolabeled compounds. PMID:17480101

  4. Prostaglandin synthesis by chicken and rat lung microsomes

    SciTech Connect

    Craig-Schmidt, M.C.; Faircloth, S.A.; Wu-Wang, C.Y.

    1986-03-01

    A comparison between chicken and rat lung was made for microsomal prostaglandin (PG) synthesis from 1-/sup 14/C-arachidonic acid. Microsomal protein (2.0 mg) from chicken or rat lung was incubated in the presence of 20 ..mu..g of 1-/sup 14/C-arachidonic acid (specific activity = 3 x 10/sup 6/ dpm/..mu..mol for chicken; 6 x 10/sup 6/ dpm/..mu..mol for rat), 0.05 M Tris-HCl buffer (pH = 8.0), 0.5 mM epinephrine, and 1 mM reduced glutathione in a total volume of 0.5 ml in a 37/sup 0/C water bath with shaking for 15 min. After acidification with 1 M HCl to pH 3, prostaglandins were extracted with ethyl acetate. The products of the reactions were separated by reversed phase chromatography, and the radioactivity of each prostanoid fraction was determined. The predominant prostanoid synthesized by chicken lung microsomes was PGE/sub 2/, followed by much lower amounts of thromboxane B/sub 2/ (TXB/sub 2/), PGF/sub 2//sub ..cap alpha../ and PGD/sub 2/. In at lung, 6-keto-PGF/sub 1//sub ..cap alpha../ was the predominant product formed, with minor amounts of 6-keto-PGE/sub 1/, TXB/sub 2/, PGF/sub 2//sub ..cap alpha../ and PGD/sub 2/. In rat lung, 6-keto-FGF/sub 1//sub ..cap alpha../ was the predominant product formed, with minor amounts of 6-keto-PGF/sub 1//sub ..cap alpha../ was the predominant product formed, with minor amounts of 6-keto-PGE/sub 1/, TXB/sub 2/, PGF/sub 2//sub ..cap alpha../, PGE/sub 2/ and PGD/sub 2/ being formed. Enzyme specific activity (pmol of PG produced per mg microsomal protein per min) was 11.9 for PGE/sub 2/ produced by chicken lung and 16. 7 for 6-keto-P/sub 1//sub ..cap alpha../ produced by rat lung. Thus, there appears to be a species variation in chicken compared to rat for the lung prostanoids which are known to cause bronchial dilation.

  5. Detection on immunoblot of new proteins from the microsomal fraction recognized by anti-liver-kidney microsome antibodies type 1.

    PubMed

    Ballot, E; Desbos, A; Auger, C; Monier, J C

    1996-09-01

    Previous studies have demonstrated that sera from patients with autoimmune hepatitis type 1 contain antibodies which react with proteins other than the endoplasmic reticulum integral membrane protein of apparent Mr 50,000, now known to be a cytochrome P450 of the IID subfamily. Sera from 141 patients found by immunofluorescence to be positive for anti-liver-kidney microsome antibodies type 1, and sera from 50 blood donors used as controls, were analyzed by immunoblotting experiments on rat liver microsomes, microsomal subfractions, and also microsomes subjected to various treatments, as described in the text. These fractions were characterized morphologically by electronic microscopy and biochemically by different enzymatic activities. Five bands were found to be stained more often by the patients' sera than by the controls' and with a statistically significant difference in frequency. These antigenic proteins were located at apparent Mr 62,000, 58,000, 50,000, 40,000, and 35,000. The 50,000 protein was of course more often stained than the others. Antibodies against these antigens belonged essentially to the IgG1 subclass. For some of them, subcellular localization and membrane topography are discussed. Interestingly, the 58,000 protein is not an integral membrane protein. PMID:8811045

  6. A microsomal ATP-binding protein involved in efficient protein transport into the mammalian endoplasmic reticulum.

    PubMed Central

    Dierks, T; Volkmer, J; Schlenstedt, G; Jung, C; Sandholzer, U; Zachmann, K; Schlotterhose, P; Neifer, K; Schmidt, B; Zimmermann, R

    1996-01-01

    Protein transport into the mammalian endoplasmic reticulum depends on nucleoside triphosphates. Photoaffinity labelling of microsomes with azido-ATP prevents protein transport at the level of association of precursor proteins with the components of the transport machinery, Sec61alpha and TRAM proteins. The same phenotype of inactivation was observed after depleting a microsomal detergent extract of ATP-binding proteins by passage through ATP-agarose and subsequent reconstitution of the pass-through into proteoliposomes. Transport was restored by co-reconstitution of the ATP eluate. This eluate showed eight distinct bands in SDS gels. We identified five lumenal proteins (Grp170, Grp94, BiP/Grp78, calreticulin and protein disulfide isomerase), one membrane protein (ribophorin I) and two ribosomal proteins (L4 and L5). In addition to BiP (Grp78), Grp170 was most efficiently retained on ATP-agarose. Purified BiP did not stimulate transport activity. Sequence analysis revealed a striking similarity of Grp170 and the yeast microsomal protein Lhs1p which was recently shown to be involved in protein transport into yeast microsomes. We suggest that Grp170 mediates efficient insertion of polypeptides into the microsomal membrane at the expense of nucleoside triphosphates. Images PMID:9003769

  7. The late addition of core lipids to nascent apolipoprotein B100, resulting in the assembly and secretion of triglyceride-rich lipoproteins, is independent of both microsomal triglyceride transfer protein activity and new triglyceride synthesis.

    PubMed

    Pan, Meihui; Liang Js, Jun-shan; Fisher, Edward A; Ginsberg, Henry N

    2002-02-01

    Although microsomal triglyceride transfer protein (MTP) and newly synthesized triglyceride (TG) are critical for co-translational targeting of apolipoprotein B (apoB100) to lipoprotein assembly in hepatoma cell lines, their roles in the later stages of lipoprotein assembly remain unclear. Using N-acetyl-Leu-Leu-norleucinal to prevent proteasomal degradation, HepG2 cells were radiolabeled and chased for 0-90 min (chase I). The medium was changed and cells chased for another 150 min (chase II) in the absence (control) or presence of Pfizer MTP inhibitor CP-10447 (CP). As chase I was extended, inhibition of apoB100 secretion by CP during chase II decreased from 75.9% to only 15% of control (no CP during chase II). Additional studies were conducted in which chase I was either 0 or 90 min, and chase II was in the presence of [(3)H]glycerol and either BSA (control), CP (inhibits both MTP activity and TG synthesis),BMS-1976360-1) (BMS) (inhibits only MTP activity), or triacsin C (TC) (inhibits only TG synthesis). When chase I was 0 min, CP, BMS, and TC reduced apoB100 secretion during chase II by 75.3, 73.9, and 53.9%. However, when chase I was 90 min, those agents reduced apoB100 secretion during chase II by only 16.0, 19.2, and 13.9%. Of note, all three inhibited secretion of newly synthesized TG during chase II by 80, 80, and 40%, whether chase I was 0 or 90 min. In both HepG2 cells and McA-RH7777 cells, if chase I was at least 60 min, inhibition of TG synthesis and/or MTP activity did not affect the density of secreted apoB100-lipoproteins under basal conditions. Oleic acid increased secretion of TG-enriched apoB100-lipoproteins similarly in the absence or presence of either of CP, BMS, or TC. We conclude that neither MTP nor newly synthesized TG is necessary for the later stages of apoB100-lipoprotein assembly and secretion in either HepG2 or McA-RH7777 cells. PMID:11704664

  8. New approaches to target microsomal triglyceride transfer protein

    PubMed Central

    Hussain, M.M.; Bakillah, Ahmed

    2009-01-01

    Purpose of review Microsomal triglyceride transfer protein (MTP), a chaperone for the biosynthesis of apolipoprotein B lipoproteins and CD1d, is a therapeutic candidate to decrease plasma lipids and to diminish inflammation. MTP inhibition increases plasma transaminases and tissue lipids, and therefore new approaches are needed to avoid them. Recent findings Inositol requiring enzyme 1β has been identified as a novel intestine-specific regulator of MTP. A new function of MTP in cholesterol ester biosynthesis has been reported. The importance of the phospholipid transfer activity of MTP in the lipidation of apolipoprotein B and CD1d has been indicated. Diurnal variations in MTP expression and its induction by food availability have been observed. On the basis of these and other findings, we propose that upregulation of inositol requiring enzyme 1β, a combined reduction of cellular free cholesterol or triglyceride or both and MTP activity, specific inhibition of phospholipid or triglyceride transfer activities, and targeting of apolipoprotein B–-MTP protein–protein interactions might be pursued to avoid some of the side effects associated with the inhibition of triglyceride transfer activity of MTP. We further speculate that short-lived MTP antagonists may be useful in controlling plasma and tissue lipids and in avoiding steatosis. Summary We have highlighted the importance of addressing the causal relationship between MTP inhibition and aberrant elevations in plasma liver enzymes. The proposed approaches may show that MTP targeting is a viable approach to lower plasma lipids. PMID:18957879

  9. Sphingolipid Long-Chain Base Synthesis in Plants (Characterization of Serine Palmitoyltransferase Activity in Squash Fruit Microsomes).

    PubMed Central

    Lynch, D. V.; Fairfield, S. R.

    1993-01-01

    The activity of serine palmitoyltransferase (palmitoyl-coenzyme A [CoA]:L-serine [Ser]-C-palmitoyltransferase [decarboxylating], EC 2.3.1.50), the enzyme catalyzing the first step in the synthesis of the long-chain base required for sphingolipid assembly, has been characterized in a plant system. Enzyme activity in a microsomal membrane fraction from summer squash fruit (Cucurbita pepo L. cv Early Prolific Straightneck) was assayed by monitoring the incorporation of L-[3H]Ser into the chloroform-soluble product, 3-ketosphinganine. Addition of NADPH to the assay system resulted in the conversion of 3-ketosphinganine to sphinganine. The apparent Km for Ser was approximately 1.8 mM. The enzyme exhibited a strong preference for palmitoyl-CoA, with optimal activity at a substrate concentration of 200 [mu]M. Pyridoxal 5[prime]-phosphate was required as a coenzyme. The pH optimum was 7.6, and the temperature optimum was 36 to 40[deg]C. Enzyme activity was greatest in the microsomal fraction obtained by differential centrifugation and was localized to the endoplasmic reticulum using marker enzymes. Two known mechanism-based inhibitors of the mammalian enzyme, L-cycloserine and [beta]-chloro-L-alanine, were effective inhibitors of enzyme activity in squash microsomes. Changes in enzyme activity with size (age) of squash fruit were observed. The results from this study suggest that the properties and catalytic mechanism of Ser palmitoyltransferase from squash are similar to those of the animal, fungal, and bacterial enzyme in most respects. The specific activity of the enzyme in squash microsomes ranged from 0.57 to 0.84 nmol min-1 mg-1 of protein, values 2- to 20-fold higher than those previously reported for preparations from animal tissues. PMID:12232036

  10. Stabilization of glucose-6-phosphatase activity by a 21 000-dalton hepatic microsomal protein.

    PubMed Central

    Burchell, A; Burchell, B; Monaco, M; Walls, H E; Arion, W J

    1985-01-01

    Hepatic microsomal glucose-6-phosphatase activity was rendered extremely unstable by a variety of techniques: (a) incubation at pH 5.0; (b) extraction of the microsomal fraction in the presence of 1% Lubrol; (c) various purification procedures. These techniques all result in the removal of a 21 kDa polypeptide from the fraction containing glucose-6-phosphatase activity. The 21 kDa protein was purified to apparent homogeneity by solubilization in the detergent Lubrol 12A-9 and chromatography on Fractogel TSK DEAE-650(S) and centrifugation at 105 000 g. The 21 kDa protein stabilizes glucose-6-phosphatase activity, whereas other purified hepatic microsomal proteins do not. The 21 kDa protein appears to be a potential regulator of glucose-6-phosphatase activity. Images Fig. 1. Fig. 2. Fig. 3. Fig. 4. PMID:2996501

  11. Changes in phylloquinone epoxidase activity related to prothrombin synthesis and microsomal clotting activity in the rat

    PubMed Central

    Willingham, Allan K.; Matschiner, John T.

    1974-01-01

    The oxidation of phylloquinone to the 2,3-epoxide (by phylloquinone epoxidase) was studied in liver from control and warfarin-resistant rats. The reaction requires microsomal fraction, soluble protein, a heat-stable soluble factor and O2. It is not inhibited by CO or CN−. Epoxidase activity was stimulated if plasma prothrombin was lowered either by anticoagulants or the absence of vitamin K. The activity of the enzyme rapidly returned to normal values after the administration of vitamin K to hypoprothrombinaemic rats. These differences in the activity of the enzyme occur in the microsomal fraction and not the cytosol. A thrombin-generating polypeptide that accumulates in microsomal fraction of hypothrombinaemic rats correlated directly with epoxidase activity. These data support the view that enzymic interconversion of phylloquinone and its 2,3-epoxide participates in the biological activity of vitamin K. PMID:4155625

  12. A two-dimensional protein map of Pleurotus ostreatus microsomes-proteome dynamics.

    PubMed

    Petráčková, Denisa; Halada, Petr; Bezoušková, Silvia; Křesinová, Zdena; Svobodová, Kateřina

    2016-01-01

    Recent studies documented that several processes in filamentous fungi are connected with microsomal enzyme activities. In this work, microsomal subproteomes of Pleurotus ostreatus were analyzed by two-dimensional (2-D) polyacrylamide gel electrophoresis and matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometry analysis. To assess proteome dynamics, microsomal proteins were isolated from fungal cultures after 7 and 12 days of cultivation. Additionally, 10 mg/L of 17α-ethinylestradiol (EE2) was treated with the cultures during 2 days. Despite the EE2 degradation by the fungus reached 97 and 76.3 % in 7- and 12-day-old cultures, respectively, only a minor effect on the composition of microsomal proteins was observed. The changes in protein maps related to ageing prevailed over those induced by EE2. Epoxide hydrolase, known to metabolize EE2, was detected in 12-day-old cultures only which suggests differences in EE2 degradation pathways utilized by fungal cultures of different age. The majority (32 %) of identified microsomal proteins were parts of mitochondrial energy metabolism. PMID:26122365

  13. Sulforaphane Inhibits Prostaglandin E2 Synthesis by Suppressing Microsomal Prostaglandin E Synthase 1

    PubMed Central

    Zhou, Jiping; Joplin, Denise G.; Cross, Janet V.; Templeton, Dennis J.

    2012-01-01

    Sulforaphane (SFN) is a dietary cancer preventive with incompletely characterized mechanism(s) of cancer prevention. Since prostaglandin E2 (PGE2) promotes cancer progression, we hypothesized that SFN may block PGE2 synthesis in cancer cells. We found that SFN indeed blocked PGE2 production in human A549 cancer cells not by inhibiting COX-2, but rather by suppressing the expression of microsomal prostaglandin E synthase (mPGES-1), the enzyme that directly synthesizes PGE2. We identified the Hypoxia Inducible Factor 1 alpha (HIF-1α) as the target of SFN-mediated mPGES-1 suppression. SFN suppressed HIF-1α protein expression and the presence of HIF-1α at the mPGES-1 promoter, resulting in reduced transcription of mPGES-1. Finally, SFN also reduced expression of mPGES-1 and PGE2 production in A549 xenograft tumors in mice. Together, these results point to the HIF-1α, mPGES-1 and PGE2 axis as a potential mediator of the anti-cancer effects of SFN, and illustrate the potential of SFN for therapeutic control of cancer and inflammation. Harmful side effects in patients taking agents that target the more upstream COX-2 enzyme render the downstream target mPGES-1 a significant target for anti-inflammatory therapy. Thus, SFN could prove to be an important therapeutic approach to both cancer and inflammation. PMID:23166763

  14. Expression of microsomal triglyceride transfer protein in lipoprotein-synthesizing tissues of the developing chicken embryo☆

    PubMed Central

    Eresheim, Christine; Plieschnig, Julia; Ivessa, N. Erwin; Schneider, Wolfgang J.; Hermann, Marcela

    2014-01-01

    In contrast to mammals, in the chicken major sites of lipoprotein synthesis and secretion are not only the liver and intestine, but also the kidney and the embryonic yolk sac. Two key components in the assembly of triglyceride-rich lipoproteins are the microsomal triglyceride transfer protein (MTP) and apolipoprotein B (apoB). We have analyzed the expression of MTP in the embryonic liver, small intestine, and kidney, and have studied the expression of MTP in, and the secretion of apoB from, the developing yolk sac (YS). Transcript and protein levels of MTP increase during embryogenesis in YS, liver, kidney, and small intestine, and decrease in YS, embryonic liver, and kidney after hatching. In small intestine, the MTP mRNA level rises sharply during the last trimester of embryo development (after day 15), while MTP protein is detectable only after hatching (day 21). In the YS of 15- and 20-day old embryos, apoB secretion was detected by pulse-chase metabolic radiolabeling experiments and subsequent immunoprecipitation. Taken together, our data reveal the importance of coordinated production of MTP and apoB in chicken tissues capable of secreting triglyceride-rich lipoproteins even before hatching. PMID:24394625

  15. Proteins of rough microsomal membranes related to ribosome binding. I. Identification of ribophorins I and II, membrane proteins characteristics of rough microsomes

    PubMed Central

    1978-01-01

    Rat liver rough microsomes (RM) contain two integral membrane proteins which are not found in smooth microsomes (SM) and appear to be related to the presence of ribosome-binding sites. These proteins, of molecular weight 65,000 and 63,000, were designated ribophorins I and II, respectively. They were not released from the microsomal membranes by alkali or acid treatment, or when the ribosomes were detached by incubation with puromycin in a high salt medium. The anionic detergent sodium deoxycholate caused solubilization of the ribophorins, but neutral detergents led to their recovery with the sedimentable ribosomes. Ribosomal aggregates containing both ribophorins, but few other membrane proteins, were obtained from RM treated with the nonionic detergent Kyro EOB (2.5 X10(-2) M) in a low ionic strength medium. Sedimentation patterns produced by these aggregates resembled those of large polysomes but were not affected by RNase treatment. The aggregates, however, were dispersed by mild trypsinization (10 microgram trypsin for 30 min at 0 degrees C), incubation with deoxycholate, or in a medium of high salt concentration. These treatments led to a concomitant degradation or release of the ribophorins. It was estimated, from the staining intensity of protein bands in acrylamide gels, that in the Kyro EOB aggregates there were one to two molecules of each ribophorin per ribosome. Sedimentable complexes without ribosomes containing both ribophorins could also be obtained by dissolving RM previously stripped of ribosomes by puromycin- KCl using cholate, a milder detergent than DOC. Electron microscope examination of the residue obtained from RM treated with Kyro EOB showed that the rapidly sedimenting polysome-like aggregates containing the ribophorins consisted of groups of tightly packed ribosomes which were associated with remnants of the microsomal membranes. PMID:649658

  16. Proteins of rough microsomal membranes related to ribosome binding. I. Identification of ribophorins I and II, membrane proteins characteristics of rough microsomes.

    PubMed

    Kreibich, G; Ulrich, B L; Sabatini, D D

    1978-05-01

    Rat liver rough microsomes (RM) contain two integral membrane proteins which are not found in smooth microsomes (SM) and appear to be related to the presence of ribosome-binding sites. These proteins, of molecular weight 65,000 and 63,000, were designated ribophorins I and II, respectively. They were not released from the microsomal membranes by alkali or acid treatment, or when the ribosomes were detached by incubation with puromycin in a high salt medium. The anionic detergent sodium deoxycholate caused solubilization of the ribophorins, but neutral detergents led to their recovery with the sedimentable ribosomes. Ribosomal aggregates containing both ribophorins, but few other membrane proteins, were obtained from RM treated with the nonionic detergent Kyro EOB (2.5 X10(-2) M) in a low ionic strength medium. Sedimentation patterns produced by these aggregates resembled those of large polysomes but were not affected by RNase treatment. The aggregates, however, were dispersed by mild trypsinization (10 microgram trypsin for 30 min at 0 degrees C), incubation with deoxycholate, or in a medium of high salt concentration. These treatments led to a concomitant degradation or release of the ribophorins. It was estimated, from the staining intensity of protein bands in acrylamide gels, that in the Kyro EOB aggregates there were one to two molecules of each ribophorin per ribosome. Sedimentable complexes without ribosomes containing both ribophorins could also be obtained by dissolving RM previously stripped of ribosomes by puromycin-KCl using cholate, a milder detergent than DOC. Electron microscope examination of the residue obtained from RM treated with Kyro EOB showed that the rapidly sedimenting polysome-like aggregates containing the ribophorins consisted of groups of tightly packed ribosomes which were associated with remnants of the microsomal membranes. PMID:649658

  17. The cell-free synthesis of cytochrome c by a microsomal fraction from rat liver

    PubMed Central

    González-Cadavid, Néstor F.; Ortega, Juan P.; González, Magally

    1971-01-01

    Conditions were investigated for demonstrating the synthesis in vitro of the complete molecule of cytochrome c by isolated liver microsomal systems from partially hepatectomized rats. It was first found that in vivo the early labelled cytochrome c associated with the microsomal fraction required, by comparison with the mitochondrial pool, more drastic conditions of extraction and its binding was less affected by freezing and thawing of the subcellular particles. The procedure of extraction and purification of cytochrome c had to be modified accordingly, to assure the recovery of the recently synthesized molecule. Several subcellular fractions were isolated from regenerating liver with a homogenization medium containing either 5 or 10mm-Mg2+ and most of them were active in the synthesis of the cytochrome c apoprotein. The microsomal fraction, in the presence of either cell sap or pH5.0 fraction, was also able to incorporate [59Fe]haemin, δ-amino[3H]laevulic acid and 55Fe into the prosthetic group of cytochrome c. These experiments confirm firmly the conclusions of our previous results obtained in vivo showing that both the apoprotein and the haem moieties are made and linked together on cytoplasmic ribosomes and only then is the complete molecule transferred to the mitochondria. PMID:5131727

  18. Inhibition of protein carbonyl formation and lipid peroxidation by glutathione in rat liver microsomes.

    PubMed

    Palamanda, J R; Kehrer, J P

    1992-02-14

    The peroxidation of rat liver microsomal lipids is stimulated in the presence of iron by the addition of NADPH or ascorbate and is inhibited by the addition of glutathione (GSH). The fate of GSH and the oxidative modification of proteins under these conditions have not been well studied. Rat liver microsomes were incubated at 37 degrees C under 95% O2:5% CO2 in the presence of 10 microM ferric chloride, 400 microM ADP, and either 450 microM ascorbic acid or 400 microM NADPH. Lipid peroxidation was assessed in the presence 0, 0.2, 0.5, 1, or 5 mM GSH by measuring thiobarbituric acid reactive substance (TBARS) and oxidative modification of proteins by measuring protein thiol and carbonyl groups. GSH inhibited TBARS and protein carbonyl group formation in both ascorbate and NADPH systems in a dose-dependent manner. Heat denaturing of microsomes or treatment with trypsin resulted in the loss of this protection. The formation of protein carbonyl groups could be duplicated by incubating microsomes with 4-hydroxynonenal. Ascorbate-dependent peroxidation caused a loss of protein thiol groups which was diminished by GSH only in fresh microsomes. Both boiling and trypsin treatment significantly decreased the basal protein thiol content of microsomes and enhanced ascorbate-stimulated lipid peroxidation. Protection against protein carbonyl group formation by GSH correlated with the inhibition of lipid peroxidation and appeared not to be due to the formation of the GSH conjugate of 4-hydroxynonenal as only trace amounts of this conjugate were detected. Ninety percent of the GSH lost after 60 min of peroxidation was recoverable as borohydride reducible material in the supernatant fraction. The remaining 10% could be accounted for as GSH-bound protein mixed disulfides. However, only 75% of the GSH lost during peroxidation appeared as glutathione disulfide, suggesting that some was converted to other soluble borohydride reducible forms. These data support a role for protein thiol

  19. Identification of protein components of the microsomal glucose 6-phosphate transporter by photoaffinity labelling.

    PubMed Central

    Kramer, W; Burger, H J; Arion, W J; Corsiero, D; Girbig, F; Weyland, C; Hemmerle, H; Petry, S; Habermann, P; Herling, A

    1999-01-01

    The glucose-6-phosphatase system catalyses the terminal step of hepatic glucose production from both gluconeogenesis and glycogenolysis and is thus a key regulatory factor of blood glucose homoeostasis. To identify the glucose 6-phosphate transporter T1, we have performed photoaffinity labelling of human and rat liver microsomes by using the specific photoreactive glucose-6-phosphate translocase inhibitors S 0957 and S 1743. Membrane proteins of molecular mass 70, 55, 33 and 31 kDa were labelled in human microsomes by [3H]S 0957, whereas in rat liver microsomes bands at 95, 70, 57, 54, 50, 41, 33 and 31 kDa were detectable. The photoprobe [3H]S 1743 led to the predominant labelling of a 57 kDa and a 50 kDa protein in the rat. Stripping of microsomes with 0.3% CHAPS retains the specific binding of T1 inhibitors; photoaffinity labelling of such CHAPS-treated microsomes resulted in the labelling of membrane proteins of molecular mass 55, 33 and 31 kDa in human liver and 50, 33 and 31 kDa in rat liver. Photoaffinity labelling of human liver tissue samples from a healthy individual and from liver samples of patients with a diagnosed glycogen-storage disease type 1b (GSD type 1b; von Gierke's disease) revealed the absence of the 55 kDa protein from one of the patients with GSD type 1. These findings support the identity of the glucose 6-phosphate transporter T1, with endoplasmic reticulum protein of molecular mass 50 kDa in rat liver and 55 kDa in human liver. PMID:10215602

  20. Identification of protein components of the microsomal glucose 6-phosphate transporter by photoaffinity labelling.

    PubMed

    Kramer, W; Burger, H J; Arion, W J; Corsiero, D; Girbig, F; Weyland, C; Hemmerle, H; Petry, S; Habermann, P; Herling, A

    1999-05-01

    The glucose-6-phosphatase system catalyses the terminal step of hepatic glucose production from both gluconeogenesis and glycogenolysis and is thus a key regulatory factor of blood glucose homoeostasis. To identify the glucose 6-phosphate transporter T1, we have performed photoaffinity labelling of human and rat liver microsomes by using the specific photoreactive glucose-6-phosphate translocase inhibitors S 0957 and S 1743. Membrane proteins of molecular mass 70, 55, 33 and 31 kDa were labelled in human microsomes by [3H]S 0957, whereas in rat liver microsomes bands at 95, 70, 57, 54, 50, 41, 33 and 31 kDa were detectable. The photoprobe [3H]S 1743 led to the predominant labelling of a 57 kDa and a 50 kDa protein in the rat. Stripping of microsomes with 0.3% CHAPS retains the specific binding of T1 inhibitors; photoaffinity labelling of such CHAPS-treated microsomes resulted in the labelling of membrane proteins of molecular mass 55, 33 and 31 kDa in human liver and 50, 33 and 31 kDa in rat liver. Photoaffinity labelling of human liver tissue samples from a healthy individual and from liver samples of patients with a diagnosed glycogen-storage disease type 1b (GSD type 1b; von Gierke's disease) revealed the absence of the 55 kDa protein from one of the patients with GSD type 1. These findings support the identity of the glucose 6-phosphate transporter T1, with endoplasmic reticulum protein of molecular mass 50 kDa in rat liver and 55 kDa in human liver. PMID:10215602

  1. Content and activity of human liver microsomal protein and prediction of individual hepatic clearance in vivo

    PubMed Central

    Zhang, Haifeng; Gao, Na; Tian, Xin; Liu, Tingting; Fang, Yan; Zhou, Jun; Wen, Qiang; Xu, Binbin; Qi, Bing; Gao, Jie; Li, Hongmeng; Jia, Linjing; Qiao, Hailing

    2015-01-01

    The lack of information concerning individual variation in content and activity of human liver microsomal protein is one of the most important obstacles for designing personalized medicines. We demonstrated that the mean value of microsomal protein per gram of liver (MPPGL) was 39.46 mg/g in 128 human livers and up to 19-fold individual variations existed. Meanwhile, the metabolic activities of 10 cytochrome P450 (CYPs) were detected in microsomes and liver tissues, respectively, which showed huge individual variations (200-fold). Compared with microsomes, the activities of liver tissues were much suitable to express the individual variations of CYP activities. Furthermore, individual variations in the in vivo clearance of tolbutamide were successfully predicted with the individual parameter values. In conclusion, we offer the values for MPPGL contents in normal liver tissues and build a new method to assess the in vitro CYP activities. In addition, large individual variations exist in predicted hepatic clearance of tolbutamide. These findings provide important physiological parameters for physiologically-based pharmacokinetics models and thus, establish a solid foundation for future development of personalized medicines. PMID:26635233

  2. Identification of Acetaminophen Adducts of Rat Liver Microsomal Proteins using 2D-LC-MS/MS.

    PubMed

    Golizeh, Makan; LeBlanc, André; Sleno, Lekha

    2015-11-16

    Xenobiotic metabolism in the liver can give rise to reactive metabolites that covalently bind to proteins, and determining which proteins are targeted is important in drug discovery and molecular toxicology. However, there are difficulties in the analysis of these modified proteins in complex biological matrices due to their low abundance. In this study, an analytical approach was developed to systematically identify target proteins of acetaminophen (APAP) in rat liver microsomes (RLM) using two-dimensional chromatography and high-resolution tandem mass spectrometry. In vitro microsomal incubations, with and without APAP, were digested and subjected to strong cation exchange (SCX) fractionation prior to reverse-phase UHPLC-MS/MS. Four data processing strategies were combined into an efficient label-free workflow meant to eliminate potential false positives, using peptide spectral matching, statistical differential analysis, product ion screening, and a custom-built delta-mass filtering tool to pinpoint potential modified peptides. This study revealed four proteins, involved in important cellular processes, to be covalently modified by APAP. Data are available via ProteomeXchange with identifier PXD002590. PMID:26510387

  3. The activity of microsomal triglyceride transfer protein is essential for accumulation of triglyceride within microsomes in McA-RH7777 cells. A unified model for the assembly of very low density lipoproteins.

    PubMed

    Wang, Y; Tran, K; Yao, Z

    1999-09-24

    Previously, based on distinct requirement of microsomal triglyceride transfer protein (MTP) and kinetics of triglyceride (TG) utilization, we concluded that assembly of very low density lipoproteins (VLDL) containing B48 or B100 was achieved through different paths (Wang, Y. , McLeod, R. S., and Yao, Z. (1997) J. Biol. Chem. 272, 12272-12278). To test if the apparent dual mechanisms were accounted for by apolipoprotein B (apoB) length, we studied VLDL assembly using transfected cells expressing various apoB forms (e.g. B64, B72, B80, and B100). For each apoB, enlargement of lipoprotein to form VLDL via bulk TG incorporation was induced by exogenous oleate, which could be blocked by MTP inhibitor BMS-197636 treatment. While particle enlargement was readily demonstrable by density ultracentrifugation for B64- and B72-VLDL, it was not obvious for B80- and B100-VLDL unless the VLDL was further resolved by cumulative rate flotation into VLDL(1) (S(f) > 100) and VLDL(2) (S(f) 20-100). BMS-197636 diminished B100 secretion in a dose-dependent manner (0.05-0.5 microM) and also blocked the particle enlargement from small to large B100-lipoproteins. These results yield a unified model that can accommodate VLDL assembly with all apoB forms, which invalidates our previous conclusion. To gain a better understanding of the MTP action, we examined the effect of BMS-197636 on lipid and apoB synthesis during VLDL assembly. While BMS-197636 (0.2 microM) entirely abolished B100-VLDL(1) assembly/secretion, it did not affect B100 translation or translocation across the microsomal membrane, nor did it affect TG synthesis and cell TG mass. However, BMS-197636 drastically decreased accumulation of [(3)H]glycerol-labeled TG and TG mass within microsomal lumen. The decreased TG accumulation was not a result of impaired B100-VLDL assembly, because in cells treated with brefeldin A (0.2 microgram/ml), the assembly of B100-VLDL was blocked yet lumenal TG accumulation was normal. Thus, MTP plays

  4. Baculovirus expression and biochemical characterization of the human microsomal triglyceride transfer protein.

    PubMed Central

    Ritchie, P J; Decout, A; Amey, J; Mann, C J; Read, J; Rosseneu, M; Scott, J; Shoulders, C C

    1999-01-01

    The microsomal triglyceride transfer protein (MTP) complexed to protein disulphide isomerase (PDI) is obligatory for the assembly of chylomicrons and very-low-density lipoproteins. The determination of the atomic structure of the MTP-PDI heterodimer has important implications for the treatment of those forms of hyperlipidaemia associated with the overproduction of very-low-density lipoproteins, which predispose to premature coronary heart disease. To perform structural studies of the human MTP-PDI complex it was necessary to produce milligram quantities of pure protein. We chose the baculovirus expression system for this purpose. Insects cells were co-infected with recombinant viruses encoding FLAG-tagged MTP and His-tagged PDI; the resulting heterodimer was purified by affinity chromatography. From 5 litres of insect cells, 4-6 mg of more than 95% pure recombinant protein was obtained. CD and attenuated total reflection Fourier-transform infrared spectroscopy indicate that the purified protein has around 34% alpha-helical and 33% beta-structure content. The recombinant protein had a comparable triglyceride transfer activity to that of bovine MTP-PDI. The production of polyclonal antibodies raised against the MTP and PDI subunits of the purified protein is described. The present study demonstrates the feasibility of expressing two proteins at high levels in insect cells and describes a transferable methodology for the purification of the resulting protein complex. PMID:10036224

  5. Ultraviolet-induced photodegradation of cucumber (Cucumis sativus L. ) microsomal and soluble protein tryptophanyl residues in vitro

    SciTech Connect

    Caldwell, C.R. )

    1993-03-01

    The in vitro effects of ultraviolet B (280--320 nm) radiation on microsomal membrane proteins and partially purified ribulose bisphosphate carboxylase (Rubisco) from cucumber (Cucumis sativus L.) was investigated by measuring the direct photolytic reduction of tryptophan fluorescence and the formation of fluorescent photooxidation products. Exposure of microsomes and Rubisco to monochromatic 300-nm radiation resulted in the loss of intrinsic tryptophan fluorescence and the production of blue-emitting fluorophores. The major product of tryptophan photolysis was tentatively identified as N-formylkynurenine (N-FK). Even though the rates of tryptophan photodegradation and N-FK formation were similar, the amount of blue fluorescence produced was significantly higher in the microsomes relative to Rubisco. Studies with various free radical scavengers and other modifiers indicated that tryptophan photodegradation requires oxygen species. The optimum wavelengths for loss of tryptophan fluorescence were 290 nm for the microsomes and 280 nm for Rubisco. The temperature dependence of tryptophan fluorescence and rate of tryptophan photodegradation indicated an alteration in the cucumber microsomal membranes at about 24[degrees]C, which influenced protein structure and tryptophan photosensitivity. 29 refs., 6 figs., 1 tab.

  6. Comparison of the pharmacological profiles of murine antisense oligonucleotides targeting apolipoprotein B and microsomal triglyceride transfer protein.

    PubMed

    Lee, Richard G; Fu, Wuxia; Graham, Mark J; Mullick, Adam E; Sipe, Donna; Gattis, Danielle; Bell, Thomas A; Booten, Sheri; Crooke, Rosanne M

    2013-03-01

    Therapeutic agents that suppress apolipoprotein B (apoB) and microsomal triglyceride transfer protein (MTP) levels/activity are being developed in the clinic to benefit patients who are unable to reach target LDL-C levels with maximally tolerated lipid-lowering drugs. To compare and contrast the metabolic consequences of reducing these targets, murine-specific apoB or MTP antisense oligonucleotides (ASOs) were administered to chow-fed and high fat-fed C57BL/6 or to chow-fed and Western diet-fed LDLr⁻/⁻ mice for periods ranging from 2 to 12 weeks, and detailed analyses of various factors affecting fatty acid metabolism were performed. Administration of these drugs significantly reduced target hepatic mRNA and protein, leading to similar reductions in hepatic VLDL/triglyceride secretion. MTP ASO treatment consistently led to increases in hepatic triglyceride accumulation and biomarkers of hepatotoxicity relative to apoB ASO due in part to enhanced expression of peroxisome proliferator activated receptor γ target genes and the inability to reduce hepatic fatty acid synthesis. Thus, although both drugs effectively lowered LDL-C levels in mice, the apoB ASO produced a more positive liver safety profile. PMID:23220583

  7. Discovery of Novel Splice Variants and Regulatory Mechanisms for Microsomal Triglyceride Transfer Protein in Human Tissues

    PubMed Central

    Suzuki, Takashi; Swift, Larry L.

    2016-01-01

    Microsomal triglyceride transfer protein (MTP) is a unique lipid transfer protein essential for the assembly of triglyceride-rich lipoproteins by the liver and intestine. Previous studies in mice identified a splice variant of MTP with an alternate first exon. Splice variants of human MTP have not been reported. Using PCR approaches we have identified two splice variants in human tissues, which we have named MTP-B and MTP-C. MTP-B has a unique first exon (Ex1B) located 10.5 kb upstream of the first exon (Ex1A) for canonical MTP (MTP-A); MTP-C contains both first exons for MTP-A and MTP-B. MTP-B was found in a number of tissues, whereas MTP-C was prominent in brain and testis. MTP-B does not encode a protein; MTP-C encodes the same protein encoded by MTP-A, although MTP-C translation is strongly inhibited by regulatory elements within its 5′-UTR. Using luciferase assays, we demonstrate that the promoter region upstream of exon 1B is quite adequate to drive expression of MTP. We conclude that alternate splicing plays a key role in regulating cellular MTP levels by introducing distinct promoter regions and unique 5′-UTRs, which contain elements that alter translation efficiency, enabling the cell to optimize MTP activity. PMID:27256115

  8. Discovery of Novel Splice Variants and Regulatory Mechanisms for Microsomal Triglyceride Transfer Protein in Human Tissues.

    PubMed

    Suzuki, Takashi; Swift, Larry L

    2016-01-01

    Microsomal triglyceride transfer protein (MTP) is a unique lipid transfer protein essential for the assembly of triglyceride-rich lipoproteins by the liver and intestine. Previous studies in mice identified a splice variant of MTP with an alternate first exon. Splice variants of human MTP have not been reported. Using PCR approaches we have identified two splice variants in human tissues, which we have named MTP-B and MTP-C. MTP-B has a unique first exon (Ex1B) located 10.5 kb upstream of the first exon (Ex1A) for canonical MTP (MTP-A); MTP-C contains both first exons for MTP-A and MTP-B. MTP-B was found in a number of tissues, whereas MTP-C was prominent in brain and testis. MTP-B does not encode a protein; MTP-C encodes the same protein encoded by MTP-A, although MTP-C translation is strongly inhibited by regulatory elements within its 5'-UTR. Using luciferase assays, we demonstrate that the promoter region upstream of exon 1B is quite adequate to drive expression of MTP. We conclude that alternate splicing plays a key role in regulating cellular MTP levels by introducing distinct promoter regions and unique 5'-UTRs, which contain elements that alter translation efficiency, enabling the cell to optimize MTP activity. PMID:27256115

  9. Studies on the metabolism of chlorotrianisene to a reactive intermediate and subsequent covalent binding to microsomal proteins

    SciTech Connect

    Juedes, M.J.

    1989-01-01

    The studies on chlorotrianisene were conducted to determine whether metabolism of chlorotrianisene occurs via the cytochrome P450 monooxygenase system and whether a reactive intermediate is being formed that is capable of binding covalently to microsomal proteins. ({sup 3}H)-chlorotrianisene was incubated with liver microsomes supplemented with NADPH. At the termination of the incubation, the protein was trapped on a glass filter and the unbound chlorotrianisene was removed by extensive washing of the protein with organic solvent. A dramatic stimulation of covalent binding was demonstrated in microsomes from rats treated with methylcholanthrene (60 fold increase) versus control or phenobarbital treatment. Verification of covalent binding was achieved by localization of radiolabeled bands following sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) of the macromolecules in the incubation mixture. Further analysis of the radiolabeled macromolecules separated on SDS-PAGE revealed that these macromolecules were degraded by protease degradation indicating that the macromolecules were proteins. Further investigations were done to determine the cause of the dramatic stimulation of covalent binding detected in microsomes from methylcholanthrene treated rats versus control or phenobarbital treated rats. Further evidence for the participation of P-450c was obtained with a reconstituted cytochrome P-450 system. Incubations of chlorotrianisene with reconstituted P-450c and NADPH-cytochrome P-450 reductase exhibited covalent binding characteristics comparable to those seen in microsomal incubations. Investigations into the nature of the binding site and the reactive intermediate are currently being conducted. By analyzing the BSA adduct, the author intends to isolate the specific amino acid binding site(s).

  10. Microsomal triacylglycerol transfer protein prevents presecretory degradation of apolipoprotein B-100. A dithiothreitol-sensitive protease is involved.

    PubMed

    Benoist, F; Nicodeme, E; Grand-Perret, T

    1996-09-15

    The role of microsomal triacylglycerol transfer protein (MTP) in the secretion of apolipoprotein B-100 (apoB-100) has been studied using an inhibitor of MTP: 4'-bromo-3'-methylmetaqualone. In vitro, this compound inhibits trioleoylglycerol transfer between lipid vesicles mediated by MTP with an IC50 of 0.9 microM whereas it does not inhibit the lipid transfer mediated by the cholesteryl ester transfer protein. In HepG2 cells, 4'-bromo-3'-methylmetaqualone inhibits the secretion of apoB-100 with an IC50 of 0.3 microM, without affecting the secretion of several other proteins like apoA-I or albumin. Moreover, there is no accumulation of apoB-100 in treated cells. Oleic acid, which increases apoB-100 secretion, only slightly modifies the IC50 of 4'-bromo-3'-methylmetaqualone (0.5 microM). The latter has no effect on the synthesis of major lipids within the cell, but decreases the secretion of triacylglycerol into apoB-100-containing lipoproteins. Pulse/chase experiments reveal that 4'-bromo-3'-methylmetaqualone acts on apoB-100 production either at the co-translational or post-translational level. The cysteine protease inhibitor N-acetyl-leucyl-leucyl-norleucinal does not protect apoB-100 from the 4'-bromo-3'-methylmetaqualone effect but seems to be involved in a later step of apoB-100 intracellular degradation. By contrast, dithiothreitol can totally reverse the effect of the MTP inhibitor on apoB-100 production. The mechanism of MTP-mediated lipid assembly with apoB-100 is discussed. PMID:8856075

  11. Endogenous phosphorylation of microsomal proteins in bovine corpus luteum. Tenfold activation by adenosine 3′:5′-cyclic monophosphate

    PubMed Central

    Hardie, D. Grahame; Stansfield, David A.

    1977-01-01

    Free ribosomes and a smooth-microsomal fraction were prepared from bovine corpus luteum. Both preparations will self-phosphorylate when incubated with Mg2+ and ATP, but at low concentrations of Mg2+ and ATP the self-phosphorylation of the smooth-microsomal fraction was much more dependent on cyclic AMP than was that of free ribosomes, stimulation by the nucleotide being up to 10-fold in the former case. The self-phosphorylation of the smooth-microsomal fraction was studied further. The reaction bears similarities to that brought about by soluble cyclic AMP-dependent protein kinase, being inhibited by Ca2+ and the heat-stable inhibitor protein from skeletal muscle. Cyclic GMP will activate the reaction at concentrations higher than those required for full activation by cyclic AMP. In the presence of cyclic AMP, phosphate bound to protein is found almost exclusively as phosphoserine. Several proteins are phosphorylated, as judged by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis, and the phosphorylation of all of them is markedly stimulated by cyclic AMP. If the reaction is carried out at high concentrations of Mg2+ and ATP, a distinct cyclic AMP-independent phosphorylation is observed. This activity is not inhibited by the heat-stable inhibitor protein, and phosphate is found esterified with both threonine and serine residues. PMID:195580

  12. Further evidence that rat liver microsomal glutathione transferase 1 is not a cellular protein target for S-nitrosylation.

    PubMed

    Shi, Qiang; Chen, Hai-Fei; Lou, Yi-Jia

    2006-09-25

    By adopting biotin switch method, we recently reported that liver microsomal glutathione transferase 1 (MGST1) might not be a protein target for S-nitrosylation in rat microsomes or in vivo. However, alternative analytic methods are needed to confirm this observation, as a single biotin switch method in judging specific protein S-nitrosylation in biological samples is increasingly recognized as insufficient, or even unreliable. Besides, only MGST1 localized on endoplasmic reticulum (ER), but not mitochondria which favors protein S-nitrosylation was examined in the previous report. Present study was therefore carried out to address these issues. Primary cultured hepatocytes were used. A physiological existing nitric oxide (NO) donor S-nitrosoglutathione (GSNO) was adopted to trigger protein S-nitrosylation. MGST1 was immunoprecipitated and its S-nitrosothiol content was measured by the NO probe 2,3-diaminonaphthalene. In parallel, S-nitrosylated proteins were immunoprecipitated by a monoclonal anti-S-nitrosocysteine antibody and probed with an anti-MGST1 antibody. In hepatocytes, neither ER nor mitochondria were found to contain S-nitrosylated MGST1 after GSNO treatment, showing that differently distributed MGST1 was consistently un-nitrosylable in the cellular environment. But under broken cell conditions, when samples were incubated directly with GSNO, MGST1 S-nitrosylation was indeed detectable in both the microsomal and mitochondrial proteins, indicating that previous failure in detecting MGST1 S-nitrosylation in microsomes is due to the limitations of biotin switch method. These results clearly, if not definitely, demonstrate that MGST1 is not a ready candidate for S-nitrosylation in the cellular content, despite its susceptibility to S-nitrosylation under broken cell conditions. PMID:16899233

  13. Mutations of the microsomal triglyceride-transfer-protein gene in abetalipoproteinemia.

    PubMed

    Narcisi, T M; Shoulders, C C; Chester, S A; Read, J; Brett, D J; Harrison, G B; Grantham, T T; Fox, M F; Povey, S; de Bruin, T W

    1995-12-01

    Elevated plasma levels of apolipoprotein B (apoB)-containing lipoproteins constitute a major risk factor for the development of coronary heart disease. In the rare recessively inherited disorder abetalipoproteinemia (ABL) the production of apoB-containing lipoproteins is abolished, despite no abnormality of the apoB gene. In the current study we have characterized the gene encoding a microsomal triglyceride-transfer protein (MTP), localized to chromosome 4q22-24, and have identified a mutation of the MTP gene in both alleles of all individuals in a cohort of eight patients with classical ABL. Each mutant allele is predicted to encode a truncated form of MTP with a variable number of aberrant amino acids at its C-terminal end. Expression of genetically engineered forms of MTP in Cos-1 cells indicates that the C-terminal portion of MTP is necessary for triglyceride-transfer activity. Deletion of 20 amino acids from the carboxyl terminus of the 894-amino-acid protein and a missense mutation of cysteine 878 to serine both abolished activity. These results establish that defects of the MTP gene are the predominant, if not sole, cause of hereditary ABL and that an intact carboxyl terminus is necessary for activity. PMID:8533758

  14. Mutations of the Microsomal Triglyceride-Transfer–Protein Gene in Abetalipoproteinemia

    PubMed Central

    Narcisi, Teresa M. E.; Shoulders, Carol C.; Chester, S. Ann; Read, Jacqueline; Brett, David J.; Harrison, Georgina B.; Grantham, Tamsin T.; Fox, Margaret F.; Povey, Sue; de Bruin, Tjerk W. A.; Erkelens, D. Willem; Muller, David P. R.; Lloyd, June K.; Scott, James

    1995-01-01

    Elevated plasma levels of apolipoprotein B (apoB)–containing lipoproteins constitute a major risk factor for the development of coronary heart disease. In the rare recessively inherited disorder abetalipoproteinemia (ABL) the production of apoB-containing lipoproteins is abolished, despite no abnormality of the apoB gene. In the current study we have characterized the gene encoding a microsomal triglyceride-transfer protein (MTP), localized to chromosome 4q22-24, and have identified a mutation of the MTP gene in both alleles of all individuals in a cohort of eight patients with classical ABL. Each mutant allele is predicted to encode a truncated form of MTP with a variable number of aberrant amino acids at its C-terminal end. Expression of genetically engineered forms of MTP in Cos-1 cells indicates that the C-terminal portion of MTP is necessary for triglyceride-transfer activity. Deletion of 20 amino acids from the carboxyl terminus of the 894-amino-acid protein and a missense mutation of cysteine 878 to serine both abolished activity. These results establish that defects of the MTP gene are the predominant, if not sole, cause of hereditary ABL and that an intact carboxyl terminus is necessary for activity. ImagesFigure 1p1304-aFigure 3Figure 4 PMID:8533758

  15. Mutations of the microsomal triglyceride-transfer-protein gene in abetalipoproteinemia

    SciTech Connect

    Narcisi, T.M.E.; Shoulders, C.C.; Chester, S.A.

    1995-12-01

    Elevated plasma levels of apolipoprotein B (apoB)-containing lipoproteins constitute a major risk factor for the development of coronary heart disease. In the rare recessively inherited disorder abetalipoproteinemia (ABL) the production of apoB-containing lipoproteins is abolished, despite no abnormality of the apoB gene. In the current study we have characterized the gene encoding a microsomal triglyceride-transfer protein (MTP), localized to chromosome 4q22-24, and have identified a mutation of the MTP gene in both alleles of all individuals in a cohort of eight patients with classical ABL. Each mutant allele is predicted to encode a truncated form of MTP with a variable number of aberrant amino acids at its C-terminal end. Expression of genetically engineered forms of MTP in Cos-1 cells indicates that the C-terminal portion of MTP is necessary for triglyceride-transfer activity. Deletion of 20 amino acids from the carboxyl terminus of the 894-amino-acid protein and a missense mutation of cysteine 878 to serine both abolished activity. These results establish that defects of the MTP gene are the predominant, if not sole, cause of hereditary ABL and that an intact carboxyl terminus is necessary for activity. 49 refs., 4 figs., 5 tabs.

  16. Molecular cloning, expression, and hormonal regulation of the chicken microsomal triglyceride transfer protein.

    PubMed

    Ivessa, N Erwin; Rehberg, Edward; Kienzle, Bernadette; Seif, Fridolin; Hermann, Robert; Hermann, Marcela; Schneider, Wolfgang J; Gordon, David A

    2013-07-01

    During an egg-laying cycle, oviparous animals transfer massive amounts of triglycerides, the major lipid component of very low density lipoprotein (VLDL), from the liver to the developing oocytes. A major stimulus for this process is the rise in estrogen associated with the onset of an egg-laying cycle. In mammals, the microsomal triglyceride transfer protein (MTP) is required for VLDL assembly and secretion. To enable studies to determine if MTP plays a role in basal and estrogen-stimulated VLDL assembly and secretion in an oviparous vertebrate, we have cloned and sequenced the chicken MTP cDNA. This cDNA encodes a protein of 893 amino acids with an N-terminal signal sequence. The primary sequence of chicken MTP is, on average, 65% identical to that of mammalian homologs, and 23% identical to the Drosophila melanogaster protein. We have obtained a clone of chicken embryo fibroblast cells that stably express the avian MTP cDNA and show that these cells display MTP activity as measured by the transfer of a fluorescently labeled neutral lipid. As in mammals, chicken MTP is localized to the endoplasmic reticulum as revealed by indirect immunofluorescence and by the fact that its N-linked oligosaccharide moiety remains sensitive to endoglycosidase H. Endogenous, enzymatically active MTP is also expressed in an estrogen receptor-expressing chicken hepatoma cell line that secretes apolipoprotein B-containing lipoproteins. In this cell line and in vivo, the expression and activity of MTP are not influenced by estrogen. Therefore, up-regulation of MTP in the liver is not required for the increased VLDL assembly during egg production in the chicken. This indicates that MTP is not rate-limiting, even for the massive estrogen-induced secretion of VLDL accompanying an egg-laying cycle. PMID:23542778

  17. In vitro synthesis of nitroxide free radicals by hog liver microsomes

    SciTech Connect

    Valvis, I.I.; Lischick, D.; Shen, D.; Sofer, S.S. )

    1990-01-01

    The in vitro biooxidation of 4-hydroxy-2,2,6,6-tetra methylpiperidine (TEMP), 4-hydroxy-2,2,4,4-tetra methyl-1,3-oxazolidine (TEMO) and diphenylamine (DPA) by hog liver microsomes to their respective nitroxide free radicals, 4-hydroxy-2,2,6,6-tetra methylpiperidine-1-oxyl (TEMPO), 2,2,4,4-tetra methyl-1,3-oxazolidine-1-oxyl (TEMOO), and diphenylnitroxide (DPNO) has been investigated. For extending the life span of the liver microsomes, a calcium alginate immobilization procedure was used. The biooxidation rates of the above amines to their respective nitroxide metabolites were measured by means of oxygen uptake at 37 degrees C and pH 7.4. N-octylamine was found to be an activator in the biooxidation of the amines. The formation of the nitroxide radicals was identified by E.S.R. spectroscopy.

  18. Identification of a Novel Transcript and Regulatory Mechanism for Microsomal Triglyceride Transfer Protein.

    PubMed

    Suzuki, Takashi; Brown, Judy J; Swift, Larry L

    2016-01-01

    Microsomal triglyceride transfer protein (MTP) is essential for the assembly of triglyceride-rich apolipoprotein B-containing lipoproteins. Previous studies in our laboratory identified a novel splice variant of MTP in mice that we named MTP-B. MTP-B has a unique first exon (1B) located 2.7 kB upstream of the first exon (1A) for canonical MTP (MTP-A). The two mature isoforms, though nearly identical in sequence and function, have different tissue expression patterns. In this study we report the identification of a second MTP splice variant (MTP-C), which contains both exons 1B and 1A. MTP-C is expressed in all the tissues we tested. In cells transfected with MTP-C, protein expression was less than 15% of that found when the cells were transfected with MTP-A or MTP-B. In silico analysis of the 5'-UTR of MTP-C revealed seven ATGs upstream of the start site for MTP-A, which is the only viable start site in frame with the main coding sequence. One of those ATGs was located in the 5'-UTR for MTP-A. We generated reporter constructs in which the 5'-UTRs of MTP-A or MTP-C were inserted between an SV40 promoter and the coding sequence of the luciferase gene and transfected these constructs into HEK 293 cells. Luciferase activity was significantly reduced by the MTP-C 5'-UTR, but not by the MTP-A 5'-UTR. We conclude that alternative splicing plays a key role in regulating MTP expression by introducing unique 5'-UTRs, which contain elements that alter translation efficiency, enabling the cell to optimize MTP levels and activity. PMID:26771188

  19. Identification of a Novel Transcript and Regulatory Mechanism for Microsomal Triglyceride Transfer Protein

    PubMed Central

    Suzuki, Takashi; Brown, Judy J.; Swift, Larry L.

    2016-01-01

    Microsomal triglyceride transfer protein (MTP) is essential for the assembly of triglyceride-rich apolipoprotein B-containing lipoproteins. Previous studies in our laboratory identified a novel splice variant of MTP in mice that we named MTP-B. MTP-B has a unique first exon (1B) located 2.7 kB upstream of the first exon (1A) for canonical MTP (MTP-A). The two mature isoforms, though nearly identical in sequence and function, have different tissue expression patterns. In this study we report the identification of a second MTP splice variant (MTP-C), which contains both exons 1B and 1A. MTP-C is expressed in all the tissues we tested. In cells transfected with MTP-C, protein expression was less than 15% of that found when the cells were transfected with MTP-A or MTP-B. In silico analysis of the 5’-UTR of MTP-C revealed seven ATGs upstream of the start site for MTP-A, which is the only viable start site in frame with the main coding sequence. One of those ATGs was located in the 5’-UTR for MTP-A. We generated reporter constructs in which the 5’-UTRs of MTP-A or MTP-C were inserted between an SV40 promoter and the coding sequence of the luciferase gene and transfected these constructs into HEK 293 cells. Luciferase activity was significantly reduced by the MTP-C 5’-UTR, but not by the MTP-A 5’-UTR. We conclude that alternative splicing plays a key role in regulating MTP expression by introducing unique 5’-UTRs, which contain elements that alter translation efficiency, enabling the cell to optimize MTP levels and activity. PMID:26771188

  20. Promotion of beta-glucan synthase activity in corn microsomal membranes by calcium and protein phosphorylation

    NASA Technical Reports Server (NTRS)

    Paliyath, G.; Poovaiah, B. W.

    1988-01-01

    Regulation of the activity of beta-glucan synthase was studied using microsomal preparations from corn coleoptiles. The specific activity as measured by the incorporation of glucose from uridine diphospho-D-[U-14C]glucose varied between 5 to 15 pmol (mg protein)-1 min-1. Calcium promoted beta-glucan synthase activity and the promotion was observed at free calcium concentrations as low as 1 micromole. Kinetic analysis of substrate-velocity curve showed an apparent Km of 1.92 x 10(-4) M for UDPG. Calcium increased the Vmax from 5.88 x 10(-7) mol liter-1 min-1 in the absence of calcium to 9.52 x 10(-7) mol liter-1 min-1 and 1.66 x 10(-6) mol liter-1 min-1 in the presence of 0.5 mM and 1 mM calcium, respectively. The Km values remained the same under these conditions. Addition of ATP further increased the activity above the calcium-promoted level. Sodium fluoride, a phosphoprotein phosphatase inhibitor, promoted glucan synthase activity indicating that phosphorylation and dephosphorylation are involved in the regulation of the enzyme activity. Increasing the concentration of sodium fluoride from 0.25 mM to 10 mM increased glucan synthase activity five-fold over the + calcium + ATP control. Phosphorylation of membrane proteins also showed a similar increase under these conditions. Calmodulin, in the presence of calcium and ATP stimulated glucan synthase activity substantially, indicating that calmodulin could be involved in the calcium-dependent phosphorylation and promotion of beta-glucan synthase activity. The role of calcium in mediating auxin action is discussed.

  1. Characterization of the ribosomal binding site in rat liver rough microsomes: ribophorins I and II, two integral membrane proteins related to ribosome binding.

    PubMed

    Kreibich, G; Czakó-Graham, M; Grebenau, R; Mok, W; Rodriguez-Boulan, E; Sabatini, D D

    1978-01-01

    Rat liver rough endoplasmic reticulum membranes (ER) contain two characteristic transmembrane glycoproteins which have been designated ribophorins I and II and are absent from smooth ER membranes. These proteins (MW 65,000 and 63,000 respectively) are related to the binding sites for ribosomes, as suggested by the following findings: i) The ribophorin content of the rough ER membranes corresponds stoichiometrically to the number of bound ribosomes; ii) ribophorins are quantitatively recovered with the bound polysomes after most other ER membrane proteins are dissolved with the nonionic detergent Kyro EOB; iii) in intact rough microsomes ribophorins can be cross-linked chemically to the ribosomes and therefore are in close proximity to them. Treatment of rough microsomes with a low Triton-X-100 concentration leads to the lateral displacement of ribosomes on the microsomal surface and to the formation of aggregates of bound ribosomes in areas of membranes which frequently invaginate into the microsomal lumen. Subfractionation of Triton-treated microsomes containing invaginations led to the recovery of smooth and "rough-inverted" vesicles. Ribophorins were present only in the latter fraction, indicating that both proteins are displaced together with the ribosomes when these aggregate without detaching. Measurements of the ribosome-binding capacity of rough and smooth microsomal membranes reconstituted after solubilization with detergents suggest that ribophorins are necessary for in vitro ribosome binding. Ribophorin-like proteins were found in rough microsomes obtained from secretory tissues of several animal species. The two proteins present in rat lacrimal gland microsomes have the same mobility as hepatocyte ribophorins and cross-react with antisera against them. PMID:723266

  2. Intestine-Specific Deletion of Microsomal Triglyceride Transfer Protein Increases Mortality in Aged Mice

    PubMed Central

    Liang, Zhe; Xie, Yan; Dominguez, Jessica A.; Breed, Elise R.; Yoseph, Benyam P.; Burd, Eileen M.; Farris, Alton B.

    2014-01-01

    Background Mice with conditional, intestine-specific deletion of microsomal triglyceride transfer protein (Mttp-IKO) exhibit a complete block in chylomicron assembly together with lipid malabsorption. Young (8–10 week) Mttp-IKO mice have improved survival when subjected to a murine model of Pseudomonas aeruginosa-induced sepsis. However, 80% of deaths in sepsis occur in patients over age 65. The purpose of this study was to determine whether age impacts outcome in Mttp-IKO mice subjected to sepsis. Methods Aged (20–24 months) Mttp-IKO mice and WT mice underwent intratracheal injection with P. aeruginosa. Mice were either sacrificed 24 hours post-operatively for mechanistic studies or followed seven days for survival. Results In contrast to young septic Mttp-IKO mice, aged septic Mttp-IKO mice had a significantly higher mortality than aged septic WT mice (80% vs. 39%, p = 0.005). Aged septic Mttp-IKO mice exhibited increased gut epithelial apoptosis, increased jejunal Bax/Bcl-2 and Bax/Bcl-XL ratios yet simultaneously demonstrated increased crypt proliferation and villus length. Aged septic Mttp-IKO mice also manifested increased pulmonary myeloperoxidase levels, suggesting increased neutrophil infiltration, as well as decreased systemic TNFα compared to aged septic WT mice. Conclusions Blocking intestinal chylomicron secretion alters mortality following sepsis in an age-dependent manner. Increases in gut apoptosis and pulmonary neutrophil infiltration, and decreased systemic TNFα represent potential mechanisms for why intestine-specific Mttp deletion is beneficial in young septic mice but harmful in aged mice as each of these parameters are altered differently in young and aged septic WT and Mttp-IKO mice. PMID:25010671

  3. Microgravity Induces Changes in Microsome-Associated Proteins of Arabidopsis Seedlings Grown on Board the International Space Station

    PubMed Central

    Grat, Sabine; Pichereaux, Carole; Rossignol, Michel; Pereda-Loth, Veronica; Eche, Brigitte; Boucheron-Dubuisson, Elodie; Le Disquet, Isabel; Medina, Francisco Javier; Graziana, Annick; Carnero-Diaz, Eugénie

    2014-01-01

    The “GENARA A” experiment was designed to monitor global changes in the proteome of membranes of Arabidopsis thaliana seedlings subjected to microgravity on board the International Space Station (ISS). For this purpose, 12-day-old seedlings were grown either in space, in the European Modular Cultivation System (EMCS) under microgravity or on a 1 g centrifuge, or on the ground. Proteins associated to membranes were selectively extracted from microsomes and identified and quantified through LC-MS-MS using a label-free method. Among the 1484 proteins identified and quantified in the 3 conditions mentioned above, 80 membrane-associated proteins were significantly more abundant in seedlings grown under microgravity in space than under 1 g (space and ground) and 69 were less abundant. Clustering of these proteins according to their predicted function indicates that proteins associated to auxin metabolism and trafficking were depleted in the microsomal fraction in µg space conditions, whereas proteins associated to stress responses, defence and metabolism were more abundant in µg than in 1 g indicating that microgravity is perceived by plants as a stressful environment. These results clearly indicate that a global membrane proteomics approach gives a snapshot of the cell status and its signaling activity in response to microgravity and highlight the major processes affected. PMID:24618597

  4. Microgravity induces changes in microsome-associated proteins of Arabidopsis seedlings grown on board the international space station.

    PubMed

    Mazars, Christian; Brière, Christian; Grat, Sabine; Pichereaux, Carole; Rossignol, Michel; Pereda-Loth, Veronica; Eche, Brigitte; Boucheron-Dubuisson, Elodie; Le Disquet, Isabel; Medina, Francisco Javier; Graziana, Annick; Carnero-Diaz, Eugénie

    2014-01-01

    The "GENARA A" experiment was designed to monitor global changes in the proteome of membranes of Arabidopsis thaliana seedlings subjected to microgravity on board the International Space Station (ISS). For this purpose, 12-day-old seedlings were grown either in space, in the European Modular Cultivation System (EMCS) under microgravity or on a 1 g centrifuge, or on the ground. Proteins associated to membranes were selectively extracted from microsomes and identified and quantified through LC-MS-MS using a label-free method. Among the 1484 proteins identified and quantified in the 3 conditions mentioned above, 80 membrane-associated proteins were significantly more abundant in seedlings grown under microgravity in space than under 1 g (space and ground) and 69 were less abundant. Clustering of these proteins according to their predicted function indicates that proteins associated to auxin metabolism and trafficking were depleted in the microsomal fraction in µg space conditions, whereas proteins associated to stress responses, defence and metabolism were more abundant in µg than in 1 g indicating that microgravity is perceived by plants as a stressful environment. These results clearly indicate that a global membrane proteomics approach gives a snapshot of the cell status and its signaling activity in response to microgravity and highlight the major processes affected. PMID:24618597

  5. Synthesis of Lipidated Proteins.

    PubMed

    Mejuch, Tom; Waldmann, Herbert

    2016-08-17

    Protein lipidation is one of the major post-translational modifications (PTM) of proteins. The attachment of the lipid moiety frequently determines the localization and the function of the lipoproteins. Lipidated proteins participate in many essential biological processes in eukaryotic cells, including vesicular trafficking, signal transduction, and regulation of the immune response. Malfunction of these cellular processes usually leads to various diseases such as cancer. Understanding the mechanism of cellular signaling and identifying the protein-protein and protein-lipid interactions in which the lipoproteins are involved is a crucial task. To achieve these goals, fully functional lipidated proteins are required. However, access to lipoproteins by means of standard expression is often rather limited. Therefore, semisynthetic methods, involving the synthesis of lipidated peptides and their subsequent chemoselective ligation to yield full-length lipoproteins, were developed. In this Review we summarize the commonly used methods for lipoprotein synthesis and the development of the corresponding chemoselective ligation techniques. Several key studies involving full-length semisynthetic lipidated Ras, Rheb, and LC3 proteins are presented. PMID:27444727

  6. Effect of liver fatty acid binding protein on fatty acid movement between liposomes and rat liver microsomes.

    PubMed Central

    McCormack, M; Brecher, P

    1987-01-01

    Although movement of fatty acids between bilayers can occur spontaneously, it has been postulated that intracellular movement is facilitated by a class of proteins named fatty acid binding proteins (FABP). In this study we have incorporated long chain fatty acids into multilamellar liposomes made of phosphatidylcholine, incubated them with rat liver microsomes containing an active acyl-CoA synthetase, and measured formation of acyl-CoA in the absence or presence of FABP purified from rat liver. FABP increased about 2-fold the accumulation of acyl-CoA when liposomes were the fatty acid donor. Using fatty acid incorporated into liposomes made either of egg yolk lecithin or of dipalmitoylphosphatidylcholine, it was found that the temperature dependence of acyl-CoA accumulation in the presence of FABP correlated with both the physical state of phospholipid molecules in the liposomes and the binding of fatty acid to FABP, suggesting that fatty acid must first desorb from the liposomes before FABP can have an effect. An FABP-fatty acid complex incubated with microsomes, in the absence of liposomes, resulted in greater acyl-CoA formation than when liposomes were present, suggesting that desorption of fatty acid from the membrane is rate-limiting in the accumulation of acyl-CoA by this system. Finally, an equilibrium dialysis cell separating liposomes from microsomes on opposite sides of a Nuclepore filter was used to show that liver FABP was required for the movement and activation of fatty acid between the compartments. These studies show that liver FABP interacts with fatty acid that desorbs from phospholipid bilayers, and promotes movement to a membrane-bound enzyme, suggesting that FABP may act intracellularly by increasing net desorption of fatty acid from cell membranes. PMID:3446187

  7. Protein synthesis rates in rat brain regions and subcellular fractions during aging

    SciTech Connect

    Avola, R.; Condorelli, D.F.; Ragusa, N.; Renis, M.; Alberghina, M.; Giuffrida Stella, A.M.; Lajtha, A.

    1988-04-01

    In vivo protein synthesis rates in various brain regions (cerebral cortex, cerebellum, hippocampus, hypothalamus, and striatum) of 4-, 12-, and 24-month-old rats were examined after injection of a flooding dose of labeled valine. The incorporation of labeled valine into proteins of mitochondrial, microsomal, and cytosolic fractions from cerebral cortex and cerebellum was also measured. At all ages examined, the incorporation rate was 0.5% per hour in cerebral cortex, cerebellum, hippocampus, and hypothalamus and 0.4% per hour in striatum. Of the subcellular fractions examined, the microsomal proteins were synthesized at the highest rate, followed by cytosolic and mitochondrial proteins. The results obtained indicate that the average synthesis rate of proteins in the various brain regions and subcellular fractions examined is fairly constant and is not significantly altered in the 4 to 24-month period of life of rats.

  8. Role of MMP-2 in PKCdelta-mediated inhibition of Na+ dependent Ca2+ uptake in microsomes of pulmonary smooth muscle: involvement of a pertussis toxin sensitive protein.

    PubMed

    Chakraborti, Sajal; Mandal, Amritlal; Das, Sudip; Chakraborti, Tapati

    2005-12-01

    Treatment of bovine pulmonary artery smooth muscle with the O2 *- generating system hypoxanthine plus xanthine oxidase stimulated MMP-2 activity and PKC activity; and inhibited Na+ dependent Ca2+ uptake in the microsomes. Pretreatment of the smooth muscle with SOD (the O2 *- scavenger) and TIMP-2 (MMP-2 inhibitor) prevented the increase in MMP-2 activity and PKC activity, and reversed the inhibition of Na+ dependent Ca2+ uptake in the microsomes. Pretreatment with calphostin C (a general PKC inhibitor) and rottlerin (a PKCdelta inhibitor) prevented the increase in PKC activity and reversed O2 *- caused inhibition of Na+ dependent Ca2+ uptake without causing any change in MMP-2 activity in the microsomes of the smooth muscle. Treatment of the smooth muscle with the O2 *- generating system revealed, respectively, 36 kDa RACK-1 and 78 kDa PKCdelta immunoreactive protein profile along with an additional 38 kDa immunoreactive fragment in the microsomes. The 38 kDa band appeared to be the proteolytic fragment of the 78 kDa PKCdelta since pretreatment with TIMP-2 abolished the increase in the 38 kDa immunoreactive fragment. Co-immunoprecipitation of PKCdelta and RACK-1 demonstrated O2 *- dependent increase in PKCdelta-RACK-1 interaction in the microsomes. Immunoblot assay elicited an immunoreactive band of 41 kDa G(i)alpha in the microsomes. Treatment of the smooth muscle tissue with the O2 *- generating system causes phosphorylation of G(i)alpha in the microsomes and pretreatment with TIMP-2 and rottlerin prevented the phosphorylation. Pretreatment of the smooth muscle tissue with pertussis toxin reversed O2 *- caused inhibition of Na+ dependent Ca2+ uptake without affecting the protease activity and PKC activity in the microsomes. We suggest the existence of a pertussis toxin sensitive G protein mediated mechanism for inhibition of Na+ dependent Ca2+ uptake in microsomes of bovine pulmonary artery smooth muscle under O2 *- triggered condition, which is regulated by

  9. Proteins of rough microsomal membranes related to ribosome binding. II. Cross-linking of bound ribosomes to specific membrane proteins exposed at the binding sites

    PubMed Central

    1978-01-01

    Two proteins (ribophorins I and II), which are integral components of rough microsomal membranes and appear to be related to the bound ribosomes, were shown to be exposed on the surface of rat liver rough microsomes (RM) and to be in close proximity to the bound ribosomes. Both proteins were labeled when intact RM were incubated with a lactoperoxidase iodinating system, but only ribophorin I was digested during mild trypsinization of intact RM. Ribophorin II (63,000 daltons) was only proteolyzed when the luminal face of the microsomal vesicles was made accessible to trypsin by the addition of sublytical detergent concentrations. Only 30--40% of the bound ribosomes were released during trypsinization on intact RM, but ribosome release was almost complete in the presence of low detergent concentrations. Very low glutaraldehyde concentrations (0.005--0.02%) led to the preferential cross-linking of large ribosomal subunits of bound ribosomes to the microsomal membranes. This cross-linking prevented the release of subunits caused by puromycin in media of high ionic strength, but not the incorporation of [3H]puromycin into nascent polypeptide chains. SDS- acrylamide gel electrophoresis of cross-linked samples a preferential reduction in the intensity of the bands representing the ribophorins and the formation of aggregates which did not penetrate into the gels. At low methyl-4-mercaptobutyrimidate (MMB) concentrations (0.26 mg/ml) only 30% of the ribosomes were cross-linked to the microsomal membranes, as shown by the puromycin-KCl test, but membranes could still be solubilized with 1% DOC. This allowed the isolation of the ribophorins together with the sedimentable ribosomes, as was shown by electrophoresis of the sediments after disruption of the cross-links by reduction. Experiments with RM which contained only inactive ribosomes showed that the presence of nascent chains was not necessary for the reversible cross-linking of ribosomes to the membranes. These

  10. Microsomal Triglyceride Transfer Protein (MTP) Associates with Cytosolic Lipid Droplets in 3T3-L1 Adipocytes

    PubMed Central

    Robinson, Delia B.; Harris, Carla M.; Johnson, Joyce E.; Mohler, Peter J.; Jerome, W. Gray; Swift, Larry L.

    2015-01-01

    Lipid droplets are intracellular energy storage organelles composed of a hydrophobic core of neutral lipid, surrounded by a monolayer of phospholipid and a diverse array of proteins. The function of the vast majority of these proteins with regard to the formation and/or turnover of lipid droplets is unknown. Our laboratory was the first to report that microsomal triglyceride transfer protein (MTP), a lipid transfer protein essential for the assembly of triglyceride-rich lipoproteins, was expressed in adipose tissue of humans and mice. In addition, our studies suggested that MTP was associated with lipid droplets in both brown and white fat. Our observations led us to hypothesize that MTP plays a key role in lipid droplet formation and/or turnover. The objective of these studies was to gain insight into the function of MTP in adipocytes. Using molecular, biochemical, and morphologic approaches we have shown: 1) MTP protein levels increase nearly five-fold as 3T3-L1 cells differentiate into adipocytes. 2) As 3T3-L1 cells undergo differentiation, MTP moves from the juxtanuclear region of the cell to the surface of lipid droplets. MTP and perilipin 2, a major lipid droplet surface protein, are found on the same droplets; however, MTP does not co-localize with perilipin 2. 3) Inhibition of MTP activity has no effect on the movement of triglyceride out of the cell either as a lipid complex or via lipolysis. 4) MTP is found associated with lipid droplets within hepatocytes from human fatty livers, suggesting that association of MTP with lipid droplets is not restricted to adipocytes. In summary, our data demonstrate that MTP is a lipid droplet-associated protein. Its location on the surface of the droplet in adipocytes and hepatocytes, coupled with its known function as a lipid transfer protein and its increased expression during adipocyte differentiation suggest a role in lipid droplet biology. PMID:26267806

  11. Some factors determining the concentration of liver proteins for optimal mutagenicity of chemicals in the Salmonella/microsome assay.

    PubMed

    Malaveille, C; Kuroki, T; Brun, G; Hautefeuille, A; Camus, A M; Bartsch, H

    1979-12-01

    In plate assays in the presence of S. typhimurium TA100 and various amounts of liver 9000 X g supernatant (S9) from either untreated, phenobarbitone- (PB) or Aroclor-treated rats, the S9 concentration required for optimal mutagenicity of aflatoxin B1 (AFB) depended both on the source of S9 and on the concentration of the test compound. In these assays, the water-soluble procarcinogen, dimethylnitrosamine (DMN) was mutagenic in S. typhimurium TA1530 only in the presence of a 35-fold higher concentration of liver S9 from PB-treated rats than that required for AFB, a lipophilic compound. In liquid assays, a biphasic relationship was observed in the mutagenicities in S. typhimurium TA100 of benzo[a]pyrene (BP) and AFB and the concentration of liver S9. For optimal mutagenesis of BP, the concentration of liver S9 from rats treated with methylcholanthrene (MC) was 4.4% (v/v); for AFB it was 2.2% (v/v) liver S9 from either Aroclor-treated or untreated rats. At higher concentrations of S9 the mutagenicity of BP and of AFB was related inversely to the amount of S9 per assay. The effect of Aroclor treatment on the microsomemediated mutagenicity of AFB was assay-dependent: in the liquid assay, AFB mutagenicity was decreased, whereas in the plate assay it did not change or was increased. As virtually no bacteria-bound microsomes were detected by electron microscopy, after the bacteria had been incubated in a medium containing 1-34% (v/v) MC-treated rat-liver S9, it is concluded that, in mutagenicity assays, mutagenic metabolites generated by microsomal enzymes from certain pro-carcinogens have to diffuse through the assay medium before reaching the bacteria. Thus the mutagenicity of BP was dependent on both the concentration of rat-liver microsomes and that of total cytosolic proteins and other soluble nucleophiles such as glutathione. At a concentration of 4.4% (v/v) liver S9, the mutagenicity of BP was about 3.6 times higher than in assays containing a 4-fold higher

  12. Stimulation of Ca2+ uptake by cyclic AMP and protein kinase in sarcoplasmic reticulum-rich and sarcolemma-rich microsomal fractions from rabbit heart.

    PubMed

    Will, H; Schirpke, B; Wollenberger, A

    1976-01-01

    The effect of cyclic AMP on Ca2+ uptake by rabbit heart microsomal vesicular fractions representing mainly fragments of either sarcoplasmic reticulum or sarcolemma was investigated in the presence and absence of soluble cardiac protein kinase and with microsomes prephosphorylated by cyclic AMP-dependent protein kinase. The acceleration of oxalate-promoted Ca2+ uptake by fragmented sarcoplasmic reticulum following cyclic AMP-dependent membrane protein phosphorylation, observed by other authors, was confirmed. In addition it was found that the acceleration was greatest at pH 7.2 and almost negligible at pH 6.0 and pH 7.8. A very marked increase in Ca2+ uptake by cyclic AMP-dependent membrane protein phosphorylation was observed in the presence of boric acid, a reversible inhibitor of Ca2+ uptake. In addition to the microsomal fraction thought to represent mainly fragments of the sarcoplasmic reticulum, the effect of protein kinase and cyclic AMP on Ca2+ uptake was investigated in a cardiac sarcolemma-enriched membrane fraction. Ca2+ uptake by sarcolemmal vesicles, unlike Ca2+ uptake by sarcoplasmic reticulum vesicles, was inhibited by low doses of digitoxin. The acceleration of oxalate-promoted Ca2+ uptake by cyclic AMP and soluble cardiac protein kinase, however, was quite similar to what was seen in preparations of fragmented sarcoplasmic reticulum, which suggests that it may reflect an acceleration of active Ca2+ transport across the myocardial cell surface membrane. PMID:185862

  13. Proteomic and Bioinformatics Analyses of Mouse Liver Microsomes

    PubMed Central

    Peng, Fang; Zhan, Xianquan; Li, Mao-Yu; Fang, Fan; Li, Guoqing; Li, Cui; Zhang, Peng-Fei; Chen, Zhuchu

    2012-01-01

    Microsomes are derived mostly from endoplasmic reticulum and are an ideal target to investigate compound metabolism, membrane-bound enzyme functions, lipid-protein interactions, and drug-drug interactions. To better understand the molecular mechanisms of the liver and its diseases, mouse liver microsomes were isolated and enriched with differential centrifugation and sucrose gradient centrifugation, and microsome membrane proteins were further extracted from isolated microsomal fractions by the carbonate method. The enriched microsome proteins were arrayed with two-dimensional gel electrophoresis (2DE) and carbonate-extracted microsome membrane proteins with one-dimensional gel electrophoresis (1DE). A total of 183 2DE-arrayed proteins and 99 1DE-separated proteins were identified with tandem mass spectrometry. A total of 259 nonredundant microsomal proteins were obtained and represent the proteomic profile of mouse liver microsomes, including 62 definite microsome membrane proteins. The comprehensive bioinformatics analyses revealed the functional categories of those microsome proteins and provided clues into biological functions of the liver. The systematic analyses of the proteomic profile of mouse liver microsomes not only reveal essential, valuable information about the biological function of the liver, but they also provide important reference data to analyze liver disease-related microsome proteins for biomarker discovery and mechanism clarification of liver disease. PMID:22500222

  14. Water Stress and Protein Synthesis

    PubMed Central

    Dhindsa, R. S.; Cleland, R. E.

    1975-01-01

    Water stress causes a reduction in hydrostatic pressure and can cause an increase in abscisic acid in plant tissues. To assess the possible role of abscisic acid and hydrostatic pressure in water stress effects, we have compared the effects of water stress, abscisic acid, and an imposed hydrostatic pressure on the rate and pattern of protein synthesis in Avena coleoptiles. Water stress reduces the rate and changes the pattern of protein synthesis as judged by a double labeling ratio technique, Abscisic acid reduces the rate but does not alter the pattern of protein synthesis. Gibberellic acid reverses the abscisic acid-induced but not the stress-induced inhibition of protein synthesis. The effect of hydrostatic pressure depends on the gas used. With a 19: 1 N2-air mixture, the rate of protein synthesis is increased in stressed but not in turgid tissues. An imposed hydrostatic pressure alters the pattern of synthesis in stressed tissues, but does not restore the pattern to that found in turgid tissues. Because of the differences in response, we conclude that water stress does not affect protein synthesis via abscisic acid or reduced hydrostatic pressure. PMID:16659167

  15. Garlic inhibits microsomal triglyceride transfer protein gene expression in human liver and intestinal cell lines and in rat intestine.

    PubMed

    Lin, Marie C; Wang, Er-Jia; Lee, Catherine; Chin, K T; Liu, Depei; Chiu, Jen-Fu; Kung, Hsiang-Fu

    2002-06-01

    Epidemiologic studies have suggested that fresh garlic has lipid-lowering activity. Because the microsomal triglyceride transfer protein (MTP) plays a pivotal role in the assembly and secretion of apolipoprotein B (apoB)-containing lipoproteins, we evaluated the effect of garlic on the expression of the MTP gene in vitro in cell lines and in vivo in rats. Fresh garlic extract (FGE) reduced MTP mRNA levels in both the human hepatoma HepG2 and intestinal carcinoma Caco-2 cells in dose-dependent fashion; significant reductions were detected with 3 g/L FGE. Maximal 72 and 59% reductions, respectively, were observed with 6 g/L FGE. To evaluate the in vivo effect of garlic on MTP gene expression, rats were given a single oral dose of fresh garlic homogenate (FGH), with hepatic and intestinal MTP mRNA measured 3 h after dosing. Rats fed FGH had significantly (46% of the control) lower intestinal MTP mRNA levels compared with the control rats, whereas hepatic MTP mRNA levels were not affected. These results suggest a new mechanism for the hypolipidemic effect of fresh garlic. Long-term dietary supplementation of fresh garlic may exert a lipid-lowering effect partly through reducing intestinal MTP gene expression, thus suppressing the assembly and secretion of chylomicrons from intestine to the blood circulation. PMID:12042427

  16. Microsomal membrane proteome of low grade diffuse astrocytomas: Differentially expressed proteins and candidate surveillance biomarkers

    PubMed Central

    Polisetty, Ravindra Varma; Gautam, Poonam; Gupta, Manoj Kumar; Sharma, Rakesh; Gowda, Harsha; Renu, Durairaj; Shivakumar, Bhadravathi Marigowda; Lakshmikantha, Akhila; Mariswamappa, Kiran; Ankathi, Praveen; Purohit, Aniruddh K.; Uppin, Megha S.; Sundaram, Challa; Sirdeshmukh, Ravi

    2016-01-01

    Diffuse astrocytoma (DA; WHO grade II) is a low-grade, primary brain neoplasm with high potential of recurrence as higher grade malignant form. We have analyzed differentially expressed membrane proteins from these tumors, using high-resolution mass spectrometry. A total of 2803 proteins were identified, 340 of them differentially expressed with minimum of 2 fold change and based on ≥2 unique peptides. Bioinformatics analysis of this dataset also revealed important molecular networks and pathways relevant to tumorigenesis, mTOR signaling pathway being a major pathway identified. Comparison of 340 differentially expressed proteins with the transcript data from Grade II diffuse astrocytomas reported earlier, revealed about 190 of the proteins correlate in their trends in expression. Considering progressive and recurrent nature of these tumors, we have mapped the differentially expressed proteins for their secretory potential, integrated the resulting list with similar list of proteins from anaplastic astrocytoma (WHO Grade III) tumors and provide a panel of proteins along with their proteotypic peptides, as a resource that would be useful for investigation as circulatory plasma markers for post-treatment surveillance of DA patients. PMID:27246909

  17. Microsomal membrane proteome of low grade diffuse astrocytomas: Differentially expressed proteins and candidate surveillance biomarkers.

    PubMed

    Polisetty, Ravindra Varma; Gautam, Poonam; Gupta, Manoj Kumar; Sharma, Rakesh; Gowda, Harsha; Renu, Durairaj; Shivakumar, Bhadravathi Marigowda; Lakshmikantha, Akhila; Mariswamappa, Kiran; Ankathi, Praveen; Purohit, Aniruddh K; Uppin, Megha S; Sundaram, Challa; Sirdeshmukh, Ravi

    2016-01-01

    Diffuse astrocytoma (DA; WHO grade II) is a low-grade, primary brain neoplasm with high potential of recurrence as higher grade malignant form. We have analyzed differentially expressed membrane proteins from these tumors, using high-resolution mass spectrometry. A total of 2803 proteins were identified, 340 of them differentially expressed with minimum of 2 fold change and based on ≥2 unique peptides. Bioinformatics analysis of this dataset also revealed important molecular networks and pathways relevant to tumorigenesis, mTOR signaling pathway being a major pathway identified. Comparison of 340 differentially expressed proteins with the transcript data from Grade II diffuse astrocytomas reported earlier, revealed about 190 of the proteins correlate in their trends in expression. Considering progressive and recurrent nature of these tumors, we have mapped the differentially expressed proteins for their secretory potential, integrated the resulting list with similar list of proteins from anaplastic astrocytoma (WHO Grade III) tumors and provide a panel of proteins along with their proteotypic peptides, as a resource that would be useful for investigation as circulatory plasma markers for post-treatment surveillance of DA patients. PMID:27246909

  18. Comprehensive Analysis of in Vivo Phosphoproteome of Mouse Liver Microsomes.

    PubMed

    Kwon, Oh Kwang; Sim, JuHee; Kim, Sun Ju; Sung, Eunji; Kim, Jin Young; Jeong, Tae Cheon; Lee, Sangkyu

    2015-12-01

    Protein phosphorylation at serine, threonine, and tyrosine residues are some of the most widespread reversible post-translational modifications. Microsomes are vesicle-like bodies, not ordinarily present within living cells, which form from pieces of the endoplasmic reticulum (ER), plasma membrane, mitochondria, or Golgi apparatus of broken eukaryotic cells. Here we investigated the total phosphoproteome of mouse liver microsomes (MLMs) using TiO2 enrichment of phosphopeptides coupled to on-line 2D-LC-MS/MS. In total, 699 phosphorylation sites in 527 proteins were identified in MLMs. When compared with the current phosphoSitePlus database, 155 novel phosphoproteins were identified in MLM. The distributions of phosphosites were 89.4, 8.0, and 2.6% for phosphoserine, phosphotheronine, and phosphotyrosine, respectively. By Motif-X analysis, eight Ser motifs and one Thr motif were found, and five acidic, two basophilic-, and two proline-directed motifs were assigned. The potential functions of phosphoproteins in MLM were assigned by Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis. In GO annotation, phosphorylated microsomal proteins were involved in mRNA processing, mRNA metabolic processes, and RNA splicing. In the KEGG pathway analysis, phosphorylated microsomal proteins were highly enriched in ribosome protein processing in ER and ribosomes and in RNA transport. Furthermore, we determined that 52 and 23 phosphoproteins were potential substrates of cAMP-dependent protein kinase A and casein kinase II, respectively, many of which are 40S/60S ribosomal proteins. Overall, our results provide an overview of features of protein phosphorylation in MLMs that should be a valuable resource for the future understanding of protein synthesis or translation involving phosphorylation. PMID:26487105

  19. Fructose-induced steatosis in mice: role of plasminogen activator inhibitor-1, microsomal triglyceride transfer protein and NKT cells.

    PubMed

    Kanuri, Giridhar; Spruss, Astrid; Wagnerberger, Sabine; Bischoff, Stephan C; Bergheim, Ina

    2011-06-01

    Plasminogen activator inhibitor-1 (PAI-1) is an acute-phase protein known to be involved in alcoholic liver disease and hepatic fibrosis. In the present study, the hypothesis that PAI-1 is causally involved in the onset of fructose-induced hepatic steatosis was tested in a mouse model. Wild-type C57BL/6J and PAI-1⁻/⁻ mice were fed with 30% fructose solution or water for 8 weeks. Markers of hepatic steatosis, expression of PAI-1, apolipoprotein B (ApoB), cluster of differentiation 1d (CD1d), markers of natural killer T (NKT) cells, protein levels of phospho-c-Met and tumor necrosis factor-α (TNF-α) were determined. Activity of the microsomal triglyceride transfer protein (MTTP) was measured in liver tissue. In comparison with water controls, chronic intake of 30% fructose solution caused a significant increase in hepatic triglycerides, PAI-1 expression and plasma alanine aminotransferase levels in wild-type mice. This effect of fructose feeding was markedly attenuated in PAI-1⁻/⁻ mice. Despite no differences in portal endotoxin levels and hepatic TNF-α protein levels between fructose-fed groups, the protective effect of the loss of PAI-1 against the onset of fructose-induced steatosis was associated with a significant increase in phospho-c-Met, phospho Akt, expression of ApoB and activity of MTTP in livers of PAI-1⁻/⁻ mice in comparison with fructose-fed wild types. Moreover, in PAI-1⁻/⁻ mice, expressions of CD1d and markers of CD1d-reactive NKT cells were markedly higher than in wild-type mice; however, expression of markers of activation of CD1d-reactive NKT cells (eg, interleukin-15 and interferon-γ) were only found to be increased in livers of fructose-fed PAI-1⁻/⁻ mice. Taken together, these data suggest that PAI-1 has a causal role in mediating the early phase of fructose-induced liver damage in mice through signaling cascades downstream of Kupffer cells and TNF-α. PMID:21423135

  20. Synthesis, microsome-mediated metabolism, and identification of major metabolites of environmental pollutant naphtho(8,1,2-ghi)chrysene

    SciTech Connect

    Sharma, A.K.; Gowdahalli, K.; Gimbor, M.; Amin, S.

    2008-05-15

    Naphtho(8,1,2-ghi)chrysene, commonly known as naphtho(1,2-e)pyrene (N(1,2-e)P) is a widespread environmental pollutant, identified in coal tar extract, air borne particulate matter, marine sediment, cigarette smoke condensate, and vehicle exhaust. Herein, we determined the ability of rat liver microsomes to metabolize N(1,2-e)P and an unequivocal assignment of the metabolites by comparing them with independently,synthesized standards. We developed the synthesis of both the fjord region and the K-region dihydrodiols and various phenolic derivatives for metabolite identification. In summary, N(1,2-e)P trans-11, 12-dihydrodiol was the major metabolite formed along with N(1,2-e)P 4,5-trtins-dihydrodiol and 12-OH-N(1,2-e)P on exposure of rat liver microsomes to N(1,2-e)P. The presence of N(1,2-e)P in the environment and formation of fjord region dihydrodiol 14 as a major metabolite in in vitro metabolism studies strongly suggest the role of N(1,2-e)P as a potential health hazard.

  1. Extensive exchange of rat liver microsomal phospholipids.

    PubMed

    Zilversmit, D B; Hughes, M E

    1977-08-15

    Liver microsomal fractions were prepared from rats injected with a single dose of choline [14C]methylchloride or with single or multiple doses of 32Pi. Exchangeability of microsomal phospholipids was determined by incubation with an excess of mitochondria and phospholipid exchange proteins derived from beef heart, beef liver or rat liver. Labeled phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine and phosphatidylinositol were found to act as a single pool and were 85--95% exchangeable in 1--2h. High latencies of mannose-6-phosphate phosphohydrolase activities and impermeability of microsomes to EDTA proved that phospholipid exchange proteins did not have access to the intracisternal space. If microsomal membranes are largely composed of phospholipid bilayers, the experiments suggest that one or more of the phospholipid classes in microsomal membranes undergo rapid translocation between the inner and outer portions of the bilayer. PMID:889827

  2. Chloroplast ribosomes and protein synthesis.

    PubMed Central

    Harris, E H; Boynton, J E; Gillham, N W

    1994-01-01

    Consistent with their postulated origin from endosymbiotic cyanobacteria, chloroplasts of plants and algae have ribosomes whose component RNAs and proteins are strikingly similar to those of eubacteria. Comparison of the secondary structures of 16S rRNAs of chloroplasts and bacteria has been particularly useful in identifying highly conserved regions likely to have essential functions. Comparative analysis of ribosomal protein sequences may likewise prove valuable in determining their roles in protein synthesis. This review is concerned primarily with the RNAs and proteins that constitute the chloroplast ribosome, the genes that encode these components, and their expression. It begins with an overview of chloroplast genome structure in land plants and algae and then presents a brief comparison of chloroplast and prokaryotic protein-synthesizing systems and a more detailed analysis of chloroplast rRNAs and ribosomal proteins. A description of the synthesis and assembly of chloroplast ribosomes follows. The review concludes with discussion of whether chloroplast protein synthesis is essential for cell survival. PMID:7854253

  3. Regulation of microsomal triglyceride transfer protein by apolipoprotein A-IV in newborn swine intestinal epithelial cells.

    PubMed

    Yao, Ying; Lu, Song; Huang, Yue; Beeman-Black, Casey C; Lu, Rena; Pan, Xiaoyue; Hussain, M Mahmood; Black, Dennis D

    2011-02-01

    Apolipoprotein (apo) A-IV overexpression enhances chylomicron (CM) assembly and secretion in newborn swine intestinal epithelial cells by producing larger particles (Lu S, Yao Y, Cheng X, Mitchell S, Leng S, Meng S, Gallagher JW, Shelness GS, Morris GS, Mahan J, Frase S, Mansbach CM, Weinberg RB, Black DD. J Biol Chem 281: 3473-3483, 2006). To determine the impact of apo A-IV on microsomal triglyceride transfer protein (MTTP), IPEC-1 cell lines containing a tetracycline-regulatable expression system were used to overexpress native swine apo A-IV and "piglike" human apo A-IV, a mutant human apo A-IV with deletion of the EQQQ-rich COOH-terminus, previously shown to upregulate basolateral triglyceride (TG) secretion 5-fold and 25-fold, respectively. Cells were incubated 24 h with and without doxycycline and oleic acid (OA, 0.8 mM). Overexpression of the native swine apo A-IV and piglike human apo A-IV increased MTTP lipid transfer activity by 39.7% (P = 0.006) and 53.6% (P = 0.0001), respectively, compared with controls. Changes in mRNA and protein levels generally paralleled changes in activity. Interestingly, native swine apo A-IV overexpression also increased MTTP large subunit mRNA, protein levels, and lipid transfer activity in the absence of OA, suggesting a mechanism not mediated by lipid absorption. Overexpression of piglike human apo A-IV significantly increased partitioning of radiolabeled OA from endoplasmic reticulum (ER) membrane to lumen, suggesting increased net transfer of membrane TG to luminal particles. These results suggest that the increased packaging of TG into nascent CMs in the ER lumen, induced by apo A-IV, is associated with upregulation of MTTP activity at the pretranslational level. Thus MTTP is regulated by apo A-IV in a manner to promote increased packaging of TG into the CM core, which may be important in neonatal fat absorption. PMID:21127258

  4. The effects of general anesthetics on ESR spectra of spin labels in phosphatidylcholine vesicles containing purified Na,K-ATPase or microsomal protein

    NASA Astrophysics Data System (ADS)

    Shibuya, Makiko; Hiraoki, Toshifumi; Kimura, Kunie; Fukushima, Kazuaki; Suzuki, Kuniaki

    2012-12-01

    We investigated the effects of general anesthetics on liposome containing spin labels, 5-doxyl stearic acid (5-DSA) and 16-doxyl stearic acid (16-DSA), and purified Na,K-ATPase or membrane protein of microsome using an electron spin resonance (ESR) spectroscopy. The spectra of 16-DSA in liposomes with both proteins showed three sharp signals compared with 5-DSA. The difference in the order parameter S value of 5-DSA and 16-DSA suggested that the nitroxide radical location of 5-DSA and 16-DSA were different in the membrane bilayer. The results were almost the same as those obtained in liposomes without proteins. The addition of sevoflurane, isoflurane, halothane, ether, ethanol and propofol increased the intensity of the signals, but the clinical concentrations of anesthetics did not significantly alter the S and τ values, which are indices of the fluidity of the membrane. These results suggest that anesthetics remain on the surface of the lipid bilayer and do not act on both the inside hydrophobic area and the relatively hydrophilic area near the surface. These results and others also suggest that the existence of Na,K-ATPase and microsomal proteins did not affect the environment around the spin labels in the liposome and the effects of anesthetics on liposome as a model membrane.

  5. Proteomic Study of Microsomal Proteins Reveals a Key Role for Arabidopsis Annexin 1 in Mediating Heat Stress-Induced Increase in Intracellular Calcium Levels*

    PubMed Central

    Wang, Xu; Ma, Xiaolong; Wang, Hui; Li, Bingjie; Clark, Greg; Guo, Yi; Roux, Stan; Sun, Daye; Tang, Wenqiang

    2015-01-01

    To understand the early signaling steps in the response of plant cells to increased environmental temperature, 2-D difference gel electrophoresis was used to study the proteins in microsomes of Arabidopsis seedlings that are regulated early during heat stress. Using mass spectrometry, 19 microsomal proteins that showed an altered expression level within 5 min after heat treatment were identified. Among these proteins, annexin 1 (AtANN1) was one of those up-regulated rapidly after heat-shock treatment. Functional studies show loss-of-function mutants for AtANN1 and its close homolog AtANN2 were more sensitive to heat-shock treatment, whereas plants overexpressing AtANN1 showed more resistance to this treatment. Correspondingly, the heat-induced expression of heat-shock proteins and heat-shock factors is inhibited in ann1/ann2 double mutant, and the heat-activated increase in cytoplasmic calcium concentration ([Ca2+]cyt) is greatly impaired in the ann1 mutant and almost undetectable in ann1/ann2 double mutant. Taken together these results suggest that AtANN1 is important in regulating the heat-induced increase in [Ca2+]cyt and in the response of Arabidopsis seedlings to heat stress. PMID:25587034

  6. β-Apo-10'-carotenoids Modulate Placental Microsomal Triglyceride Transfer Protein Expression and Function to Optimize Transport of Intact β-Carotene to the Embryo.

    PubMed

    Costabile, Brianna K; Kim, Youn-Kyung; Iqbal, Jahangir; Zuccaro, Michael V; Wassef, Lesley; Narayanasamy, Sureshbabu; Curley, Robert W; Harrison, Earl H; Hussain, M Mahmood; Quadro, Loredana

    2016-08-26

    β-Carotene is an important source of vitamin A for the mammalian embryo, which depends on its adequate supply to achieve proper organogenesis. In mammalian tissues, β-carotene 15,15'-oxygenase (BCO1) converts β-carotene to retinaldehyde, which is then oxidized to retinoic acid, the biologically active form of vitamin A that acts as a transcription factor ligand to regulate gene expression. β-Carotene can also be cleaved by β-carotene 9',10'-oxygenase (BCO2) to form β-apo-10'-carotenal, a precursor of retinoic acid and a transcriptional regulator per se The mammalian embryo obtains β-carotene from the maternal circulation. However, the molecular mechanisms that enable its transfer across the maternal-fetal barrier are not understood. Given that β-carotene is transported in the adult bloodstream by lipoproteins and that the placenta acquires, assembles, and secretes lipoproteins, we hypothesized that the aforementioned process requires placental lipoprotein biosynthesis. Here we show that β-carotene availability regulates transcription and activity of placental microsomal triglyceride transfer protein as well as expression of placental apolipoprotein B, two key players in lipoprotein biosynthesis. We also show that β-apo-10'-carotenal mediates the transcriptional regulation of microsomal triglyceride transfer protein via hepatic nuclear factor 4α and chicken ovalbumin upstream promoter transcription factor I/II. Our data provide the first in vivo evidence of the transcriptional regulatory activity of β-apocarotenoids and identify microsomal triglyceride transfer protein and its transcription factors as the targets of their action. This study demonstrates that β-carotene induces a feed-forward mechanism in the placenta to enhance the assimilation of β-carotene for proper embryogenesis. PMID:27402843

  7. Enhancement of RNA Synthesis, Protein Synthesis, and Abscission by Ethylene

    PubMed Central

    Abeles, F. B.; Holm, R. E.

    1966-01-01

    Ethylene stimulated RNA and protein synthesis in bean (Phaseolus vulgaris L. var. Red Kidney) abscission zone explants prior to abscission. The effect of ethylene on RNA synthesis and abscission was blocked by actinomycin D. Carbon dioxide, which inhibits the effect of ethylene on abscission, also inhibited the influence of ethylene on protein synthesis. An aging period appears to be essential before bean explants respond to ethylene. Stimulation of protein synthesis by ethylene occurred only in receptive or senescent explants. Treatment of juvenile explants with ethylene, which has no effect on abscission also has no effect on protein synthesis. Evidence in favor of a hormonal role for ethylene during abscission is discussed. PMID:16656405

  8. Identification of domains in apolipoprotein B100 that confer a high requirement for the microsomal triglyceride transfer protein.

    PubMed

    Nicodeme, E; Benoist, F; McLeod, R; Yao, Z; Scott, J; Shoulders, C C; Grand-Perret, T

    1999-01-22

    The microsomal triglyceride transfer protein (MTP) is required for the assembly and secretion of apoB-containing lipoproteins. To investigate the role of MTP in lipoprotein assembly, we determined the ability of carboxyl-terminally truncated forms of apoB to be secreted from cells treated with the MTP inhibitor 4'-bromo-3'-methylmetaqualone (Benoist, F., Nicodeme, E., and Grand-Perret, T. (1996) Eur. J. Biochem. 240, 713-720). In Caco-2 and mhAT3F cells that produce apoB100 and apoB48, the inhibitor preferentially blocked apoB100 secretion. When the inhibitor was tested on McA-RH7777 cells stably transfected with cDNAs encoding human apoB100, apoB72, apoB53, apoB29, and apoB18, the secretion of apoB100, apoB72, and apoB53 was preferentially impaired relative to apoB48 and shorter forms. To delineate the region between apoB48 and apoB53 that has a high requirement for MTP, we used puromycin to generate a range of truncated forms of apoB in HepG2 cells. The secretion of apoB53 and longer forms of apoB was markedly affected by low concentrations of the MTP inhibitor (approximately 1 microM), whereas apoB51 and smaller forms of apoB were only affected at higher concentrations (> 10 microM). The size-related sensitivity to MTP inhibitor was not due to late processing or retention, since the same result was observed when nascent lipoproteins were isolated from the endoplasmic reticulum. The MTP inhibitor did not alter the density of the secreted lipoproteins, indicating that each apoB polypeptide requires a minimally defined amount of lipid to attain a secretable conformation. Our results suggest that the folding of the domain between apoB51 and apoB53 has a high requirement for lipid. This domain is predicted to form amphipathic alpha-helices and to bind lipid reversibly. It proceeds and is followed by rigid amphipathic beta-sheets that are predicted to associate with lipid irreversibly. We speculate that these domains enable apoB to switch from a stable lipid

  9. Antithyroid microsomal antibody

    MedlinePlus

    Thyroid antimicrosomal antibody; Antimicrosomal antibody; Microsomal antibody; Thyroid peroxidase antibody; TPOAb ... test is done to confirm the cause of thyroid problems, including Hashimoto thyroiditis . The test is also ...

  10. Inhibition of microsomal prostaglandin E synthase-1 by aminothiazoles decreases prostaglandin E2 synthesis in vitro and ameliorates experimental periodontitis in vivo

    PubMed Central

    Kats, Anna; Båge, Tove; Georgsson, Pierre; Jönsson, Jörgen; Quezada, Hernán Concha; Gustafsson, Anders; Jansson, Leif; Lindberg, Claes; Näsström, Karin; Yucel-Lindberg, Tülay

    2013-01-01

    The potent inflammatory mediator prostaglandin E2 (PGE2) is implicated in the pathogenesis of several chronic inflammatory conditions, including periodontitis. The inducible enzyme microsomal prostaglandin E synthase-1 (mPGES-1), catalyzing the terminal step of PGE2 biosynthesis, is an attractive target for selective PGE2 inhibition. To identify mPGES-1 inhibitors, we investigated the effect of aminothiazoles on inflammation-induced PGE2 synthesis in vitro, using human gingival fibroblasts stimulated with the cytokine IL-1β and a cell-free mPGES-1 activity assay, as well as on inflammation-induced bone resorption in vivo, using ligature-induced experimental periodontitis in Sprague-Dawley rats. Aminothiazoles 4-([4-(2-naphthyl)-1,3-thiazol-2-yl]amino)phenol (TH-848) and 4-(3-fluoro-4-methoxyphenyl)-N-(4-phenoxyphenyl)-1,3-thiazol-2-amine (TH-644) reduced IL-1β-induced PGE2 production in fibroblasts (IC50 1.1 and 1.5 μM, respectively) as well as recombinant mPGES-1 activity, without affecting activity or expression of the upstream enzyme cyclooxygenase-2. In ligature-induced experimental periodontitis, alveolar bone loss, assessed by X-ray imaging, was reduced by 46% by local treatment with TH-848, compared to vehicle, without any systemic effects on PGE2, 6-keto PGF1α, LTB4 or cytokine levels. In summary, these results demonstrate that the aminothiazoles represent novel mPGES-1 inhibitors for inhibition of PGE2 production and reduction of bone resorption in experimental periodontitis, and may be used as potential anti-inflammatory drugs for treatment of chronic inflammatory diseases, including periodontitis.—Kats, A., Båge, T., Georgsson, P., Jönsson, J., Quezada, H. C., Gustafsson, A., Jansson, L., Lindberg, C., Näsström, K., Yucel-Lindberg, T. Inhibition of microsomal prostaglandin E synthase-1 by aminothiazoles decreases prostaglandin E2 synthesis in vitro and ameliorates experimental periodontitis in vivo. PMID:23447581

  11. Rapid induction of microsomal delta 12(omega 6)-desaturase activity in chilled Acanthamoeba castellanii.

    PubMed

    Jones, A L; Lloyd, D; Harwood, J L

    1993-11-15

    The activity of microsomal delta 12-desaturase in Acanthamoeba castellanii was increased after growing cultures were chilled from the optimal growth temperature (30 degrees C) to 15 degrees C. This increase was detectable in microsomes isolated from organisms subjected to only 10 min chilling. The mechanism of induction was investigated. The increase in activity on chilling was greatly reduced when protein synthesis was blocked before the temperature shift. Thus the major mechanism for the induction of delta 12-desaturase is increased protein synthesis. delta 12-Desaturase activity was higher when assayed at 20 degrees C than when assayed at 30 degrees C, but these changes were not due to the increased solubility of O2 at 20 degrees C. The major substrate of delta 12-desaturase was found to be 1-acyl-2-oleoyl phosphatidylcholine. PMID:8250841

  12. Modulation of catechol estrogen synthesis by rat liver microsomes: effects of treatment with growth hormone or testosterone

    SciTech Connect

    Quail, J.A.; Jellinck, P.H.

    1987-09-01

    The ability of GH from various mammalian species, administered to normal mature male rats by constant infusion, to decrease the hepatic 2-hydroxylation of estradiol (E2) to female levels, as measured by the release of /sup 3/H/sub 2/O from (2-3H)E2, was determined. Rat and human GH (hGH) showed the highest activity while ovine GH was inactive. PRL (0.6 IU/h X kg) administered together with hGH (0.02 IU/h X kg) did not antagonize the feminizing action of GH. Infusion of hGH into male rats decreased the affinity of estradiol 2-hydroxylase for its steroid substrate and altered the linear Lineweaver-Burk plot towards a nonlinear hyperbolic plot characteristic of the female. The apparent Michaelis-Menten constant (Km) for the reaction was 1.69 microM for males and 2.75 microM for testosterone-treated ovariectomized females. An equal mixture of liver microsomes from male and female rats gave kinetic values similar to those observed with males alone. Neonatal imprinting with androgen did not alter the magnitude of the response of female rats to treatment with testosterone and/or GH at maturity and the androgen effect could only be shown in ovariectomized animals. The results with rats of different endocrine status were corroborated by the kinetic data and by the pattern of metabolites obtained with (4-/sup 14/C)E2 when examined by TLC and autoradiography. The hormonal control of estradiol 2-hydroxylase, the key enzyme in catechol estrogen formation, and the contribution of sex-specific multiple forms of the enzyme to this reaction are discussed.

  13. Protein Synthesis--An Interactive Game.

    ERIC Educational Resources Information Center

    Clements, Lee Ann J.; Jackson, Karen E.

    1998-01-01

    Describes an interactive game designed to help students see and understand the dynamic relationship between DNA, RNA, and proteins. Appropriate for either a class or laboratory setting, following a lecture session about protein synthesis. (DDR)

  14. Quest for the chemical synthesis of proteins.

    PubMed

    Engelhard, Martin

    2016-05-01

    The chemical synthesis of proteins has been the wish of chemists since the early 19th century. There were decisive methodological steps necessary to accomplish this aim. Cornerstones were the introduction of the Z-protecting group of Bergmann and Zervas, the development of Solid-phase Peptide Synthesis of Merrifield, and the establishment of Native Chemical Ligation by Kent. Chemical synthesis of proteins has now become generally applicable technique for the synthesis of proteins with tailor made properties which can be applied not only in vitro but also in vivo .Copyright © 2016 European Peptide Society and John Wiley & Sons, Ltd. PMID:27114253

  15. Interrogating Circulating Microsomes and Exosomes Using Metal Nanoparticles.

    PubMed

    Zhou, Yi-Ge; Mohamadi, Reza M; Poudineh, Mahla; Kermanshah, Leyla; Ahmed, Sharif; Safaei, Tina Saberi; Stojcic, Jessica; Nam, Robert K; Sargent, Edward H; Kelley, Shana O

    2016-02-10

    A chip-based approach for electrochemical characterization and detection of microsomes and exosomes based on direct electro-oxidation of metal nanoparticles (MNPs) that specifically recognize surface markers of these vesicles is reported. It is found that exosomes and microsomes derived from prostate cancer cells can be identified by their surface proteins EpCAM and PSMA, suggesting the potential of exosomes and microsomes for use as diagnostic biomarkers. PMID:26707703

  16. Cell-free protein synthesis systems derived from cultured mammalian cells.

    PubMed

    Brödel, Andreas K; Wüstenhagen, Doreen A; Kubick, Stefan

    2015-01-01

    We present a technology for the production of target proteins using novel cell-free systems derived from cultured human K562 cells and Chinese hamster ovary (CHO) cells. The protocol includes the cultivation of cells, the preparation of translationally active lysates, and the cell-free synthesis of desired proteins. An efficient expression vector based on the internal ribosome entry site (IRES) from the intergenic region (IGR) of the cricket paralysis virus (CrPV) was constructed for both systems. The coupled batch-based platforms enable the synthesis of a broad range of target proteins such as cytosolic proteins, secreted proteins, membrane proteins embedded into endogenous microsomes, and glycoproteins. The glycosylation of erythropoietin demonstrates the successful performance of posttranslational modifications in the novel cell-free systems. Protein yields of approximately 20 μg/ml (K562-based cell-free system) and 50 μg/ml (CHO-based cell-free system) of active firefly luciferase are obtained in the coupled transcription-translation systems within 3 h. As a result, both cell-free protein synthesis systems serve as powerful tools for high-throughput proteomics. PMID:25502197

  17. Storage Protein Synthesis in Maize

    PubMed Central

    Larkins, Brian A.; Bracker, Charles E.; Tsai, C. Y.

    1976-01-01

    Undegraded free and membrane-bound polysomes were isolated from developing kernels of Zea mays L. frozen in liquid nitrogen. Freezing in liquid nitrogen was a prerequisite for preserving polysome structure in stored kernels. Membrane-bound polysomes from 22-day post-pollination kernels ground in high pH buffers containing 50 mm Mg2+ contained unique classes of large polysomes. These large polysomes were sensitive to ribonuclease, and electron micrographs verified that they were not formed by aggregation. The membrane-bound polysomes were the principal site of zein synthesis, since the major protein synthesized in vitro was similar to purified zein in its ethanol solubility and mobility on sodium dodecyl sulfate polyacrylamide gels. Images PMID:16659563

  18. T-2 mycotoxin inhibits mitochondrial protein synthesis

    SciTech Connect

    Pace, J.G.; Watts, M.R.; Canterbury, W.J.

    1988-01-01

    The authors investigated the effect of T-2 toxin on rat liver mitochondrial protein synthesis. Isolated rat liver mitochondria were supplemented with an S-100 supernatant from rat liver and an external ATP-generating system. An in-vitro assay employing cycloheximide, and inhibitor of cytoplasmic protein synthesis, and chloramphenicol, and inhibitor of mitochondrial protein synthesis, to distinguish mitochondrial protein synthesis from the cytoplasmic process. Amino acid incorporation into mitochondria was dependent on the concentration of mitochondria and was inhibited by chloramphenicol. The rate of uptake of tritium leucine into mitochondrial protein was unaffected by the addition of T-2 toxin and was not a rate-limiting step in incorporation. However, 0.02 micrograms/ml of T-2 toxin decreased the rate of protein synthesis inhibition correlated with the amount of T-2 toxin taken up by the mitochondria. While T-2 toxin is known to inhibit eukaryotic protein synthesis, this is the first time T-2 was shown to inhibit mitochondrial protein synthesis.

  19. Antithyroid microsomal antibody

    MedlinePlus

    ... Thyroid antimicrosomal antibody; Antimicrosomal antibody; Microsomal antibody; Thyroid peroxidase antibody; TPOAb Images Blood test References Guber HA, Faraq AF. Evaluation of endocrine function. In: McPherson RA, Pincus MR, eds. Henry's Clinical ...

  20. Mechanism of action of hypoglycemic effects of an intestine-specific inhibitor of microsomal triglyceride transfer protein (MTP) in obese rats.

    PubMed

    Sakata, Shohei; Katsumi, Sohei; Mera, Yasuko; Kuroki, Yukiharu; Nashida, Reiko; Kakutani, Makoto; Ohta, Takeshi

    2015-01-01

    Diminished insulin sensitivity in the peripheral tissues and failure of pancreatic beta cells to secrete insulin are known major determinants of type 2 diabetes mellitus. JTT-130, an intestine-specific microsomal transfer protein inhibitor, has been shown to suppress high fat-induced obesity and ameliorate impaired glucose tolerance while enhancing glucagon-like peptide-1 (GLP-1) secretion. We investigated the effects of JTT-130 on glucose metabolism and elucidated the mechanism of action, direct effects on insulin sensitivity and glucose-stimulated insulin secretion in a high fat diet-induced obesity rat model. Male Sprague Dawley rats fed a high-fat diet were treated with a single administration of JTT-130. Glucose tolerance, hyperglycemic clamp and hyperinsulinemic-euglycemic testing were performed to assess effects on insulin sensitivity and glucose-stimulated insulin secretion, respectively. Plasma GLP-1 and tissue triglyceride content were also determined under the same conditions. A single administration of JTT-130 suppressed plasma glucose elevations after oral glucose loading and increased the disposition index while elevating GLP-1. JTT-130 also enhanced glucose-stimulated insulin secretion in hyperglycemic clamp tests, whereas increased insulin sensitivity was observed in hyperinsulinemic-euglycemic clamp tests. Single-dose administration of JTT-130 decreased lipid content in the liver and skeletal muscle. JTT-130 demonstrated acute and direct hypoglycemic effects by enhancing insulin secretion and/or insulin sensitivity. PMID:25704025

  1. Adaptation of low-resolution methods for the study of yeast microsomal polytopic membrane proteins: a methodological review.

    PubMed

    Bochud, Arlette; Ramachandra, Nagaraju; Conzelmann, Andreas

    2013-02-01

    Most integral membrane proteins of yeast with two or more membrane-spanning sequences have not yet been crystallized and for many of them the side on which the active sites or ligand-binding domains reside is unknown. Also, bioinformatic topology predictions are not yet fully reliable. However, so-called low-resolution biochemical methods can be used to locate hydrophilic loops or individual residues of polytopic membrane proteins at one or the other side of the membrane. The advantages and limitations of several such methods for topological studies with yeast ER integral membrane proteins are discussed. We also describe new tools that allow us to better control and validate results obtained with SCAM (substituted cysteine accessibility method), an approach that determines the position of individual residues with respect to the membrane plane, whereby only minimal changes in the primary sequence have to be introduced into the protein of interest. PMID:23356255

  2. Protein synthesis in geostimulated root caps

    NASA Technical Reports Server (NTRS)

    Feldman, L. J.

    1982-01-01

    A study is presented of the processes occurring in the root cap of corn which are requisite for the formation of root cap inhibitor and which can be triggered or modulated by both light and gravity. The results of this study indicate the importance of protein synthesis for light-induced gravitropic bending in roots. Root caps in which protein synthesis is prevented are unable to induce downward bending. This suggests that light acts by stimulating proteins which are necessary for the translation of the gravitropic stimulus into a growth response (downward bending). The turnover of protein with time was also examined in order to determine whether light acts by stimulating the synthesis of unique proteins required for downward growth. It is found that auxin in combination with light allows for the translation of the gravitropic stimulus into a growth response at least in part through the modification of protein synthesis. It is concluded that unique proteins are stimulated by light and are involved in promoting the downward growth in roots which are responding to gravity.

  3. Carotenoid incorporation into microsomes: yields, stability and membrane dynamics

    NASA Astrophysics Data System (ADS)

    Socaciu, Carmen; Jessel, Robert; Diehl, Horst A.

    2000-12-01

    The carotenoids β-carotene (BC), lycopene (LYC), lutein (LUT), zeaxanthin (ZEA), canthaxanthin (CTX) and astaxanthin (ASTA) have been incorporated into pig liver microsomes. Effective incorporation concentrations in the range of about 1-6 nmol/mg microsomal protein were obtained. A stability test at room temperature revealed that after 3 h BC and LYC had decayed totally whereas, gradually, CTX (46%), LUT (21%), ASTA (17%) and ZEA (5%) decayed. Biophysical parameters of the microsomal membrane were changed hardly by the incorporation of carotenoids. A small rigidification may occur. Membrane anisotropy seems to offer only a small tolerance for incorporation of carotenoids and seems to limit the achievable incorporation concentrations of the carotenoids into microsomes. Microsomes instead of liposomes should be preferred as a membrane model to study mutual effects of carotenoids and membrane dynamics.

  4. Expression and methylation of microsomal triglyceride transfer protein and acetyl-CoA carboxylase are associated with fatty liver syndrome in chicken.

    PubMed

    Liu, Zhen; Li, Qinghe; Liu, Ranran; Zhao, Guiping; Zhang, Yonghong; Zheng, Maiqing; Cui, Huanxian; Li, Peng; Cui, Xiaoyan; Liu, Jie; Wen, Jie

    2016-06-01

    The typical characteristic of fatty liver syndrome (FLS) is an increased hepatic triacylglycerol content, and a sudden decline in egg production often occurs. FLS may develop into fatty liver hemorrhagic syndrome (FLHS), characterized by sudden death from hepatic rupture and hemorrhage. DNA methylation is associated with transcriptional silencing, leading to the etiology and pathogenesis of some animal diseases. The roles of DNA methylation in the genesis of FLS, however, are largely unknown. The lipogenic methyl-deficient diet (MDD) caused FLS similar to human nonalcoholic steatohepatitis (NASH). After 16 Jingxing-Huang (JXH) hens were fed MDD for 10 wk, eight exhibited FLS (designated as FLS-susceptible birds); the remainder, without FLS, served as controls (NFLS). Physiological and biochemical variables, gene expression levels, and DNA methylation were determined in the liver. The development of FLS in JXH hens was accompanied by abnormal lipid accumulation. Relative expression of acetyl-CoA carboxylase (ACC), fatty acid synthase (FAS), and microsomal triglyceride transfer protein (MTTP) were significantly up-regulated in the FLS group in comparison with the NFLS group. The transcript abundance of sterol regulatory element binding protein 1 (SREBP-1c), stearoyl-CoA desaturase (SCD), liver X receptor alpha (LXRα), peroxisome proliferator-activated receptor alpha (PPARα), and peroxisome proliferator-activated receptor gamma (PPARγ) did not differ between the two groups. Interestingly, MTTP and ACC mRNA abundance were negatively correlated with the level of promoter methylation. The extent of DNA methylation of the cytosine-guanine (CpG) sites in the SREBP-1c, FAS, PPARα, and LXRα promoter regions was also analyzed by direct sequencing but none differed between FLS and NFLS birds. Taken together, these results specify link DNA methylation to the pathogenesis of FLS in chickens. PMID:27083546

  5. In Vitro Enhancement of Carvedilol Glucuronidation by Amiodarone-Mediated Altered Protein Binding in Incubation Mixture of Human Liver Microsomes with Bovine Serum Albumin.

    PubMed

    Sekimoto, Makoto; Takamori, Toru; Nakamura, Saki; Taguchi, Masato

    2016-01-01

    Carvedilol is mainly metabolized in the liver to O-glucuronide (O-Glu). We previously found that the glucuronidation activity of racemic carvedilol in pooled human liver microsomes (HLM) was increased, R-selectively, in the presence of amiodarone. The aim of this study was to clarify the mechanisms for the enhancing effect of amiodarone on R- and S-carvedilol glucuronidation. We evaluated O-Glu formation of R- and S-carvedilol enantiomers in a reaction mixture of HLM including 0.2% bovine serum albumin (BSA). In the absence of amiodarone, glucuronidation activity of R- and S-carvedilol for 25 min was 0.026, and 0.51 pmol/min/mg protein, and that was increased by 6.15 and 1.60-fold in the presence of 50 µM amiodarone, respectively. On the other hand, in the absence of BSA, or when BSA was replaced with human serum albumin, no enhancing effect of amiodarone on glucuronidation activity was observed, suggesting that BSA played a role in the mechanisms for the enhancement of glucuronidation activity. Unbound fraction of S-carvedilol in the reaction mixture was greater than that of R-carvedilol in the absence of amiodarone. Also, the addition of amiodarone caused a greater increase of unbound fraction of R-carvedilol than that of S-carvedilol. These results suggest that the altered protein binding by amiodarone is a key mechanism for R-selective stimulation of carvedilol glucuronidation. PMID:27476943

  6. Cloning and characterization of microsomal triglyceride transfer protein gene and its potential connection with peroxisome proliferator-activated receptor (PPAR) in blunt snout bream (Megalobrama amblycephala).

    PubMed

    Li, Jun-Yi; Zhang, Ding-Dong; Jiang, Guang-Zhen; Li, Xiang-Fei; Zhang, Chun-Nuan; Zhou, Man; Liu, Wen-Bin; Xu, Wei-Na

    2015-11-01

    Microsomal triglyceride transfer protein (MTTP), a major intracellular protein capable of transferring neutral lipids, plays a pivotal role in the assembly and secretion of apolipoprotein B-containing lipoproteins. In this study, MTTP cDNA was firstly cloned from the liver of blunt snout bream (Megalobrama amblycephala), the full-length cDNA covered 3457-bp with an open reading frame of 2661-bp, which encodes 886 amino acids, including a putative signal peptide of 24 amino acids long. After the feeding trial, a graded tissue-specific expression pattern of MTTP was observed and high expression abundance in the liver and intestine indicated its major function in lipid transport in this fish species. In addition, expression of genes encoding MTTP as well as peroxisome proliferator-activated receptor (PPAR), which are transcription factors and serve as key regulators in lipid homoeostasis, was all affected by dietary lipid and choline supplementations. Elevated dietary lipid levels significantly increased the liver, intestinal and muscle MTTP mRNA abundance. Additionally, the down-regulation of MTTP expression in the liver and muscle was observed when fish were fed with inadequate choline supplementation in high-fat diet, yet up-regulated as supplementing extra choline in diet. Expressions of PPARα and PPARβ in the liver and muscle showed similar trend of MTTP expression. The results suggested the potential connection of MTTP and PPAR in response to different dietary nutritional factors. Furthermore, extra choline supplementations could promote lipid transfer and enhance fatty acid oxidation, which indicated a molecular mechanism of choline on diminishing fat accumulation in blunt snout bream. PMID:26210738

  7. Mitochondrial nucleoid interacting proteins support mitochondrial protein synthesis

    PubMed Central

    He, J.; Cooper, H. M.; Reyes, A.; Di Re, M.; Sembongi, H.; Litwin, T. R.; Gao, J.; Neuman, K. C.; Fearnley, I. M.; Spinazzola, A.; Walker, J. E.; Holt, I. J.

    2012-01-01

    Mitochondrial ribosomes and translation factors co-purify with mitochondrial nucleoids of human cells, based on affinity protein purification of tagged mitochondrial DNA binding proteins. Among the most frequently identified proteins were ATAD3 and prohibitin, which have been identified previously as nucleoid components, using a variety of methods. Both proteins are demonstrated to be required for mitochondrial protein synthesis in human cultured cells, and the major binding partner of ATAD3 is the mitochondrial ribosome. Altered ATAD3 expression also perturbs mtDNA maintenance and replication. These findings suggest an intimate association between nucleoids and the machinery of protein synthesis in mitochondria. ATAD3 and prohibitin are tightly associated with the mitochondrial membranes and so we propose that they support nucleic acid complexes at the inner membrane of the mitochondrion. PMID:22453275

  8. Protein Synthesis Initiation Factors: Phosphorylation and Regulation

    SciTech Connect

    Karen S. Browning

    2009-06-15

    The initiation of the synthesis of proteins is a fundamental process shared by all living organisms. Each organism has both shared and unique mechanisms for regulation of this vital process. Higher plants provide for a major amount of fixation of carbon from the environment and turn this carbon into food and fuel sources for our use. However, we have very little understanding of how plants regulate the synthesis of the proteins necessary for these metabolic processes. The research carried out during the grant period sought to address some of these unknowns in the regulation of protein synthesis initiation. Our first goal was to determine if phosphorylation plays a significant role in plant initiation of protein synthesis. The role of phosphorylation, although well documented in mammalian protein synthesis regulation, is not well studied in plants. We showed that several of the factors necessary for the initiation of protein synthesis were targets of plant casein kinase and showed differential phosphorylation by the plant specific isoforms of this kinase. In addition, we identified and confirmed the phosphorylation sites in five of the plant initiation factors. Further, we showed that phosphorylation of one of these factors, eIF5, affected the ability of the factor to participate in the initiation process. Our second goal was to develop a method to make initiation factor 3 (eIF3) using recombinant methods. To date, we successfully cloned and expressed 13/13 subunits of wheat eIF3 in E. coli using de novo gene construction methods. The final step in this process is to place the subunits into three different plasmid operons for co-expression. Successful completion of expression of eIF3 will be an invaluable tool to the plant translation community.

  9. Co-ordination between membrane phospholipid synthesis and accelerated biosynthesis of cytoplasmic ribonucleic acid and protein

    PubMed Central

    Tata, J. R.

    1970-01-01

    1. The rate of synthesis of membrane phospholipid was studied in rat liver and seminal vesicles by following the incorporation of [32P]orthophosphate, [14C]choline and [14C]glycerol. Particular emphasis was laid on the endoplasmic reticulum, which was fractionated into smooth microsomal membranes, heavy rough membranes, light rough membranes and free polyribosomes. 2. Phospholipid labelling patterns suggested a heterogeneity in the synthesis and turnover of the different lipid moieties of smooth and rough endoplasmic membranes. The major phospholipids, phosphatidylcholine and phosphatidylethanolamine, were labelled relatively rapidly with 32P over a short period of time whereas incorporation of radioisotope into the minor phospholipids, sphingomyelin, lysolecithin and phosphatidylinositol proceeded slowly but over a longer period of time. 3. The incorporation of orotic acid into RNA and labelled amino acids into protein of the four submicrosomal fractions was also studied. 4. Rapid growth of the liver was induced by the administration of growth hormone and tri-iodothyronine to hypophysectomized and thyroidectomized rats and by partial hepatectomy. Growth of seminal vesicles of castrated rats was stimulated with testosterone propionate. 5. The rate of labelling of membrane phospholipids was enhanced in all major subcellular particulate fractions (nuclear, mitochondrial and microsomal) during induced growth. However, it was in the rough endoplasmic reticulum that the accumulation of phospholipids, RNA and protein was most marked. The effect of hormone administration was also to accelerate preferentially the labelling with 32P of sphingomyelin relative to that of phosphatidylcholine or phosphatidylethanolamine. 6. Time-course analyses showed that, in all four growth systems studied, the enhancement of the rate of membrane phospholipid synthesis coincided with the rather abrupt increase in the synthesis of RNA and protein of the rough endoplasmic reticulum. Growth

  10. Chlorolissoclimides: New inhibitors of eukaryotic protein synthesis

    PubMed Central

    Robert, Francis; Gao, Hong Qing; Donia, Marwa; Merrick, William C.; Hamann, Mark T.; Pelletier, Jerry

    2006-01-01

    Lissoclimides are cytotoxic compounds produced by shell-less molluscs through chemical secretions to deter predators. Chlorinated lissoclimides were identified as the active component of a marine extract from Pleurobranchus forskalii found during a high-throughput screening campaign to characterize new protein synthesis inhibitors. It was demonstrated that these compounds inhibit protein synthesis in vitro, in extracts prepared from mammalian and plant cells, as well as in vivo against mammalian cells. Our results suggest that they block translation elongation by inhibiting translocation, leading to an accumulation of ribosomes on mRNA. These data provide a rationale for the cytotoxic nature of this class of small molecule natural products. PMID:16540697

  11. Brain microsomal fatty acid elongation is increased in abcd1-deficient mouse during active myelination phase.

    PubMed

    Morita, Masashi; Kawamichi, Misato; Shimura, Yusuke; Kawaguchi, Kosuke; Watanabe, Shiro; Imanaka, Tsuneo

    2015-12-01

    The dysfunction of ABCD1, a peroxisomal ABC protein, leads to the perturbation of very long chain fatty acid (VLCFA) metabolism and is the cause of X-linked adrenoleukodystrophy. Abcd1-deficient mice exhibit an accumulation of saturated VLCFAs, such as C26:0, in all tissues, especially the brain. The present study sought to measure microsomal fatty acid elongation activity in the brain of wild-type (WT) and abcd1-deficient mice during the course of development. The fatty acid elongation activity in the microsomal fraction was measured by the incorporation of [2-(14)C]malonyl-CoA into fatty acids in the presence of C16:0-CoA or C20:0-CoA. Cytosolic fatty acid synthesis activity was completely inhibited by the addition of N-ethylmaleimide (NEM). The microsomal fatty acid elongation activity in the brain was significantly high at 3 weeks after birth and decreased substantially at 3 months after birth. Furthermore, we detected two different types of microsomal fatty acid elongation activity by using C16:0-CoA or C20:0-CoA as the substrate and found the activity toward C20:0-CoA in abcd1-deficient mice was higher than the WT 3-week-old animals. These results suggest that during the active myelination phase the microsomal fatty acid elongation activity is stimulated in abcd1-deficient mice, which in turn perturbs the lipid composition in myelin. PMID:26108493

  12. A Polymorphism in the Microsomal Triglyceride Transfer Protein Can Predict the Response to Antiviral Therapy in Egyptian Patients with Chronic Hepatitis C Virus Genotype 4 Infection

    PubMed Central

    Saad, Yasmin; Shaker, Olfat; Nassar, Yasser; Ahmad, Lama; Said, Mohamed; Esmat, Gamal

    2014-01-01

    Background/Aims A polymorphism in the microsomal triglyceride transfer protein (MTP) is associated with hepatic fibrosis, and carriers showed higher levels of steatosis, higher levels of hepatitis C virus (HCV) RNA and advanced fibrosis. The aim of this study was to study MTP expression pattern in HCV patients and impact of the MTP polymorphism on the response to antiviral therapy. Methods One hundred consecutive naive HCV genotype 4 patients were recruited to receive antiviral therapy, and 40 control subjects were also recruited. Demographic, laboratory, and histopathology data were collected. DNA was isolated, and the samples were subjected to polymerase chain reaction analysis and genotyping for MTP by restriction fragment length polymorphism analysis. Results Patients and controls were age- and sex-matched (male/female, 56/44, age, 39.2±7.8 years for patients with HCV; male/female, 18/22, age, 38.1±8.1 years for controls). MTP single nucleotide polymorphisms (SNPs) (GG, GT, TT) and alleles (G, T) in the patients versus the controls were 70%, 21%, 9% & 80.5%, 19.5% versus 10%, 87.5%, 2.5% & 53.8%, 46.3%, respectively (p=0.0001). The sustained viral response (SVR) of the patients was 60%. SNPs in MTP genotypes (GG, GT, and TT) and alleles (G and T) in the responders and nonresponders were 71.7%, 25%, 3.3% & 84.2%, 15.8% versus 67.5%, 15%, 17.5% & 75%, 25% (p=0.038 and p=0.109, respectively). A multivariate analysis showed that the GT genotype was an independent predictor of SVR (area under the curve 90% and p=0.0001). Conclusions MTP could be a new predictor for SVR to antiviral therapy in patients with HCV genotype 4 infection. PMID:25287167

  13. EFFECT OF ANTIBIOTICS AND INHIBITORS ON M PROTEIN SYNTHESIS

    PubMed Central

    Brock, Thomas D.

    1963-01-01

    Brock, Thomas D. (Western Reserve University, Cleveland, Ohio). Effect of antibiotics and inhibitors on M protein synthesis. J. Bacteriol. 85:527–531. 1963.—This work extends the observations of Fox and Krampitz on M protein synthesis in nongrowing cells of streptococci. A survey of a large number of antibiotics and other potential inhibitors was made. Some substances bring about inhibition of fermentation and inhibit M protein synthesis because they deprive the cell of the energy needed for this process. A second group of substances inhibit growth at concentrations tenfold or more lower than they inhibit M protein synthesis. These are the antibiotics which inhibit synthesis of cell wall or other structures in growing cells, but do not affect protein synthesis. A third group of substances inhibit growth and M protein synthesis at the same concentration. These substances probably inhibit growth because they inhibit general protein synthesis, and are therefore specific inhibitors of protein synthesis. In this class are chloramphenicol, erythromycin, and the tetracyclines. Several other antibiotics of previously unknown mode of action are in this class. A fourth group of substances had no effect on M protein synthesis. No substances were found which inhibited M protein synthesis at a lower concentration than that which inhibited growth. M protein synthesis in nongrowing cells may be a useful model system for obtaining a detailed understanding of protein synthesis. PMID:14042928

  14. Solution structure of oxidized rat microsomal cytochrome b5 in the presence of 2 M guanidinium chloride: monitoring the early steps in protein unfolding.

    PubMed

    Arnesano, F; Banci, L; Bertini, I; Koulougliotis, D

    1998-12-01

    One- and two-dimensional proton NMR spectroscopy has been employed in order to study the denaturation effect of guanidinium chloride (GdmCl) on the oxidized state of the A-form of rat microsomal cytochrome b5 (cyt b5). The protein rapidly starts losing the heme at denaturant concentrations larger than approximately 2.0 M and a largely unfolded protein is eventually obtained. An estimate of the unfolding kinetics is obtained and, by use of a two-state model (folded left and right arrow unfolded), a value for DeltaG degrees. Below this concentration, small (protein core maintains its overall structure. The analysis of the two-dimensional nuclear Overhauser effect spectroscopy (2D NOESY) maps has allowed the determination of the solution structure of the protein in the presence of 2 M GdmCl. By use of 1199 meaningful NOESY constraints (obtained from the assignment of 75% of the total protons) and 166 pseudocontact shifts, a family of 40 structures has been obtained through the program PSEUDYANA. The family was further refined through restrained energy minimization and the final root mean square deviation (RMSD) values with respect to the average structure are 0.67 +/- 0.10 A and 1.14 +/- 0.11 A for the backbone and heavy atoms, respectively. The quality of the present structure is equivalent to that of the one obtained recently for the native form [Arnesano et al. (1998) Biochemistry 37, 173-184], thus allowing a meaningful comparison between the two structures. Upon addition of 2 M GdmCl, significant local structural differences are induced to the protein backbone segments comprising residues 33-38 (helix alpha2) and 62-64 (end of helix alpha4-beginning of helix alpha5) while the overall folding scheme of the protein is still maintained. These protein regions form part of the "pocket

  15. Origins of the protein synthesis cycle

    NASA Technical Reports Server (NTRS)

    Fox, S. W.

    1981-01-01

    Largely derived from experiments in molecular evolution, a theory of protein synthesis cycles has been constructed. The sequence begins with ordered thermal proteins resulting from the self-sequencing of mixed amino acids. Ordered thermal proteins then aggregate to cell-like structures. When they contained proteinoids sufficiently rich in lysine, the structures were able to synthesize offspring peptides. Since lysine-rich proteinoid (LRP) also catalyzes the polymerization of nucleoside triphosphate to polynucleotides, the same microspheres containing LRP could have synthesized both original cellular proteins and cellular nucleic acids. The LRP within protocells would have provided proximity advantageous for the origin and evolution of the genetic code.

  16. HT-2 toxin 4-glucuronide as new T-2 toxin metabolite: enzymatic synthesis, analysis, and species specific formation of T-2 and HT-2 toxin glucuronides by rat, mouse, pig, and human liver microsomes.

    PubMed

    Welsch, Tanja; Humpf, Hans-Ulrich

    2012-10-10

    Glucuronides of the mycotoxin T-2 toxin and its phase I metabolite HT-2 toxin are important phase II metabolites under in vivo and in vitro conditions. Since standard substances are essential for the direct quantitation of these glucuronides, a method for the enzymatic synthesis of T-2 and HT-2 toxin glucuronides employing liver microsomes was optimized. Structure elucidation by nuclear magnetic resonance spectroscopy (NMR) and mass spectrometry revealed that besides T-2 toxin glucuronide and HT-2 toxin 3-glucuronide also the newly identified isomer HT-2 toxin 4-glucuronide was formed. Glucuronidation of T-2 and HT-2 toxin in liver microsomes of rat, mouse, pig, and human was compared and metabolites were analyzed directly by liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS). A distinct, species specific pattern of glucuronidation of T-2 and HT-2 toxin was observed with interesting interindividual differences. Until recently, glucuronides have frequently been analyzed indirectly by quantitation of the aglycone after enzymatic cleavage of the glucuronides by β-glucuronidase. Therefore, the hydrolysis efficiencies of T-2 and HT-2 toxin glucuronides using β-glucuronidases from Helix pomatia, bovine liver, and Escherichia coli were compared. PMID:22967261

  17. Postnatal ontogeny of skeletal muscle protein synthesis in pigs

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The neonatal period is characterized by rapid growth and elevated rates of synthesis and accretion of skeletal muscle proteins. The fractional rate of muscle protein synthesis is very high at birth and declines rapidly with age. The elevated capacity for muscle protein synthesis in the neonatal pig ...

  18. Postnatal ontogeny of skeletal muscle protein synthesis in pigs

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The neonatal period is characterized by rapid growth and elevated rates of synthesis and accretion of skeletal muscle proteins. The fractional rate of muscle protein synthesis is very high at birth and declines rapidly with development. The elevated capacity for muscle protein synthesis in the neo...

  19. Cumulative Muscle Protein Synthesis and Protein Intake Requirements.

    PubMed

    Simmons, Erin; Fluckey, James D; Riechman, Steven E

    2016-07-17

    Muscle protein synthesis (MPS) fluctuates widely over the course of a day and is influenced by many factors. The time course of MPS responses to exercise and the influence of training and nutrition can only be pieced together from several different investigations and methods, many of which create unnatural experimental conditions. Measurements of cumulative MPS, the sum synthesis over an extended period, using deuterium oxide have been shown to accurately reflect muscle responses and may allow investigations of the response to exercise, total protein intake requirements, and interaction with protein timing in free-living experimental conditions; these factors have yet to be carefully integrated. Such studies could include clinical and athletic populations to integrate nutritional and exercise recommendations and help guide their revisions to optimize the skeletal muscle function that is so important to overall health. PMID:27215586

  20. Effects of salt on the pattern of protein synthesis in barley roots

    SciTech Connect

    Hurkman, W.J.; Tanaka, C.K.

    1987-03-01

    The effect of salt stress on the incorporation of (/sup 3//sub 5/S)methionine into protein was examined in roots of barley (Hordeum vulgare L.cv California Mariout 72). Plants were grown in nutrient solution with or without 200 millimolar NaCl. Roots of intact plants were labeled in vivo and proteins were extracted and analyzed by fluorography of two-dimensional gels. Although the protein patterns for control and salt-stressed plants were qualitatively similar, the net synthesis of a number of proteins was quantitatively changed. The most striking change was a significant increase of label in two protein pairs that had pls of approximately 6.3 and 6.5. Each pair consisted of proteins of approximately 26 and 27 kilodaltons (kD). In roots of control plants, the 27-kD proteins were more heavily labeled in the microsomal fraction relative to the 26-kD proteins, whereas the 26-kD proteins were enriched in the post 178,000g supernatant fraction; in roots of salt treated plants, the 26- and 27-kD proteins were more intensely labeled in both fractions. Labeling of the 26- and 27-kD proteins returned to control levels when salt-stressed plants were transferred to nutrient solution without NaCl. No cross-reaction was detected between the antibody to the 26-kD protein from salt-adapted tobacco cells and the 26- and 27-kD proteins of barley.

  1. Protein Synthesis in Relation to Ripening of Pome Fruits 1

    PubMed Central

    Frenkel, Chaim; Klein, Isaac; Dilley, D. R.

    1968-01-01

    Protein synthesis by intact Bartlett pear fruits was studied with ripening as measured by flesh softening, chlorophyll degradation, respiration, ethylene synthesis, and malic enzyme activity. Protein synthesis is required for normal ripening, and the proteins synthesized early in the ripening process are, in fact, enzymes required for ripening. 14C-Phenylalanine is differentially incorporated into fruit proteins separated by acrylamide gel electrophoresis of pome fruits taken at successive ripening stages. Capacity for malic enzyme synthesis increases during the early stage of ripening. Fruit ripening and ethylene synthesis are inhibited when protein synthesis is blocked by treatment with cycloheximide at the early-climacteric stage. Cycloheximide became less effective as the climacteric developed. Ethylene did not overcome inhibition of ripening by cycloheximide. The respiratory climacteric is not inhibited by cycloheximide. It is concluded that normal ripening of pome fruits is a highly coordinated process of biochemical differentiation involving directed protein synthesis. PMID:16656897

  2. Radiation inactivation analysis of rat liver microsomal glucose 6-phosphatase

    SciTech Connect

    Ness, G.C.; Sample, C.E.; McCreery, M.J.; Sukalski, K.A.; Nordlie, R.C.

    1986-05-01

    Attempts to obtain the molecular weight of microsomal glucose-6-phosphatase based on solubilization and purification have yielded widely divergent results. Since radiation inactivation analysis can be used to obtain molecular weights of proteins within the native membrane environments, this technique was applied. Identical target sizes of about 70 kd for both glucose 6-phosphate phosphohydrolase and carbamyl phosphate:glucose phosphotransferase were observed. This value was unaffected by adding deoxycholate, which disrupts the microsomal membranes, to the microsomal suspensions prior to irradiation. The data suggest that the glucose 6-phosphate transport function and the glucose 6-phosphate phosphohydrolase activity of microsomal glucose 6-phosphatase either residue on a single polypeptide or on two covalently linked polypeptides.

  3. Protein synthesis inhibitor from potato tuber

    SciTech Connect

    Romaen, R. )

    1989-04-01

    A protein fraction capable of inhibit in vitro protein synthesis was found in potato tubers in fresh and wounded tissue. Inhibitor activity from fresh tissue decays with wounding. Inhibition activity was detected absorbed to ribsomal fraction and cytosol of potato tuber tissue by a partially reconstituted in vitro system from potato tuber and wheat germ. Adsorbed ribosomal fraction was more suitable of purification. This fraction was washed from ribosomes with 0.3M KCl, concentrated with ammonium sulfate precipitation and purified through sephadex G100 and sephadex G-75 columns chromatography. After 61 fold purification adsorbed protein fraction can inhibit germination of maize, wheat and sesame seeds, as well as {sup 3}H-leucine incorporation into protein by imbibed maize embryos. Inhibition activity was lost by temperature, alkali and protease-K hydrolysis. Preliminar analysis could not show presence of reductor sugars. Physiological role of this inhibitor in relation to rest and active tissue remains to be studied.

  4. Solubilization and partial characterization of a microsomal high affinity GTPase

    SciTech Connect

    Nicchitta, C.; Williamson, J.R.

    1987-05-01

    Isolated rat liver microsomes release sequestered Ca/sup 2 +/ following addition of GTP. In contrast to permeabilized cells, GTP dependent microsomal Ca/sup 2 +/ release requires low concentrations of polyethylene glycol (PEG). They have identified a microsomal, PEG-sensitive high affinity GTPase which shares a number of characteristics with the GTP-dependent Ca/sup 2 +/ release system. To aid in further characterization of this activity they have initiated studies on the solubilization and purification of the microsomal GTPases. When microsomes are solubilized under the following conditions (150 mM NaCl, 5 mg protein/ml, 1% Triton X-114) PEG sensitive GTPase activity selectively partitions into the detergent rich phase of the Triton X-114 extract. As observed in intact microsomal membranes the Triton X-114 soluble GTPase is maximally stimulated by 3% PEG. Half maximal stimulation is observed at 1% PEG. PEG increases the Vmax of this activity; no effects on Km were observed. The Km for GTP of the detergent soluble GTPase is 5 ..mu..M. This GTPase is sensitive to inhibition by sulfhydryl reagents. PEG-sensitive GTPase activity was completely inhibited in the presence of 25 ..mu..M p-hydroxymercuribenzoate (PHMB); half maximal inhibition was observed at 5 ..mu..M. Labeling of the Triton X-114 extract with the photosensitive compound (/sup 32/P) 8-azido GTP indicated the presence of two prominent GTP binding proteins of approximate molecular weights 17 and 54 kD.

  5. Metabolic activation of 2-methylfuran by rat microsomal systems

    SciTech Connect

    Ravindranath, V.; Boyd, M.R.

    1985-05-01

    2-Methylfuran (2-MF), a constituent of cigarette smoke and coffee, causes necrosis of liver, lungs, and kidneys in rodents. 2-MF is metabolically activated by mixed-function oxidases to acetylacrolein, a reactive metabolite that binds covalently to microsomal protein. The hepatic microsomal metabolism of 2-MF to reactive metabolite required the presence of NADPH and oxygen and was dependent on incubation time and substrate concentration. The microsomal metabolism of 2-MF was inducible by pretreatment of rats with phenobarbital and was inhibited by piperonyl butoxide and N-octyl imidazole, which indicates that the metabolism of 2-MF may be mediated by cytochrome P-450. Acetylacrolein was a potent inhibitor of mixed-function oxidase and completely inhibited the microsomal metabolism of 2-MF, indicating that 2-MF is a suicide substrate for the enzyme. The sulfhydryl nucleophile cysteine was a better trapping agent of the reactive metabolite of 2-MF than N-acetylcysteine or glutathione. Lysine decreased the covalent binding of 2-MF metabolites, presumably by reacting with the aldehyde group of acetylacrolein. In addition, in the presence of NADPH, 2-MF was bioactivated by both pulmonary and renal cortical microsomes to reactive metabolites that were covalently bound to microsomal proteins.

  6. Cyclosporin metabolism by human gastrointestinal mucosal microsomes.

    PubMed Central

    Webber, I R; Peters, W H; Back, D J

    1992-01-01

    The in vitro metabolism of the immunosuppressant cyclosporin (CsA) by human gastrointestinal mucosal microsomes has been studied. Macroscopically normal intestinal (n = 4) and liver (n = 2) tissue was obtained from kidney transplant donors, and microsomes prepared. Intestinal metabolism was most extensive with duodenal protein (15% conversion to metabolites M1/M17 after 2 h incubation at 37 degrees C; metabolite measurement by h.p.l.c). Western blotting confirmed the presence of P-4503A (enzyme subfamily responsible for CsA metabolism) in duodenum and ileum tissue, but not in colon tissue. The results of this study indicate that the gut wall may play a role in the first-pass metabolism of CsA, and could therefore be a contributory factor to the highly variable oral bioavailability of CsA. PMID:1389941

  7. The evolution of the protein synthesis system. I - A model of a primitive protein synthesis system

    NASA Technical Reports Server (NTRS)

    Mizutani, H.; Ponnamperuma, C.

    1977-01-01

    A model is developed to describe the evolution of the protein synthesis system. The model is comprised of two independent autocatalytic systems, one including one gene (A-gene) and two activated amino acid polymerases (O and A-polymerases), and the other including the addition of another gene (N-gene) and a nucleotide polymerase. Simulation results have suggested that even a small enzymic activity and polymerase specificity could lead the system to the most accurate protein synthesis, as far as permitted by transitions to systems with higher accuracy.

  8. High-yield cell-free synthesis of human EGFR by IRES-mediated protein translation in a continuous exchange cell-free reaction format

    PubMed Central

    Quast, Robert B.; Sonnabend, Andrei; Stech, Marlitt; Wüstenhagen, Doreen A.; Kubick, Stefan

    2016-01-01

    Cell-free protein synthesis systems derived from eukaryotic sources often provide comparatively low amounts of several μg per ml of de novo synthesized membrane protein. In order to overcome this, we herein demonstrate the high-yield cell-free synthesis of the human EGFR in a microsome-containing system derived from cultured Sf21 cells. Yields were increased more than 100-fold to more than 285 μg/ml by combination of IRES-mediated protein translation with a continuous exchange cell-free reaction format that allowed for prolonged reaction lifetimes exceeding 24 hours. In addition, an orthogonal cell-free translation system is presented that enabled the site-directed incorporation of p-Azido-L-phenylalanine by amber suppression. Functionality of cell-free synthesized receptor molecules is demonstrated by investigation of autophosphorylation activity in the absence of ligand and interaction with the cell-free synthesized adapter molecule Grb2. PMID:27456041

  9. Mitochondrial Protein Synthesis, Import, and Assembly

    PubMed Central

    Fox, Thomas D.

    2012-01-01

    The mitochondrion is arguably the most complex organelle in the budding yeast cell cytoplasm. It is essential for viability as well as respiratory growth. Its innermost aqueous compartment, the matrix, is bounded by the highly structured inner membrane, which in turn is bounded by the intermembrane space and the outer membrane. Approximately 1000 proteins are present in these organelles, of which eight major constituents are coded and synthesized in the matrix. The import of mitochondrial proteins synthesized in the cytoplasm, and their direction to the correct soluble compartments, correct membranes, and correct membrane surfaces/topologies, involves multiple pathways and macromolecular machines. The targeting of some, but not all, cytoplasmically synthesized mitochondrial proteins begins with translation of messenger RNAs localized to the organelle. Most proteins then pass through the translocase of the outer membrane to the intermembrane space, where divergent pathways sort them to the outer membrane, inner membrane, and matrix or trap them in the intermembrane space. Roughly 25% of mitochondrial proteins participate in maintenance or expression of the organellar genome at the inner surface of the inner membrane, providing 7 membrane proteins whose synthesis nucleates the assembly of three respiratory complexes. PMID:23212899

  10. Tools for Characterizing Bacterial Protein Synthesis Inhibitors

    PubMed Central

    Orelle, Cédric; Carlson, Skylar; Kaushal, Bindiya; Almutairi, Mashal M.; Liu, Haipeng; Ochabowicz, Anna; Quan, Selwyn; Pham, Van Cuong; Squires, Catherine L.; Murphy, Brian T.

    2013-01-01

    Many antibiotics inhibit the growth of sensitive bacteria by interfering with ribosome function. However, discovery of new protein synthesis inhibitors is curbed by the lack of facile techniques capable of readily identifying antibiotic target sites and modes of action. Furthermore, the frequent rediscovery of known antibiotic scaffolds, especially in natural product extracts, is time-consuming and expensive and diverts resources that could be used toward the isolation of novel lead molecules. In order to avoid these pitfalls and improve the process of dereplication of chemically complex extracts, we designed a two-pronged approach for the characterization of inhibitors of protein synthesis (ChIPS) that is suitable for the rapid identification of the site and mode of action on the bacterial ribosome. First, we engineered antibiotic-hypersensitive Escherichia coli strains that contain only one rRNA operon. These strains are used for the rapid isolation of resistance mutants in which rRNA mutations identify the site of the antibiotic action. Second, we show that patterns of drug-induced ribosome stalling on mRNA, monitored by primer extension, can be used to elucidate the mode of antibiotic action. These analyses can be performed within a few days and provide a rapid and efficient approach for identifying the site and mode of action of translation inhibitors targeting the bacterial ribosome. Both techniques were validated using a bacterial strain whose culture extract, composed of unknown metabolites, exhibited protein synthesis inhibitory activity; we were able to rapidly detect the presence of the antibiotic chloramphenicol. PMID:24041905

  11. Paracetamol hepatotoxicity and microsomal function.

    PubMed

    Kaushal, R; Dave, K R; Katyare, S S

    1999-03-01

    The effect of paracetamol-induced hepatotoxicity in rats (650 mg/kg) on microsomal function was examined. Paracetamol treatment resulted in lowered Na(+),K(+)-ATPase activity in the microsomes with decrease in V(max) of the low affinity high V(max) component II. However, the temperature kinetics was not influenced significantly. The total phospholipid and cholesterol contents as well as lipid peroxidation in the microsomes were unchanged. However, content of acidic phospholipids: phosphatidylserine and phosphatidylinositol decreased by 50% with a reciprocal increase in the sphingomyelin content; the lysophosphoglyceride content increased by 12-fold. The microsomal membrane appeared to be more fluidized following paracetamol treatment. Paracetamol treatment also resulted in a significant reduction in the sulfhydryl groups content. PMID:21781911

  12. ON-COLUMN ENRICHMENT OF HYDROPHOBIC CYP450 PROTEINS IN HPLC FRACTIONATION OF MOUSE MICROSOMES PRIOR TO PROTEIN DIGESTION AND NANOSPRAY-LC/MSMS ANALYSIS

    EPA Science Inventory

    Introduction

    Membrane proteins play crucial role in many cellular processes and are promising candidates for biomarker discovery but are under-represented in the field of proteomics due to their hydrophobic nature. Although standard reversed-phase LC methods often exhibit ...

  13. Organization and Regulation of Mitochondrial Protein Synthesis.

    PubMed

    Ott, Martin; Amunts, Alexey; Brown, Alan

    2016-06-01

    Mitochondria are essential organelles of endosymbiotic origin that are responsible for oxidative phosphorylation within eukaryotic cells. Independent evolution between species has generated mitochondrial genomes that are extremely diverse, with the composition of the vestigial genome determining their translational requirements. Typically, translation within mitochondria is restricted to a few key subunits of the oxidative phosphorylation complexes that are synthesized by dedicated ribosomes (mitoribosomes). The dramatically rearranged mitochondrial genomes, the limited set of transcripts, and the need for the synthesized proteins to coassemble with nuclear-encoded subunits have had substantial consequences for the translation machinery. Recent high-resolution cryo-electron microscopy has revealed the effect of coevolution on the mitoribosome with the mitochondrial genome. In this review, we place the new structural information in the context of the molecular mechanisms of mitochondrial translation and focus on the novel ways protein synthesis is organized and regulated in mitochondria. PMID:26789594

  14. Cell-free protein synthesis: applications come of age.

    PubMed

    Carlson, Erik D; Gan, Rui; Hodgman, C Eric; Jewett, Michael C

    2012-01-01

    Cell-free protein synthesis has emerged as a powerful technology platform to help satisfy the growing demand for simple and efficient protein production. While used for decades as a foundational research tool for understanding transcription and translation, recent advances have made possible cost-effective microscale to manufacturing scale synthesis of complex proteins. Protein yields exceed grams protein produced per liter reaction volume, batch reactions last for multiple hours, costs have been reduced orders of magnitude, and reaction scale has reached the 100-liter milestone. These advances have inspired new applications in the synthesis of protein libraries for functional genomics and structural biology, the production of personalized medicines, and the expression of virus-like particles, among others. In the coming years, cell-free protein synthesis promises new industrial processes where short protein production timelines are crucial as well as innovative approaches to a wide range of applications. PMID:22008973

  15. Understanding Protein Synthesis: An Interactive Card Game Discussion

    ERIC Educational Resources Information Center

    Lewis, Alison; Peat, Mary; Franklin, Sue

    2005-01-01

    Protein synthesis is a complex process and students find it difficult to understand. This article describes an interactive discussion "game" used by first year biology students at the University of Sydney. The students, in small groups, use the game in which the processes of protein synthesis are actioned by the students during a practical…

  16. SHORT-TERM MEMORY IS INDEPENDENT OF BRAIN PROTEIN SYNTHESIS

    SciTech Connect

    Davis, Hasker P.; Rosenzweig, Mark R.; Jones, Oliver W.

    1980-09-01

    Male Swiss albino CD-1 mice given a single injection of a cerebral protein synthesis inhibitor, anisomycin (ANI) (1 mg/animal), 20 min prior to single trial passive avoidance training demonstrated impaired retention at tests given 3 hr, 6 hr, 1 day, and 7 days after training. Retention was not significantly different from saline controls when tests were given 0.5 or 1.5 hr after training. Prolonging inhibition of brain protein synthesis by giving either 1 or 2 additional injections of ANI 2 or 2 and 4 hr after training did not prolong short-term retention performance. The temporal development of impaired retention in ANI treated mice could not be accounted for by drug dosage, duration of protein synthesis inhibition, or nonspecific sickness at test. In contrast to the suggestion that protein synthesis inhibition prolongs short-term memory (Quinton, 1978), the results of this experiment indicate that short-term memory is not prolonged by antibiotic drugs that inhibit cerebral protein synthesis. All evidence seems consistent with the hypothesis that short-term memory is protein synthesis independent and that the establishment of long-term memory depends upon protein synthesis during or shortly after training. Evidence for a role of protein synthesis in memory maintenance is discussed.

  17. Functional characterization of two microsomal fatty acid desaturases from Jatropha curcas L.

    PubMed

    Wu, Pingzhi; Zhang, Sheng; Zhang, Lin; Chen, Yaping; Li, Meiru; Jiang, Huawu; Wu, Guojiang

    2013-10-15

    Linoleic acid (LA, C18:2) and α-linolenic acid (ALA, C18:3) are polyunsaturated fatty acids (PUFAs) and major storage compounds in plant seed oils. Microsomal ω-6 and ω-3 fatty acid (FA) desaturases catalyze the synthesis of seed oil LA and ALA, respectively. Jatropha curcas L. seed oils contain large proportions of LA, but very little ALA. In this study, two microsomal desaturase genes, named JcFAD2 and JcFAD3, were isolated from J. curcas. Both deduced amino acid sequences possessed eight histidines shown to be essential for desaturases activity, and contained motif in the C-terminal for endoplasmic reticulum localization. Heterologous expression in Saccharomyces cerevisiae and Arabidopsis thaliana confirmed that the isolated JcFAD2 and JcFAD3 proteins could catalyze LA and ALA synthesis, respectively. The results indicate that JcFAD2 and JcFAD3 are functional in controlling PUFA contents of seed oils and could be exploited in the genetic engineering of J. curcas, and potentially other plants. PMID:23796520

  18. Inhibition of mammalian mitochondrial protein synthesis by oxazolidinones.

    PubMed

    McKee, E E; Ferguson, M; Bentley, A T; Marks, T A

    2006-06-01

    The effects of a variety of oxazolidinones, with different antibacterial potencies, including linezolid, on mitochondrial protein synthesis were determined in intact mitochondria isolated from rat heart and liver and rabbit heart and bone marrow. The results demonstrate that a general feature of the oxazolidinone class of antibiotics is the inhibition of mammalian mitochondrial protein synthesis. Inhibition was similar in mitochondria from all tissues studied. Further, oxazolidinones that were very potent as antibiotics were uniformly potent in inhibiting mitochondrial protein synthesis. These results were compared to the inhibitory profiles of other antibiotics that function by inhibiting bacterial protein synthesis. Of these, chloramphenicol and tetracycline were significant inhibitors of mammalian mitochondrial protein synthesis while the macrolides, lincosamides, and aminoglycosides were not. Development of future antibiotics from the oxazolidinone class will have to evaluate potential mitochondrial toxicity. PMID:16723564

  19. DIETARY PROTEIN AND LACTOSE INCREASE TRANSLATION INITIATION FACTOR ACTIVATION AND TISSUE PROTEIN SYNTHESIS IN NEONATAL PIGS

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Protein synthesis and eukaryotic initiation factor (eIF) activation are increased in muscle and liver of pigs parenterally infused with amino acids and insulin. To examine the effects of enteral protein and carbohydrate on protein synthesis, pigs (n = 42, 1.7 kg body wt) were fed isocaloric milk die...

  20. Modeling protein synthesis from a physicist's perspective: A toy model

    NASA Astrophysics Data System (ADS)

    Basu, Aakash; Chowdhury, Debashish

    2007-10-01

    Proteins are polymers of amino acids. These macromolecules are synthesized by intracellular machines called ribosomes. Although the experimental investigation of protein synthesis has been a traditional area of research in molecular cell biology, important quantitative models of protein synthesis have been reported in research journals devoted to statistical physics and related interdisciplinary topics. From the perspective of a physicist, protein synthesis is the classical transport of interacting ribosomes on a messenger RNA (mRNA) template that dictates the sequence of the amino acids on the protein. We discuss appropriate simplification of the models and methods. In particular, we develop and analyze a simple toy model using some elementary techniques of nonequilibrium statistical mechanics and predict the average rate of protein synthesis and the spatial organization of the ribosomes in the steady state.

  1. NADPH- and iron-dependent lipid peroxidation inhibit aromatase activity in human placental microsomes.

    PubMed

    Milczarek, Ryszard; Sokołowska, Ewa; Hallmann, Anna; Kaletha, Krystian; Klimek, Jerzy

    2008-06-01

    During pregnancy placenta is the most significant source of lipid hydroperoxides and other reactive oxygen species (ROS). The increased production of lipid peroxides and other ROS is often linked to pre-eclampsia. It is already proved that placental endoplasmic reticulum may be an important place of lipid peroxides and superoxide radical production. In the present study we revealed that NADPH- and iron-dependent lipid peroxidation in human placental microsomes (HPM) inhibit placental aromatase--a key enzyme of estrogen biosynthesis in human placenta. We showed that significant inhibition of this enzyme is caused by small lipid peroxidation (TBARS (thiobarbituric acid-reactive substances)<4nmol/mg microsomal protein (m.p.)). More intensive lipid peroxidation (TBARS>9nmol/mg microsomal protein) diminishes aromatase activity to value being less than 5% of initial value. NADPH- and iron-dependent lipid peroxidation also causes disappearance of cytochrome P450 parallel to observed aromatase activity inhibition. EDTA, alpha-tocopherol, MgCl(2) and superoxide dismutase (SOD) prevent aromatase activity inhibition and cytochrome P450(AROM) degradation. Mannitol and catalase have not effect on TBARS synthesis, aromatase activity and cytochrome P450 degradation. In view of the above we postulate that the inhibition of aromatase activity observed is mainly a consequence of cytochrome P450(AROM) degradation induced by lipid radicals. The role of hydroxyl radical in cytochrome P450 degradation is negligible in our experimental conditions. The results presented here also suggest that the inhibition of aromatase activity can also take place in placenta at in vivo conditions. PMID:18499441

  2. Role of RNA and Protein Synthesis in Abscission

    PubMed Central

    Abeles, F. B.

    1968-01-01

    The cell separation aspect of abscission is thought to involve the action of specific cell wall degrading enzymes. Enzymes represent synthesis which in turn is preceded by the synthesis of specific RNA molecules, and it follows that inhibition of either of these processes would also block abscission. Since abscission is a localized phenomenon usually involving 2 or 3 cell layers, RNA and protein synthesis should also be localized. Manipulations of plant material which either accelerate or retard abscission may be due to the regulation of RNA and protein synthesis. This paper is a review of literature concerned with these and related questions. Images PMID:16657020

  3. Interrelation between protein synthesis, proteostasis and life span.

    PubMed

    Arnsburg, Kristin; Kirstein-Miles, Janine

    2014-02-01

    The production of newly synthesized proteins is a key process of protein homeostasis that initiates the biosynthetic flux of proteins and thereby determines the composition, stability and functionality of the proteome. Protein synthesis is highly regulated on multiple levels to adapt the proteome to environmental and physiological challenges such as aging and proteotoxic conditions. Imbalances of protein folding conditions are sensed by the cell that then trigger a cascade of signaling pathways aiming to restore the protein folding equilibrium. One regulatory node to rebalance proteostasis upon stress is the control of protein synthesis itself. Translation is reduced as an immediate response to perturbations of the protein folding equilibrium that can be observed in the cytosol as well as in the organelles such as the endoplasmatic reticulum and mitochondria. As reduction of protein synthesis is linked to life span increase, the signaling pathways regu-lating protein synthesis might be putative targets for treatments of age-related diseases. Eukaryotic cells have evolved a complex system for protein synthesis regulation and this review will summarize cellular strategies to regulate mRNA translation upon stress and its impact on longevity. PMID:24653664

  4. Detection on immunoblot of new proteins from the soluble fraction of the cell recognized either by anti-liver-kidney microsome antibodies type 1 or by anti-liver cytosol antibodies type 1--relationship with hepatitis C virus infection.

    PubMed

    Ballot, E; Desbos, A; Monier, J C

    1996-09-01

    Antibodies directed against liver cytosol protein, called anti-liver cytosol type 1 (LC1 Ab), have been described by both immunofluorescence (IF) and immunodiffusion techniques in sera from patients with autoimmune hepatitis (AIH). They have never been found in association with antibodies directed against the hepatitis C virus (HCV), unlike the anti-liver-kidney microsome antibodies type 1 (LKM1 Ab), the serological marker of AIH type 2. This suggests that there are two subgroups of AIH type 2, i.e., HCV-related and non-HCV-related. In this study, immunoblotting experiments were performed using proteins from the soluble phase of the rat liver cell; 141 sera which tested positive for LKM1 Ab by IF, 24 identified as having LC1 Ab by IF, and 50 from blood donors as controls were analyzed. Three bands were stained by LC1 Ab sera more often than by the control sera, and with a statistically significant frequency. These 3 proteins were located at apparent Mr 50,000, 55,000, and 60,000. The LKM1 Ab-positive sera as defined by IF stained six bands with a statistically significant frequency compared to the controls. Their apparent Mr were 35,000, 39,000, 47,000, 50,000, 55,000, and 60,000. LKM1 Ab-positive sera which were anti-HCV negative recognized a 60,000 protein belonging to the soluble phase of the cell, with a statistically significant frequency compared to LKM1 Ab-positive sera which were anti-HCV positive. This 60,000 protein was also recognized by LC1 Ab-positive sera, which were almost always anti-HCV negative. The presence of antibodies against a 60,000 protein from the soluble phase of the cell is discussed in terms of the anti-HCV serological markers found in the sera from patients with AIH. PMID:8811044

  5. MATERNAL PROTEIN HOMEOSTASIS AND MILK PROTEIN SYNTHESIS DURING FEEDING AND FASTING IN HUMANS

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Little is known about amino acid (aa) and protein metabolism in lactating women. We hypothesized: 1) aa sources other than the plasma acid pool provide substrate for milk protein synthesis in humans; and 2) if albumin was one such source, then albumin fractional synthesis rate (FSR) is higher in th...

  6. Predictors of Muscle Protein Synthesis after Severe Pediatric Burns

    PubMed Central

    Diaz, Eva C.; Herndon, David N.; Lee, Jinhyung; Porter, Craig; Cotter, Matthew; Suman, Oscar E.; Sidossis, Labros S.; Børsheim, Elisabet

    2015-01-01

    Background Following a major burn, skeletal muscle protein synthesis rate increases, but is often insufficient to compensate for massively elevated muscle protein breakdown rates. Given the long-term nature of the pathophysiologic response to burn injury, we hypothesized that muscle protein synthesis rate would be chronically elevated in severely burned children. The objectives of this study were to characterize muscle protein synthesis rate of burned children over a period of 24 months post-injury, and identify predictors that influence this response. Study design 87 children with ≥40% total body surface area (TBSA) burn were included. Patients participated in stable isotope infusion studies at 1, 2 and ~ 4 weeks post-burn, and at 6, 12 and 24 months post-injury to determine skeletal muscle fractional synthesis rate. Generalized estimating equations with log link normal distribution were applied to account for clustering of patients and control for patient characteristics. Results Patients (8±6 years) had large (62, 51–72% TBSA) and deep (47±21% TBSA third degree) burns. Muscle fractional synthesis rate was elevated throughout the first 12 months post-burn compared to established values from healthy young adults. Muscle fractional synthesis rate was lower in boys, children >3 years old, and when burns were >80% TBSA. Conclusions Muscle protein synthesis is elevated for at least one year after injury, suggesting that greater muscle protein turnover is a component of the long-term pathophysiological response to burn trauma. Muscle protein synthesis is highly affected by gender, age and burn size in severely burned children. These findings may explain the divergence in net protein balance and lean body mass in different populations of burn victims. PMID:25807408

  7. Protein chemical synthesis by α-ketoacid-hydroxylamine ligation.

    PubMed

    Harmand, Thibault J; Murar, Claudia E; Bode, Jeffrey W

    2016-06-01

    Total chemical synthesis of proteins allows researchers to custom design proteins without the complex molecular biology that is required to insert non-natural amino acids or the biocontamination that arises from methods relying on overexpression in cells. We describe a detailed procedure for the chemical synthesis of proteins with the α-ketoacid-hydroxylamine (KAHA ligation), using (S)-5-oxaproline (Opr) as a key building block. This protocol comprises two main parts: (i) the synthesis of peptide fragments by standard fluorenylmethoxycarbonyl (Fmoc) chemistry and (ii) the KAHA ligation between fragments containing Opr and a C-terminal peptide α-ketoacid. This procedure provides an alternative to native chemical ligation (NCL) that could be valuable for the synthesis of proteins, particularly targets that do not contain cysteine residues. The ligation conditions-acidic DMSO/H2O or N-methyl-2-pyrrolidinone (NMP)/H2O-are ideally suited for solubilizing peptide segments, including many hydrophobic examples. The utility and efficiency of the protocol is demonstrated by the total chemical synthesis of the mature betatrophin (also called ANGPTL8), a 177-residue protein that contains no cysteine residues. With this protocol, the total synthesis of the betatrophin protein has been achieved in around 35 working days on a multimilligram scale. PMID:27227514

  8. Circulating protein synthesis rates reveal skeletal muscle proteome dynamics.

    PubMed

    Shankaran, Mahalakshmi; King, Chelsea L; Angel, Thomas E; Holmes, William E; Li, Kelvin W; Colangelo, Marc; Price, John C; Turner, Scott M; Bell, Christopher; Hamilton, Karyn L; Miller, Benjamin F; Hellerstein, Marc K

    2016-01-01

    Here, we have described and validated a strategy for monitoring skeletal muscle protein synthesis rates in rodents and humans over days or weeks from blood samples. We based this approach on label incorporation into proteins that are synthesized specifically in skeletal muscle and escape into the circulation. Heavy water labeling combined with sensitive tandem mass spectrometric analysis allowed integrated synthesis rates of proteins in muscle tissue across the proteome to be measured over several weeks. Fractional synthesis rate (FSR) of plasma creatine kinase M-type (CK-M) and carbonic anhydrase 3 (CA-3) in the blood, more than 90% of which is derived from skeletal muscle, correlated closely with FSR of CK-M, CA-3, and other proteins of various ontologies in skeletal muscle tissue in both rodents and humans. Protein synthesis rates across the muscle proteome generally changed in a coordinate manner in response to a sprint interval exercise training regimen in humans and to denervation or clenbuterol treatment in rodents. FSR of plasma CK-M and CA-3 revealed changes and interindividual differences in muscle tissue proteome dynamics. In human subjects, sprint interval training primarily stimulated synthesis of structural and glycolytic proteins. Together, our results indicate that this approach provides a virtual biopsy, sensitively revealing individualized changes in proteome-wide synthesis rates in skeletal muscle without a muscle biopsy. Accordingly, this approach has potential applications for the diagnosis, management, and treatment of muscle disorders. PMID:26657858

  9. Circulating protein synthesis rates reveal skeletal muscle proteome dynamics

    PubMed Central

    Shankaran, Mahalakshmi; King, Chelsea L.; Angel, Thomas E.; Holmes, William E.; Li, Kelvin W.; Colangelo, Marc; Price, John C.; Turner, Scott M.; Bell, Christopher; Hamilton, Karyn L.; Miller, Benjamin F.; Hellerstein, Marc K.

    2015-01-01

    Here, we have described and validated a strategy for monitoring skeletal muscle protein synthesis rates in rodents and humans over days or weeks from blood samples. We based this approach on label incorporation into proteins that are synthesized specifically in skeletal muscle and escape into the circulation. Heavy water labeling combined with sensitive tandem mass spectrometric analysis allowed integrated synthesis rates of proteins in muscle tissue across the proteome to be measured over several weeks. Fractional synthesis rate (FSR) of plasma creatine kinase M-type (CK-M) and carbonic anhydrase 3 (CA-3) in the blood, more than 90% of which is derived from skeletal muscle, correlated closely with FSR of CK-M, CA-3, and other proteins of various ontologies in skeletal muscle tissue in both rodents and humans. Protein synthesis rates across the muscle proteome generally changed in a coordinate manner in response to a sprint interval exercise training regimen in humans and to denervation or clenbuterol treatment in rodents. FSR of plasma CK-M and CA-3 revealed changes and interindividual differences in muscle tissue proteome dynamics. In human subjects, sprint interval training primarily stimulated synthesis of structural and glycolytic proteins. Together, our results indicate that this approach provides a virtual biopsy, sensitively revealing individualized changes in proteome-wide synthesis rates in skeletal muscle without a muscle biopsy. Accordingly, this approach has potential applications for the diagnosis, management, and treatment of muscle disorders. PMID:26657858

  10. Recovery of ribophorins and ribosomes in "inverted rough" vesicles derived from rat liver rough microsomes

    PubMed Central

    1982-01-01

    Treatment of rat liver rough microsomes (3.5 mg of protein/ml) with sublytical concentrations (0.08%) of the neutral detergent Triton X-100 caused a lateral displacement of bound ribosomes and the formation of ribosomal aggregates on the microsomal surface. At slightly higher detergent concentrations (0.12-0.16%) membrane areas bearing ribosomal aggregates invaginated into the microsomal lumen and separated from the rest of the membrane. Two distinct classes of vesicles could be isolated by density gradient centrifugation from microsomes treated with 0.16% Triton X-100: one with ribosomes bound to the inner membrane surfaces ("inverted rough" vesicles) and another with no ribosomes attached to the membranes. Analysis of the fractions showed that approximately 30% of the phospholipids and 20-30% of the total membrane protein were released from the membranes by this treatment. Labeling with avidin-ferritin conjugates demonstrated that concanavalin A binding sites, which in native rough microsomes are found in the luminal face of the membranes, were present on the outer surface of the inverted rough vesicles. Freeze-fracture electron microscopy showed that both fracture faces had similar concentrations of intramembrane particles. SDS PAGE analysis of the two vesicle subfractions demonstrated that, of all the integral microsomal membrane proteins, only ribophorins I and II were found exclusively in the inverted rough vesicles bearing ribosomes. These observations are consistent with the proposal that ribophorins are associated with the ribosomal binding sites characteristic of rough microsomal membranes. PMID:7068749

  11. Temperature-Regulated Protein Synthesis by Leptospira interrogans

    PubMed Central

    Nally, Jarlath E.; Timoney, John F.; Stevenson, Brian

    2001-01-01

    Leptospira interrogans is an important mammalian pathogen. Transmission from an environmental source requires adaptations to a range of new environmental conditions in the organs and tissues of the infected host. Since many pathogenic bacteria utilize temperature to discern their environment and regulate the synthesis of appropriate proteins, we investigated the effects of temperature on protein synthesis in L. interrogans. Bacteria were grown for several days after culture temperatures were shifted from 30 to 37°C. Triton X-114 cellular fractionation identified several proteins of the cytoplasm, periplasm, and outer membrane for which synthesis was dependent on the culture temperature. Synthesis of a cytoplasmic protein of 20 kDa was switched off at 37°C, whereas synthesis of a 66-kDa periplasmic protein was increased at the higher temperature. Increased synthesis of a 25-kDa outer membrane protein was observed when the organisms were shifted from 30 to 37°C. A 36-kDa protein synthesized at 30 but not at 37°C was identified as LipL36, an outer membrane lipoprotein. In contrast, expression of another lipoprotein, LipL41, was the same at either temperature. Immunoblotting with convalescent equine sera revealed that some proteins exhibiting thermoregulation of synthesis elicited antibody responses during infection. Our results show that sera from horses which aborted as a result of naturally acquired infection with L. interrogans serovar pomona type kennewicki recognize periplasmic and outer membrane proteins which are differentially synthesized in response to temperature and which therefore may be important in the host-pathogen interaction during infection. PMID:11119530

  12. Discovery, Synthesis and Biological Evaluation of Novel SMN Protein Modulators

    PubMed Central

    Xiao, Jingbo; Marugan, Juan J.; Zheng, Wei; Titus, Steve; Southall, Noel; Cherry, Jonathan J.; Evans, Matthew; Androphy, Elliot J.; Austin, Christopher P.

    2011-01-01

    Spinal Muscular Atrophy (SMA) is an autosomal recessive disorder affecting the expression or function of survival motor neuron protein (SMN) due to the homozygous deletion or rare point mutations in the survival motor neuron gene 1 (SMN1). The human genome includes a second nearly identical gene called SMN2 that is retained in SMA. SMN2 transcripts undergo alternative splicing with reduced levels of SMN. Up-regulation of SMN2 expression, modification of its splicing, or inhibition of proteolysis of the truncated protein derived from SMN2 have been discussed as potential therapeutic strategies for SMA. In this manuscript, we detail the discovery of a series of arylpiperidines as novel modulators of SMN protein. Systematic hit-to-lead efforts significantly improved potency and efficacy of the series in the primary and orthogonal assays. Structure property relationships including microsomal stability, cell permeability and in vivo pharmacokinetics (PK) studies were also investigated. We anticipate that a lead candidate chosen from this series may serve as a useful probe for exploring the therapeutic benefits of SMN protein up-regulation in SMA animal models, and a starting point for clinical development. PMID:21819082

  13. The origin of polynucleotide-directed protein synthesis

    NASA Technical Reports Server (NTRS)

    Orgel, Leslie E.

    1989-01-01

    If protein synthesis evolved in an RNA world it was probably preceded by simpler processes by means of which interaction with amino acids conferred selective advantage on replicating RNA molecules. It is suggested that at first the simple attachment of amino acids to the 2'(3') termini of RNA templates favored initiation of replication at the end of the template rather than at internal positions. The second stage in the evolution of protein synthesis would probably have been the association of pairs of charged RNA adaptors in such a way as to favor noncoded formation of peptides. Only after this process had become efficient could coded synthesis have begun.

  14. Biotransformation of phenol to hydroquinone and catechol by rat liver microsomes.

    PubMed

    Sawahata, T; Neal, R A

    1983-03-01

    Hepatic microsomal biotransformation of phenol to hydroquinone and catechol has been investigated with special reference to the covalent binding to microsomal protein of reactive metabolites formed during microsomal metabolism of phenol. Incubation of [14C]phenol with microsomes from phenobarbital-treated rat liver in the presence of an NADPH-generating system resulted in the formation of hydroquinone and catechol in the ratio of 20:1. No significant formation of 1,2,4-benzenetriol was observed. The biotransformation of phenol to both hydroquinone and catechol required NADPH and molecular oxygen. NADH was much less effective than NADPH as an electron donor and exhibited no significant synergistic effect when used together with NADPH. The biotransformation was inhibited by typical cytochrome P-450 inhibitors such as carbon monoxide, SKF 525-A, and metyrapone. These results indicated the involvement of cytochrome P-450 in the microsomal hydroxylation of phenol at both the ortho- and para-positions. Covalent binding of radioactivity to microsomal protein was observed when [14C]phenol was incubated with rat liver microsomes in the presence of an NADPH-generating system. The covalent binding was also found to require NADPH and molecular oxygen. Inclusion of cytochrome P-450 inhibitors in the incubation mixture resulted in a decrease in the covalent binding. These results indicated that at least one step in the metabolic activation of phenol to the metabolites responsible for covalent binding to microsomal protein was mediated by cytochrome P-450. Inclusion of N-acetylcysteine in the incubation mixture resulted in the complete inhibition of the covalent binding of radioactivity derived from [14C]phenol to microsomal protein, and there was a concomitant formation of N-acetylcysteine adducts of hydroquinone and catechol. These results indicated that hydroquinone and catechol were both precursors to reactive metabolites responsible for the covalent binding. PMID:6835203

  15. Effect of chronic ethanol consumption on glycosylation processes in rat liver microsomes and Golgi apparatus.

    PubMed

    Cottalasso, D; Gazzo, P; Dapino, D; Domenicotti, C; Pronzato, M A; Traverso, N; Bellocchio, A; Nanni, G; Marinari, U M

    1996-01-01

    Previous studies have demonstrated that acute ethanol intoxication affects various steps of protein glycosylation at the level of rat liver endoplasmic reticulum and Golgi apparatus. The aim of this investigation was to demonstrate whether chronic ethanol intake can induce definitive changes of liver glycoprotein processing. Rats were given ethanol by liquid diet for 8 weeks. At the end of this period the triglyceride levels in liver homogenate and microsomes were significantly higher than in controls. Isolated hepatocytes prelabelled with [3H]Na palmitate and [14C]glucosamine showed a significant storage of the lipid and carbohydrate radioactivity in microsomes and Golgi apparatus and a significant impairment of labelled glycolipoprotein secretion. Changes of the glycosylation steps were observed both in endoplasmic reticulum and in Golgi apparatus: in the former the levels of dolichyl phosphate, which is rate-limiting for the synthesis of glycoprotein, showed a significant reduction; in the latter the activity of the main enzymes responsible for the terminal glycosylation process was significantly decreased. These data suggest that an impairment of glycoprotein maturation may be involved in the pathogenesis of liver injury induced by chronic ethanol intake. PMID:8672174

  16. Regulation of platelet activating factor synthesis: modulation of 1-alkyl-2-lyso-sn-glycero-3-phosphocholine:acetyl-CoA acetyltransferase by phosphorylation and dephosphorylation in rat spleen microsomes

    SciTech Connect

    Lenihan, D.J.; Lee, T.C.

    1984-05-16

    1-Alkyl-2-lyso-sn-glycero-3-phosphocholine:acetyl-CoA acetyltransferase plays an important regulatory role in the biosynthesis of platelet activating factor, a potent bioactive mediator. The authors tested the hypothesis that the activity of acetyltransferase may be modulated by enzymatic phosphorylation and dephosphorylation. The results showed that acetyltransferase activity in rat spleens was 2- to 3-fold higher in microsomes isolated in the presence of F/sup -/ than in those isolated in the presence of Cl/sup -/. The microsomal acetyltransferase could be activated by preincubation of microsomes, isolated in the presence of Cl/sup -/, with ATP, Mg/sup 2 +/, and the soluble fraction from rat spleen. Addition of phosphatidylserine, diacylglycerols, plus Ca/sup 2 +/ further enhanced the activity. The increase in the activity of acetyltranferase was abolished by treatment of the activated microsomes with alkaline phosphatase. Conversely, the activity of acetyltransferase can be reactivated in the alkaline phosphatase-treated microsomes with incubation conditions that favor phosphorylation. Therefore, the findings suggest that acetyltransferase activity is regulated by reversible activation/inactivation through phosphorylation/dephosphorylation.

  17. Adeno-associated virus rep protein synthesis during productive infection

    SciTech Connect

    Redemann, B.E.; Mendelson, E.; Carter, B.J.

    1989-02-01

    Adeno-associated virus (AAV) Rep proteins mediate viral DNA replication and can regulate expression from AAV genes. The authors studied the kinetics of synthesis of the four Rep proteins, Rep78, Rep68, Rep52, and Rep40, during infection of human 293 or KB cells with AAV and helper adenovirus by in vivo labeling with (/sup 35/S)methionine, immunoprecipitation, and immunoblotting analyses. Rep78 and Rep52 were readily detected concomitantly with detection of viral monomer duplex DNA replicating about 10 to 12 h after infection, and Rep68 and Rep40 were detected 2 h later. Rep78 and Rep52 were more abundant than Rep68 and Rep40 owing to a higher synthesis rate throughout the infectious cycle. In some experiments, very low levels of Rep78 could be detected as early as 4 h after infection. The synthesis rates of Rep proteins were maximal between 14 and 24 h and then decreased later after infection. Isotopic pulse-chase experiments showed that each of the Rep proteins was synthesized independently and was stable for at least 15 h. A slower-migrating, modified form of Rep78 was identified late after infection. AAV capsid protein synthesis was detected at 10 to 12 h after infection and also exhibited synthesis kinetics similar to those of the Rep proteins. AAV DNA replication showed at least two clearly defined stages. Bulk duplex replicating DNA accumulation began around 10 to 12 h and reached a maximum level at about 20 h when Rep and capsid protein synthesis was maximal. Progeny single-stranded DNA accumulation began about 12 to 13 h, but most of this DNA accumulated after 24 h when Rep and capsid protein synthesis had decreased.

  18. Glutathione depletion by aniline analogs in vitro associated with liver microsomal cytochrome P-450.

    PubMed

    Aikawa, K; Satoh, T; Kobayashi, K; Kitagawa, H

    1978-10-01

    Enzymic depletion of glutathione (GSH) in vitro by aniline analogs was mostly dependent on the cytochrome P-450 level in liver microsomes. In a case of acetaminophen (AAP), active metabolite of AAP formed through liver microsomal drug metabolizing enzymes consumed GSH. The active metabolite formed binds, at least in part, covalently to liver microsomal proteins. In addition, species differences in the extent of GSH depletion by AAP in vitro was related to the amounts of the active metabolite of AAP bound covalently to liver microsomal protein(s) by experiments using 14C-AAP. Similar depletion of GSH was also seen with other aniline analogs such as aniline itself and p-chloroaniline, but not with acetanilide, in four animal species. These in vitro results obtained here strongly support the well-known findings concerning both GSH depletion and covalent binding in vivo of the active metabolite after AAP treatment. PMID:722999

  19. Cell-free protein synthesis in microfluidic array devices.

    PubMed

    Mei, Qian; Fredrickson, Carl K; Simon, Andrew; Khnouf, Ruba; Fan, Z Hugh

    2007-01-01

    We report the development of a microfluidic array device for continuous-exchange, cell-free protein synthesis. The advantages of protein expression in the microfluidic array include (1) the potential to achieve high-throughput protein expression, matching the throughput of gene discovery; (2) more than 2 orders of magnitude reduction in reagent consumption, decreasing the cost of protein synthesis; and (3) the possibility to integrate with detection for rapid protein analysis, eliminating the need to harvest proteins. The device consists of an array of units, and each unit can be used for production of an individual protein. The unit comprises a tray chamber for in vitro protein expression and a well chamber as a nutrient reservoir. The tray is nested in the well, and they are separated by a dialysis membrane and connected through a microfluidic connection that provides a means to supply nutrients and remove the reaction byproducts. The device is demonstrated by synthesis of green fluorescent protein, chloramphenicol acetyl-transferase, and luciferase. Protein expression in the device lasts 5-10 times longer and the production yield is 13-22 times higher than in a microcentrifuge tube. In addition, we studied the effects of the operation temperature and hydrostatic flow on the protein production yield. PMID:17924644

  20. Multiple Post-translational Modifications Affect Heterologous Protein Synthesis*

    PubMed Central

    Tokmakov, Alexander A.; Kurotani, Atsushi; Takagi, Tetsuo; Toyama, Mitsutoshi; Shirouzu, Mikako; Fukami, Yasuo; Yokoyama, Shigeyuki

    2012-01-01

    Post-translational modifications (PTMs) are required for proper folding of many proteins. The low capacity for PTMs hinders the production of heterologous proteins in the widely used prokaryotic systems of protein synthesis. Until now, a systematic and comprehensive study concerning the specific effects of individual PTMs on heterologous protein synthesis has not been presented. To address this issue, we expressed 1488 human proteins and their domains in a bacterial cell-free system, and we examined the correlation of the expression yields with the presence of multiple PTM sites bioinformatically predicted in these proteins. This approach revealed a number of previously unknown statistically significant correlations. Prediction of some PTMs, such as myristoylation, glycosylation, palmitoylation, and disulfide bond formation, was found to significantly worsen protein amenability to soluble expression. The presence of other PTMs, such as aspartyl hydroxylation, C-terminal amidation, and Tyr sulfation, did not correlate with the yield of heterologous protein expression. Surprisingly, the predicted presence of several PTMs, such as phosphorylation, ubiquitination, SUMOylation, and prenylation, was associated with the increased production of properly folded soluble proteins. The plausible rationales for the existence of the observed correlations are presented. Our findings suggest that identification of potential PTMs in polypeptide sequences can be of practical use for predicting expression success and optimizing heterologous protein synthesis. In sum, this study provides the most compelling evidence so far for the role of multiple PTMs in the stability and solubility of heterologously expressed recombinant proteins. PMID:22674579

  1. Monitoring protein synthesis in single live cancer cells.

    PubMed

    Tu, Chengyi; Santo, Loredana; Mishima, Yuko; Raje, Noopur; Smilansky, Zeev; Zoldan, Janet

    2016-05-16

    Protein synthesis is generally under sophisticated and dynamic regulation to meet the ever-changing demands of a cell. Global up or down-regulation of protein synthesis and the shift of protein synthesis location (as shown, for example, during cellular stress or viral infection) are recognized as cellular responses to environmental changes such as nutrient/oxygen deprivation or to alterations such as pathological mutations in cancer cells. Monitoring protein synthesis in single live cells can be a powerful tool for cancer research. Here we employed a microfluidic platform to perform high throughput delivery of fluorescent labeled tRNAs into multiple myeloma cells with high transfection efficiency (∼45%) and high viability (>80%). We show that the delivered tRNAs were actively recruited to the ER for protein synthesis and that treatment with puromycin effectively disrupted this process. Interestingly, we observed the scattered distribution of tRNAs in cells undergoing mitosis, which has not been previously reported. Fluorescence lifetime analysis detected extensive FRET signals generated from tRNAs labeled as FRET pairs, further confirming that the delivered tRNAs were used by active ribosomes for protein translation. Our work demonstrates that the microfluidic delivery of FRET labeled tRNAs into living cancer cells can provide new insights into basic cancer metabolism and has the potential to serve as a platform for drug screening, diagnostics, or personalized medication. PMID:26956582

  2. Regulation of protein synthesis during early limitation of Saccharomyces cerevisiae.

    PubMed Central

    Swedes, J S; Dial, M E; McLaughlin, C S

    1979-01-01

    Arsenate, a competitive inhibitor with phosphate in phosphorylation reactions, has been used to lower adenine and guanine nucleotide levels in Saccharomyces cerevisiae to study nucleotide effects on protein synthesis. By measuring polysome levels, we have shown that initiation of protein synthesis is much more sensitive than elongation or termination to inhibition when the ATP/ADP, GTP/GDP ratios are low. When the arsenate-phosphate molar ratio was 0.27, protein synthesis was inhibited by about 85% and the kinetics of polysome decay was similar to that observed with the initiation inhibitor, verrucarin-76, or with the protein synthesis initiation mutant, ts187, at the restrictive temperature. With this level of arsenate, the adenylate energy charge dropped from 0.9 to 0.7 and the ATP/ADP and GTP/GDP ratios dropped from 6 to 2. The observed correlations between nucleotide ratio changes and inhibition of protein synthesis suggest that the former may be a control signal for the latter. The significance of these in vivo correlations will have to be tested with an in vitro protein synthesizing system. Higher arsenate levels resulted in even lower ATP/ADP, GTP/GDP ratios and in a slower decay of polysomes, implying that, eventually, elongation (in addition to initiation) was being inhibited. PMID:374362

  3. Energizing eukaryotic cell-free protein synthesis with glucose metabolism.

    PubMed

    Anderson, Mark J; Stark, Jessica C; Hodgman, C Eric; Jewett, Michael C

    2015-07-01

    Eukaryotic cell-free protein synthesis (CFPS) is limited by the dependence on costly high-energy phosphate compounds and exogenous enzymes to power protein synthesis (e.g., creatine phosphate and creatine kinase, CrP/CrK). Here, we report the ability to use glucose as a secondary energy substrate to regenerate ATP in a Saccharomyces cerevisiae crude extract CFPS platform. We observed synthesis of 3.64±0.35 μg mL(-1) active luciferase in batch reactions with 16 mM glucose and 25 mM phosphate, resulting in a 16% increase in relative protein yield (μg protein/$ reagents) compared to the CrP/CrK system. Our demonstration provides the foundation for development of cost-effective eukaryotic CFPS platforms. PMID:26054976

  4. Leucine-Enriched Essential Amino Acids Augment Mixed Protein Synthesis, But Not Collagen Protein Synthesis, in Rat Skeletal Muscle after Downhill Running.

    PubMed

    Kato, Hiroyuki; Suzuki, Hiromi; Inoue, Yoshiko; Suzuki, Katsuya; Kobayashi, Hisamine

    2016-01-01

    Mixed and collagen protein synthesis is elevated for as many as 3 days following exercise. Immediately after exercise, enhanced amino acid availability increases synthesis of mixed muscle protein, but not muscle collagen protein. However, the potential for synergic effects of amino acid ingestion with exercise on both mixed and collagen protein synthesis remains unclear. We investigated muscle collagen protein synthesis in rats following post-exercise ingestion of leucine-enriched essential amino acids. We determined fractional protein synthesis rates (FSR) at different time points following exercise. Mixed protein and collagen protein FSRs in skeletal muscle were determined by measuring protein-bound enrichments of hydroxyproline and proline, and by measuring the intracellular enrichment of proline, using injections of flooding d₃-proline doses. A leucine-enriched mixture of essential amino acids (or distilled water as a control) was administrated 30 min or 1 day post-exercise. The collagen protein synthesis in the vastus lateralis was elevated for 2 days after exercise. Although amino acid administration did not increase muscle collagen protein synthesis, it did lead to augmented mixed muscle protein synthesis 1 day following exercise. Thus, contrary to the regulation of mixed muscle protein synthesis, muscle collagen protein synthesis is not affected by amino acid availability after damage-inducing exercise. PMID:27367725

  5. Leucine-Enriched Essential Amino Acids Augment Mixed Protein Synthesis, But Not Collagen Protein Synthesis, in Rat Skeletal Muscle after Downhill Running

    PubMed Central

    Kato, Hiroyuki; Suzuki, Hiromi; Inoue, Yoshiko; Suzuki, Katsuya; Kobayashi, Hisamine

    2016-01-01

    Mixed and collagen protein synthesis is elevated for as many as 3 days following exercise. Immediately after exercise, enhanced amino acid availability increases synthesis of mixed muscle protein, but not muscle collagen protein. However, the potential for synergic effects of amino acid ingestion with exercise on both mixed and collagen protein synthesis remains unclear. We investigated muscle collagen protein synthesis in rats following post-exercise ingestion of leucine-enriched essential amino acids. We determined fractional protein synthesis rates (FSR) at different time points following exercise. Mixed protein and collagen protein FSRs in skeletal muscle were determined by measuring protein-bound enrichments of hydroxyproline and proline, and by measuring the intracellular enrichment of proline, using injections of flooding d3-proline doses. A leucine-enriched mixture of essential amino acids (or distilled water as a control) was administrated 30 min or 1 day post-exercise. The collagen protein synthesis in the vastus lateralis was elevated for 2 days after exercise. Although amino acid administration did not increase muscle collagen protein synthesis, it did lead to augmented mixed muscle protein synthesis 1 day following exercise. Thus, contrary to the regulation of mixed muscle protein synthesis, muscle collagen protein synthesis is not affected by amino acid availability after damage-inducing exercise. PMID:27367725

  6. Glucose Synthesis in a Protein-Based Artificial Photosynthesis System.

    PubMed

    Lu, Hao; Yuan, Wenqiao; Zhou, Jack; Chong, Parkson Lee-Gau

    2015-09-01

    The objective of this study was to understand glucose synthesis of a protein-based artificial photosynthesis system affected by operating conditions, including the concentrations of reactants, reaction temperature, and illumination. Results from non-vesicle-based glyceraldehyde-3-phosphate (GAP) and glucose synthesis showed that the initial concentrations of ribulose-1,5-bisphosphate (RuBP) and adenosine triphosphate (ATP), lighting source, and temperature significantly affected glucose synthesis. Higher initial concentrations of RuBP and ATP significantly enhanced GAP synthesis, which was linearly correlated to glucose synthesis, confirming the proper functions of all catalyzing enzymes in the system. White fluorescent light inhibited artificial photosynthesis and reduced glucose synthesis by 79.2 % compared to in the dark. The reaction temperature of 40 °C was optimum, whereas lower or higher temperature reduced glucose synthesis. Glucose synthesis in the vesicle-based artificial photosynthesis system reconstituted with bacteriorhodopsin, F 0 F 1 ATP synthase, and polydimethylsiloxane-methyloxazoline-polydimethylsiloxane triblock copolymer was successfully demonstrated. This system efficiently utilized light-induced ATP to drive glucose synthesis, and 5.2 μg ml(-1) glucose was synthesized in 0.78-ml reaction buffer in 7 h. Light-dependent reactions were found to be the bottleneck of the studied artificial photosynthesis system. PMID:26170084

  7. Acetaldehyde inhibition of protein synthesis in isolated rat pancreatic acini

    SciTech Connect

    Majumdar, A.P.; Haiman, M.J.; Zylbert, B.A.; Billy, H.T.; Vesenka, G.D.; Geokas, M.C.

    1986-03-30

    Exposure of isolated dispersed pancreatic acini to increasing concentrations of ethanol (5 to 500 mM) or acetaldehyde (0.5 to 100 mM) produced a progressive inhibition of (3H)leucine incorporation into both cellular (those remaining in the cell) and secretory (those released into the medium) proteins. Whereas 500 mM ethanol caused 90-95% inhibition in the synthesis of cellular and secretory proteins, the concentration of acetaldehyde needed to produce a similar inhibition was found to be 50 mM. All subsequent experiments were performed with 12.5 mM acetaldehyde, a concentration that consistently inhibited acinar protein synthesis by about 50%. The acetaldehyde-mediated inhibition of acinar protein synthesis was partially normalized when this metabolite was removed after 30 min during a 90-min incubation period. In the presence of acetaldehyde, the secretion of 3H-pulse-labeled proteins, but not amylase, trypsinogen, or chymotrypsinogen, was greatly depressed. Acetaldehyde also caused a marked reduction in (3H)uridine incorporation into acinar RNA. The entry of (3H)uridine, (3H)leucine, and (3H)aminoisobutyric acid into isolated acini was found to be slightly (15-25%) decreased by acetaldehyde. It is concluded that acetaldehyde exerts a direct toxic effect on isolated dispersed pancreatic acini as evidenced by diminution of both protein and RNA synthesis and decreased secretion of the newly synthesized proteins. This inhibitory effect of acetaldehyde could be partially reversed.

  8. His6 tag-assisted chemical protein synthesis

    NASA Astrophysics Data System (ADS)

    Bang, Duhee; Kent, Stephen B. H.

    2005-04-01

    To make more practical the total chemical synthesis of proteins by the ligation of unprotected peptide building blocks, we have developed a method to facilitate the isolation and handling of intermediate products. The synthetic technique makes use of a His6 tag at the C terminus of the target polypeptide chain, introduced during the synthesis of the C-terminal peptide segment building block. The presence of a His6 tag enables the isolation of peptide or protein products directly from ligation reaction mixtures by Ni-NTA affinity column purification. This simple approach enables facile buffer exchange to alternate reaction conditions and is compatible with direct analytical control by protein MS of the multiple ligation steps involved in protein synthesis. We used syntheses of crambin and a modular tetratricopeptide repeat protein of 17 kDa as models to examine the utility of this affinity purification approach. The results show that His6 tag-assisted chemical protein synthesis is a useful method that substantially reduces handling losses and provides for rapid chemical protein syntheses. affinity purification | native chemical ligation

  9. Protein synthesis rates in atrophied gastrocnemius muscles after limb immobilization

    NASA Technical Reports Server (NTRS)

    Tucker, K. R.; Seider, M. J.; Booth, F. W.

    1981-01-01

    Noting that protein synthesis declines in the gastrocnemius 6 hr after immobilization, the study sought to detect an increase of protein synthesis when the limb was freed, and to examine the effects of exercise on the rate of increase. Rats were used as subjects, with their hind legs in plaster of Paris in plantar flexion to eliminate strain on the gastrocnemius. Periods of immobilization were varied and samples of blood from the muscle were taken to track protein synthesis rates for different groups in immobilization and exercise regimens (running and weightlifting). Synthesis rates declined 3.6% during time in the cast, then increased 6.3%/day after the casts were removed. Both running and weightlifting were found to increase the fractional rate of protein formation in the gastrocnemius muscle when compared with contralateral muscles that were not exercised and were used as controls, suggesting that the mechanism controlling protein synthesis in skeletal muscles is rapidly responsive to changes in muscular contractile activity.

  10. Regulation of protein synthesis during sea urchin early development

    SciTech Connect

    Kelso, L.C.

    1989-01-01

    Fertilization of the sea urchin egg results in a 20-40 fold increase in the rate of protein synthesis. The masked message hypothesis proposes that mRNAs are masked or unavailable for translation in the egg. We devised an in vivo assay to test this hypothesis. Our results show that masked mRNAs limit protein synthesis in the unfertilized egg. In addition, we show that protein synthesis is also regulated at the level of translational machinery. Following fertilization is a period of rapid cell divisions. This period, known as the rapid cleavage stage, is characterized by the transient synthesis of a novel set of proteins. The synthesis of these proteins is programmed by maternal mRNAs stored in the unfertilized egg. To study the behavior of these mRNAs, we prepared a cDNA library from polysomal poly (A+) RNA from 2-hour embryos. ({sup 32}P) labeled probes, prepared from the cDNA library, were used to monitor the levels of individual mRNAs in polysomes at fertilization and during early development.

  11. The Role of Protein Synthesis in the Senescence of Leaves

    PubMed Central

    Martin, Colin; Thimann, Kenneth V.

    1972-01-01

    The senescence of oat leaves has been studied by following the loss of chlorophyll and protein and the increase of α-amino nitrogen, after detachment and darkening. Protein synthesis and the amounts of proteolytic enzymes in the leaves have been determined directly. The process of senescence is shown to be a sequential one in which protein synthesis,most probably the formation of a proteolytic enzyme with l-serine in its active center, is of prime importance. The evidence is as follows. Firstly, l-serine specifically enhances senescence, especially in presence of kinetin. Secondly, cycloheximide, which inhibits protein synthesis in other systems, delays senescence and prevents the serine enhancement. Although requiring higher concentrations, cycloheximide can be as effective as kinetin in inhibiting senescence. It is shown directly that cycloheximide prevents protein synthesis in oat leaves under the same conditions as when it prevents senescence. Thirdly, leaves have been shown to contain two proteinases, with pH optima at 3 and 7.5, whose activity increases during senescence, even though the total leaf protein is decreasing. The amounts of both these enzymes present after 3 days are clearly increased by serine, and are greatly decreased by cycloheximide or by kinetin. The role of kinetin in delaying senescence thus may rest on its ability to suppress protease formation. PMID:16657898

  12. DNA Nanoparticles for Improved Protein Synthesis In Vitro

    PubMed Central

    Galinis, Robertas; Stonyte, Greta; Kiseliovas, Vaidotas; Zilionis, Rapolas; Studer, Sabine; Hilvert, Donald; Janulaitis, Arvydas

    2016-01-01

    Abstract The amplification and digital quantification of single DNA molecules are important in biomedicine and diagnostics. Beyond quantifying DNA molecules in a sample, the ability to express proteins from the amplified DNA would open even broader applications in synthetic biology, directed evolution, and proteomics. Herein, a microfluidic approach is reported for the production of condensed DNA nanoparticles that can serve as efficient templates for in vitro protein synthesis. Using phi29 DNA polymerase and a multiple displacement amplification reaction, single DNA molecules were converted into DNA nanoparticles containing up to about 104 clonal gene copies of the starting template. DNA nanoparticle formation was triggered by accumulation of inorganic pyrophosphate (produced during DNA synthesis) and magnesium ions from the buffer. Transcription–translation reactions performed in vitro showed that individual DNA nanoparticles can serve as efficient templates for protein synthesis in vitro. PMID:26821778

  13. Prolonged inhibition of bacterial protein synthesis abolishes Salmonella invasion.

    PubMed Central

    MacBeth, K J; Lee, C A

    1993-01-01

    We have found that prolonged inhibition of bacterial protein synthesis abolishes the ability of Salmonella typhimurium to enter HEp-2 cells. Our results suggest that an essential invasion factor has a functional half-life that is seen as a gradual loss of invasiveness in the absence of protein synthesis. Therefore, Salmonella invasiveness appears to be a transient phenotype that is lost unless protein synthesis is maintained. This finding may explain why salmonellae grown to stationary phase lose their ability to enter cultured cells. In addition, a short-lived capacity to enter cells may be important during infection so that bacterial invasiveness is limited to certain times and host sites during pathogenesis. PMID:8454361

  14. Bacterial Protein Synthesis as a Target for Antibiotic Inhibition.

    PubMed

    Arenz, Stefan; Wilson, Daniel N

    2016-01-01

    Protein synthesis occurs on macromolecular machines, called ribosomes. Bacterial ribosomes and the translational machinery represent one of the major targets for antibiotics in the cell. Therefore, structural and biochemical investigations into ribosome-targeting antibiotics provide not only insight into the mechanism of action and resistance of antibiotics, but also insight into the fundamental process of protein synthesis. This review summarizes the recent advances in our understanding of protein synthesis, particularly with respect to X-ray and cryoelectron microscopy (cryo-EM) structures of ribosome complexes, and highlights the different steps of translation that are targeted by the diverse array of known antibiotics. Such findings will be important for the ongoing development of novel and improved antimicrobial agents to combat the rapid emergence of multidrug resistant pathogenic bacteria. PMID:27481773

  15. Quantifying elongation rhythm during full-length protein synthesis.

    PubMed

    Rosenblum, Gabriel; Chen, Chunlai; Kaur, Jaskiran; Cui, Xiaonan; Zhang, Haibo; Asahara, Haruichi; Chong, Shaorong; Smilansky, Zeev; Goldman, Yale E; Cooperman, Barry S

    2013-07-31

    Pauses regulate the rhythm of ribosomal protein synthesis. Mutations disrupting even minor pauses can give rise to improperly formed proteins and human disease. Such minor pauses are difficult to characterize by ensemble methods, but can be readily examined by single-molecule (sm) approaches. Here we use smFRET to carry out real-time monitoring of the expression of a full-length protein, the green fluorescent protein variant Emerald GFP. We demonstrate significant correlations between measured elongation rates and codon and isoacceptor tRNA usage, and provide a quantitative estimate of the effect on elongation rate of replacing a codon recognizing an abundant tRNA with a synonymous codon cognate to a rarer tRNA. Our results suggest that tRNA selection plays an important general role in modulating the rates and rhythms of protein synthesis, potentially influencing simultaneous co-translational processes such as folding and chemical modification. PMID:23822614

  16. Selective memory generalization by spatial patterning of protein synthesis

    PubMed Central

    O’Donnell, Cian; Sejnowski, Terrence J.

    2014-01-01

    Summary Protein synthesis is crucial for both persistent synaptic plasticity and long-term memory. De novo protein expression can be restricted to specific neurons within a population, and to specific dendrites within a single neuron. Despite its ubiquity, the functional benefits of spatial protein regulation for learning are unknown. We used computational modeling to study this problem. We found that spatially patterned protein synthesis can enable selective consolidation of some memories but forgetting of others, even for simultaneous events that are represented by the same neural population. Key factors regulating selectivity include the functional clustering of synapses on dendrites, and the sparsity and overlap of neural activity patterns at the circuit level. Based on these findings we proposed a novel two-step model for selective memory generalization during REM and slow-wave sleep. The pattern-matching framework we propose may be broadly applicable to spatial protein signaling throughout cortex and hippocampus. PMID:24742462

  17. Cyclin B synthesis and rapamycin-sensitive regulation of protein synthesis during starfish oocyte meiotic divisions.

    PubMed

    Lapasset, Laure; Pradet-Balade, Bérengère; Vergé, Valérie; Lozano, Jean-Claude; Oulhen, Nathalie; Cormier, Patrick; Peaucellier, Gérard

    2008-11-01

    Translation of cyclin mRNAs represents an important event for proper meiotic maturation and post-fertilization mitoses in many species. Translational control of cyclin B mRNA has been described to be achieved through two separate but related mechanisms: translational repression and polyadenylation. In this paper, we evaluated the contribution of global translational regulation by the cap-dependent translation repressor 4E-BP (eukaryotic initiation factor 4E-binding protein) on the cyclin B protein synthesis during meiotic maturation of the starfish oocytes. We used the immunosupressant drug rapamycin, a strong inhibitor of cap-dependent translation, to check for the involvement of this protein synthesis during this physiological process. Rapamycin was found to prevent dissociation of 4E-BP from the initiation factor eIF4E and to suppress correlatively a burst of global protein synthesis occurring at the G2/M transition. The drug had no effect on first meiotic division but defects in meiotic spindle formation prevented second polar body emission, demonstrating that a rapamycin-sensitive pathway is involved in this mechanism. While rapamycin affected the global protein synthesis, the drug altered neither the specific translation of cyclin B mRNA nor the expression of the Mos protein. The expression of these two proteins was correlated with the phosphorylation and the dissociation of the cytoplasmic polyadenylation element-binding protein from eIF4E. PMID:18361417

  18. Effects of Whey, Caseinate, or Milk Protein Ingestion on Muscle Protein Synthesis after Exercise

    PubMed Central

    Kanda, Atsushi; Nakayama, Kyosuke; Sanbongi, Chiaki; Nagata, Masashi; Ikegami, Shuji; Itoh, Hiroyuki

    2016-01-01

    Whey protein (WP) is characterized as a “fast” protein and caseinate (CA) as a “slow” protein according to their digestion and absorption rates. We hypothesized that co-ingestion of milk proteins (WP and CA) may be effective for prolonging the muscle protein synthesis response compared to either protein alone. We therefore compared the effect of ingesting milk protein (MP) to either WP or CA alone on muscle protein synthesis after exercise in rats. We also compared the effects of these milk-derived proteins to a control, soy protein (SP). Male Sprague-Dawley rats swam for two hours. Immediately after exercise, one of the following four solutions was administered: WP, CA, MP, or SP. Individual rats were euthanized at designated postprandial time points and triceps muscle samples collected for measurement of the protein fractional synthesis rate (FSR). FSR tended to increase in all groups post-ingestion, although the initial peaks of FSR occurred at different times (WP, peak time = 60 min, FSR = 7.76%/day; MP, peak time = 90 min, FSR = 8.34%/day; CA, peak time = 120 min, FSR = 7.85%/day). Milk-derived proteins caused significantly greater increases (p < 0.05) in FSR compared with SP at different times (WP, 60 min; MP, 90 and 120 min; CA, 120 min). Although statistical analysis could not be performed, the calculated the area under the curve (AUC) values for FSR following this trend were: MP, 534.61; CA, 498.22; WP, 473.46; and SP, 406.18. We conclude that ingestion of MP, CA or WP causes the initial peak time in muscle protein synthesis to occur at different times (WP, fast; MP, intermediate; CA, slow) and the dairy proteins have a superior effect on muscle protein synthesis after exercise compared with SP. PMID:27271661

  19. Effects of Whey, Caseinate, or Milk Protein Ingestion on Muscle Protein Synthesis after Exercise.

    PubMed

    Kanda, Atsushi; Nakayama, Kyosuke; Sanbongi, Chiaki; Nagata, Masashi; Ikegami, Shuji; Itoh, Hiroyuki

    2016-01-01

    Whey protein (WP) is characterized as a "fast" protein and caseinate (CA) as a "slow" protein according to their digestion and absorption rates. We hypothesized that co-ingestion of milk proteins (WP and CA) may be effective for prolonging the muscle protein synthesis response compared to either protein alone. We therefore compared the effect of ingesting milk protein (MP) to either WP or CA alone on muscle protein synthesis after exercise in rats. We also compared the effects of these milk-derived proteins to a control, soy protein (SP). Male Sprague-Dawley rats swam for two hours. Immediately after exercise, one of the following four solutions was administered: WP, CA, MP, or SP. Individual rats were euthanized at designated postprandial time points and triceps muscle samples collected for measurement of the protein fractional synthesis rate (FSR). FSR tended to increase in all groups post-ingestion, although the initial peaks of FSR occurred at different times (WP, peak time = 60 min, FSR = 7.76%/day; MP, peak time = 90 min, FSR = 8.34%/day; CA, peak time = 120 min, FSR = 7.85%/day). Milk-derived proteins caused significantly greater increases (p < 0.05) in FSR compared with SP at different times (WP, 60 min; MP, 90 and 120 min; CA, 120 min). Although statistical analysis could not be performed, the calculated the area under the curve (AUC) values for FSR following this trend were: MP, 534.61; CA, 498.22; WP, 473.46; and SP, 406.18. We conclude that ingestion of MP, CA or WP causes the initial peak time in muscle protein synthesis to occur at different times (WP, fast; MP, intermediate; CA, slow) and the dairy proteins have a superior effect on muscle protein synthesis after exercise compared with SP. PMID:27271661

  20. Cell-free protein synthesis and assembly on a biochip

    NASA Astrophysics Data System (ADS)

    Heyman, Yael; Buxboim, Amnon; Wolf, Sharon G.; Daube, Shirley S.; Bar-Ziv, Roy H.

    2012-06-01

    Biologically active complexes such as ribosomes and bacteriophages are formed through the self-assembly of proteins and nucleic acids. Recapitulating these biological self-assembly processes in a cell-free environment offers a way to develop synthetic biodevices. To visualize and understand the assembly process, a platform is required that enables simultaneous synthesis, assembly and imaging at the nanoscale. Here, we show that a silicon dioxide grid, used to support samples in transmission electron microscopy, can be modified into a biochip to combine in situ protein synthesis, assembly and imaging. Light is used to pattern the biochip surface with genes that encode specific proteins, and antibody traps that bind and assemble the nascent proteins. Using transmission electron microscopy imaging we show that protein nanotubes synthesized on the biochip surface in the presence of antibody traps efficiently assembled on these traps, but pre-assembled nanotubes were not effectively captured. Moreover, synthesis of green fluorescent protein from its immobilized gene generated a gradient of captured proteins decreasing in concentration away from the gene source. This biochip could be used to create spatial patterns of proteins assembled on surfaces.

  1. Semi-synthesis of labeled proteins for spectroscopic applications.

    PubMed

    De Rosa, Lucia; Russomanno, Anna; Romanelli, Alessandra; D'Andrea, Luca Domenico

    2013-01-01

    Since the introduction of SPPS by Merrifield in the 60s, peptide chemists have considered the possibility of preparing large proteins. The introduction of native chemical ligation in the 90s and then of expressed protein ligation have opened the way to the preparation of synthetic proteins without size limitations. This review focuses on semi-synthetic strategies useful to prepare proteins decorated with spectroscopic probes, like fluorescent labels and stable isotopes, and their biophysical applications. We show that expressed protein ligation, combining the advantages of organic chemistry with the easy and size limitless recombinant protein expression, is an excellent strategy for the chemical synthesis of labeled proteins, enabling a single protein to be functionalized at one or even more distinct positions with different probes. PMID:23282535

  2. Thyroid hormone stimulation of plasma protein synthesis in cultured hepatocytes.

    PubMed

    Hertzberg, K M; Pindyck, J; Mosesson, M W; Grieninger, G

    1981-01-25

    The direct effect of thyroid hormones on hepatocellular plasma protein synthesis has been studied in primary monolayer cultures derived from chick embryo liver. The chemically defined medium used for plating and maintaining the cultures contained no other hormones, protein, or serum supplement. Addition of physiological concentrations (10 nM) of triiodothyronine or thyroxine produced 3-fold or greater increases in the rates of synthesis of fibrinogen and three other major secreted proteins. By comparison albumin, transferrin, and total protein synthesis were not substantially increased. The enhanced synthesis of selected plasma proteins could be detected 6 h after initial addition of triiodothyronine. Exposure of the cells to the hormone for only 30 min was nearly as effective as continuous exposure in eliciting the ultimate response. Triiodothyronine exerted its half-maximal effect at a concentration of 1 nM. Diminished potency was associated with less iodination of the hormone; a marked reduction was noted with di-iodinated thyronine and no stimulatory activity at all with either mono- or non-iodinated thyronine. PMID:7451459

  3. Encapsulation of liver microsomes into a thermosensitive hydrogel for characterization of drug metabolism and toxicity.

    PubMed

    Yang, Huiying; Zheng, Yuanting; Zhao, Bei; Shao, Tengfei; Shi, Qingling; Zhou, Ning; Cai, Weimin

    2013-12-01

    This study reported the encapsulation of liver microsomes into a thermosensitive hydrogel to characterize drug metabolism and predict drug effects. Pluronic(®)F-127 (F127) and acrylamide-bisacrylamide (Acr-Bis) were utilized as the two precursors. After chemical crosslinking catalyzed by ammonium persulfate (APS) and N,N,N',N'-tetramethylethylenediamine (TEMED), the resulting Pluronic F127-acrylamide-bisacrylamide (FAB) hydrogel could encapsulate microsomes at 4 °C and facilitate metabolic reactions at 37 °C. The gel morphology at different Acr-Bis concentrations was characterized using field emission scanning electron microscopy (FE-SEM). Higher concentrations of Acr-Bis could lead to higher degrees of cross-linking of the gel. A fluorescent staining assay was subsequently used to demonstrate successful encapsulation of microsomes into the gel as well as the free diffusion process of micromolecular substrates. The thermosensitivity of the FAB gel was studied using swelling ratio and protein release assay to verify its ability to encapsulate microsomes. The metabolic activity of microsomes encapsulated in gels was investigated by detecting the metabolites of FDA-approved substrates, including dextromethorphan, chlorzoxazone and testosterone. Compared with the traditional method of microsomal incubation, the FAB gel maintained 60%-70% of microsome activity. Lastly, the classic anticancer prodrug cyclophosphamide (CTX) was chosen as a model drug for the study of drug metabolism and the prediction of drug effects. When the microsomes encapsulated in the FAB gel were used in the cell culture system, CTX induced a higher level of apoptosis in MCF-7 cells compared with traditional microsomes. PMID:24075480

  4. Insulin accelerates global and mitochondrial protein synthesis rates in neonatal muscle during sepsis

    Technology Transfer Automated Retrieval System (TEKTRAN)

    In neonatal pigs, sepsis decreases protein synthesis in skeletal muscle by decreasing translation initiation. However, insulin stimulates muscle protein synthesis despite persistent repression of translation initiation signaling. To determine whether the insulin-induced increase in global rates of m...

  5. A Working Model of Protein Synthesis Using Lego(TM) Building Blocks.

    ERIC Educational Resources Information Center

    Templin, Mark A.; Fetters, Marcia K.

    2002-01-01

    Uses Lego building blocks to improve the effectiveness of teaching about protein synthesis. Provides diagrams and pictures for a 2-3 day student activity. Discusses mRNA, transfer RNA, and a protein synthesis model. (MVL)

  6. Effects of 1,2-dichloroethane intoxication on dolichol levels and glycosyltransferase activities in rat liver microsomes and Golgi apparatus.

    PubMed

    Cottalasso, D; Fontana, L; Gazzo, P; Dapino, D; Domenicotti, C; Pronzato, M A; Nanni, G

    1995-12-15

    Rat intoxication with a single dose of 1,2-dichloroethane (DCE) (50 microliters/100 g b.w) is able to induce a significant modification of protein glycosylation in the liver endoplasmic reticulum and Golgi apparatus. HPLC analysis shows that within 5-60 min after DCE-intoxication, the levels of total dolichol, free dolichol and dolichyl phosphate strongly decreased in the microsomes and Golgi apparatus. Particularly in total microsomes, dolichyl phosphate, which is rate-limiting for the biosynthesis of the N-linked oligosaccharide chains, drops to values significantly lower than in the control group 15 min after DCE poisoning. In the Golgi apparatus, the total dolichol, essential to enhance the fluidity and permeability of these membranes, early and significantly decreases already 5 min after DCE poisoning. Moreover, in the Golgi apparatus galactosyl- and sialyltransferase activities, the main enzymatic activities of terminal protein glycosylation, are significantly reduced, as measured 15 min after DCE intoxication. These data suggest that the impairment of glycoprotein synthesis, maturation and secretion may be involved in the pathogenesis of liver injury induced by acute DCE-intoxication. PMID:8560503

  7. Tinkering with Translation: Protein Synthesis in Virus-Infected Cells

    PubMed Central

    Walsh, Derek; Mathews, Michael B.; Mohr, Ian

    2013-01-01

    Viruses are obligate intracellular parasites, and their replication requires host cell functions. Although the size, composition, complexity, and functions encoded by their genomes are remarkably diverse, all viruses rely absolutely on the protein synthesis machinery of their host cells. Lacking their own translational apparatus, they must recruit cellular ribosomes in order to translate viral mRNAs and produce the protein products required for their replication. In addition, there are other constraints on viral protein production. Crucially, host innate defenses and stress responses capable of inactivating the translation machinery must be effectively neutralized. Furthermore, the limited coding capacity of the viral genome needs to be used optimally. These demands have resulted in complex interactions between virus and host that exploit ostensibly virus-specific mechanisms and, at the same time, illuminate the functioning of the cellular protein synthesis apparatus. PMID:23209131

  8. Evaluation of the global protein synthesis in Mytilus galloprovincialis in marine pollution monitoring: seasonal variability and correlations with other biomarkers.

    PubMed

    Pytharopoulou, Sofia; Kouvela, Ekaterini C; Sazakli, Eleni; Leotsinidis, Michel; Kalpaxis, Dimitrios L

    2006-10-25

    Protein synthesis down-regulation is a life-saving mechanism for many organisms exposed to xenobiotics that threaten normal life. The present study was designed to assess the spatial and seasonal variability of global protein synthesis, determined in the microsomal fraction of digestive glands from caged Mytilus galloprovincialis mussels exposed for 30 days in a relatively clean region and two unevenly polluted areas (Stations 1 and 2) along the Gulf of Patras (Greece). The in vivo activity of translating ribosomes was evaluated by analyzing the translating ribosomes, polysome content, which may serve as an indicator of the efficiency of the protein-synthesizing machinery. To correlate with classical biomonitoring strategies, various biomarkers were measured in digestive glands, including metallothionein content, heavy-metal content, and lysosomal membrane stability. In parallel, gill cells were examined for micronucleus frequency. Metal ion concentrations were also estimated in the surrounding waters as a measure of metal exposure. Substantially lower polysome content was recorded in caged mussels collected from Station 1, in particular during the winter and spring sampling. As verified by chemical analysis of the seawater and measurement of other biomarkers, Station 1 was more contaminated than Station 2. Polysome content was found negatively correlated with metallothionein levels, micronucleus frequency and cytosolic Cu and Hg in all seasons. In addition, negative correlations were obtained between polysome content and lysosomal membrane stability in winter and spring. A progressive increase in polysomes was observed from winter to autumn, in particular in samples from Station 1. A non-uniform trend was detected in 80S ribosomal monosomes, whereas the seasonal changes in ribosomal subunits were opposite to those found in polysome content. Comparisons between seasonal and local site-specific influences on polysome content provides evidence that winter and spring

  9. The Development of an Interactive Videodisc Program on Protein Synthesis.

    ERIC Educational Resources Information Center

    Hazan, Charlene Corey

    An interactive videodisk (IVD) program was developed to reinforce learning of the biological concept of protein synthesis for high school students. The laser videodisc "The Living Textbook Life Science" was the source of frames, and the authoring system of G. Smith was used to create the disc. The interactive program was designed to make the…

  10. Protein Synthesis Inhibition Blocks Consolidation of an Acrobatic Motor Skill

    ERIC Educational Resources Information Center

    Kaelin-Lang, Alain; Dichgans, Johannes; Schulz, Jorg B.; Luft, Andreas R.; Buitrago, Manuel M.

    2004-01-01

    To investigate whether motor skill learning depends on de novo protein synthesis, adult rats were trained in an acrobatic locomotor task (accelerating rotarod) for 7 d. Animals were systemically injected with cycloheximide (CHX, 0.5 mg/kg, i.p.) 1 h before sessions 1 and 2 or sessions 2 and 3. Control rats received vehicle injections before…

  11. Leucine acts as a nutrient signal to stimulate protein synthesis

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The postprandial rise in amino acids and insulin independently stimulates protein synthesis in skeletal muscle of piglets. Leucine is an important mediator of the response to amino acids. We have shown that the postprandial rise in leucine, but not isoleucine or valine, acutely stimulates muscle pro...

  12. The Teaching of Protein Synthesis--A Microcomputer Based Method.

    ERIC Educational Resources Information Center

    Goodridge, Frank

    1983-01-01

    Describes two computer programs (BASIC for 32K Commodore PET) for teaching protein synthesis. The first is an interactive test of base-pairing knowledge, and the second generates random DNA nucleotide sequences, with instructions for substitution, insertion, and deletion printed out for each student. (JN)

  13. Problem-Solving Test: The Mechanism of Protein Synthesis

    ERIC Educational Resources Information Center

    Szeberenyi, Jozsef

    2009-01-01

    Terms to be familiar with before you start to solve the test: protein synthesis, ribosomes, amino acids, peptides, peptide bond, polypeptide chain, N- and C-terminus, hemoglobin, [alpha]- and [beta]-globin chains, radioactive labeling, [[to the third power]H] and [[to the fourteenth power]C]leucine, cytosol, differential centrifugation, density…

  14. AMINO ACIDS AUGMENT MUSCLE PROTEIN SYNTHESIS IN NEONATAL PIGS DURING ENDOTOXEMIA BY MODULATING TRANSLATION INITIATION

    Technology Transfer Automated Retrieval System (TEKTRAN)

    In adults, sepsis reduces protein synthesis in skeletal muscle by restraining translation. The effect of sepsis on amino acid-stimulated muscle protein synthesis has not been determined in neonates, a population who is highly anabolic and whose muscle protein synthesis rates are uniquely sensitive ...

  15. Enhanced skeletal muscle protein synthesis rates in pigs treated with somatotropin requires fed amino acids levels

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Chronic somatotropin (pST) treatment in pigs increases skeletal muscle protein synthesis and circulating insulin, a known promoter of protein synthesis. Previously, we showed that the pST-mediated rise in insulin alone could not account for the pST-induced increase in protein synthesis. This study...

  16. Dietary protein distribution positively influences 24-h muscle protein synthesis in healthy adults.

    PubMed

    Mamerow, Madonna M; Mettler, Joni A; English, Kirk L; Casperson, Shanon L; Arentson-Lantz, Emily; Sheffield-Moore, Melinda; Layman, Donald K; Paddon-Jones, Douglas

    2014-06-01

    The RDA for protein describes the quantity that should be consumed daily to meet population needs and to prevent deficiency. Protein consumption in many countries exceeds the RDA; however, intake is often skewed toward the evening meal, whereas breakfast is typically carbohydrate rich and low in protein. We examined the effects of protein distribution on 24-h skeletal muscle protein synthesis in healthy adult men and women (n = 8; age: 36.9 ± 3.1 y; BMI: 25.7 ± 0.8 kg/m2). By using a 7-d crossover feeding design with a 30-d washout period, we measured changes in muscle protein synthesis in response to isoenergetic and isonitrogenous diets with protein at breakfast, lunch, and dinner distributed evenly (EVEN; 31.5 ± 1.3, 29.9 ± 1.6, and 32.7 ± 1.6 g protein, respectively) or skewed (SKEW; 10.7 ± 0.8, 16.0 ± 0.5, and 63.4 ± 3.7 g protein, respectively). Over 24-h periods on days 1 and 7, venous blood samples and vastus lateralis muscle biopsy samples were obtained during primed (2.0 μmol/kg) constant infusion [0.06 μmol/(kg⋅min)] of l-[ring-(13)C6]phenylalanine. The 24-h mixed muscle protein fractional synthesis rate was 25% higher in the EVEN (0.075 ± 0.006%/h) vs. the SKEW (0.056 ± 0.006%/h) protein distribution groups (P = 0.003). This pattern was maintained after 7 d of habituation to each diet (EVEN vs. SKEW: 0.077 ± 0.006 vs. 0.056 ± 0.006%/h; P = 0.001). The consumption of a moderate amount of protein at each meal stimulated 24-h muscle protein synthesis more effectively than skewing protein intake toward the evening meal. PMID:24477298

  17. An oxygen-regulated switch in the protein synthesis machinery

    PubMed Central

    Uniacke, James; Holterman, Chet E.; Lachance, Gabriel; Franovic, Aleksandra; Jacob, Mathieu D.; Fabian, Marc R.; Payette, Josianne; Holcik, Martin; Pause, Arnim; Lee, Stephen

    2016-01-01

    SUMMARY Protein synthesis involves the translation of ribonucleic acid information into proteins, the building blocks of life. The initial step of protein synthesis consists of the eukaryotic translation initiation factor 4E (eIF4E) binding to the 7-methylguanosine (m7-GpppG) 5′cap of mRNAs1,2. Low oxygen tension (hypoxia) represses cap-mediated translation by sequestering eIF4E through mammalian target of rapamycin (mTOR)-dependent mechanisms3–6. While the internal ribosome entry site is an alternative translation initiation mechanism, this pathway alone cannot account for the translational capacity of hypoxic cells7,8. This raises a fundamental question in biology as to how proteins are synthesized in periods of oxygen scarcity and eIF4E inhibition9. Here, we uncover an oxygen-regulated translation initiation complex that mediates selective cap-dependent protein synthesis. Hypoxia stimulates the formation of a complex that includes the oxygen-regulated hypoxia-inducible factor 2α (HIF-2α), the RNA binding protein RBM4 and the cap-binding eIF4E2, an eIF4E homologue. PAR-CLIP10 analysis identified an RNA hypoxia response element (rHRE) that recruits this complex to a wide array mRNAs, including the epidermal growth factor receptor (EGFR). Once assembled at the rHRE, HIF-2α/RBM4/eIF4E2 captures the 5′cap and targets mRNAs to polysomes for active translation thereby evading hypoxia-induced repression of protein synthesis. These findings demonstrate that cells have evolved a program whereby oxygen tension switches the basic translation initiation machinery. PMID:22678294

  18. Characterization of a novel ACTH inducible cytochrome P-450 from rat adrenal microsomes

    SciTech Connect

    Otto, S.A.; Marcus, C.M.; Jefcoate, C.R. )

    1990-02-26

    In rat adrenal cortex 7,12 dimethylbenz(a)anthracene (DMBA) causes massive necrosis that is dependent of ACTH. This is related to an ACTH inducible adrenal microsomal cytochrome P-450 that catalyzes hydrocarbon metabolism. Rat adrenal microsomes, catalyze the formation of DMBA 3,4 diol a precursor of the bay region reactive electrophile DMBA 3,4 diol 1,2 oxide. Both DMBA metabolism and a 57Kd protein have disappeared from microsomes 30 days after hypophysectomy, but are restored by 14 days treatment with ACTH. Dexamethasone which fully suppresses ACTH only partially suppresses this activity. The 57 Kd protein was partially purified to a single major band in one step from solubilized microsomes by h.p.l.c. chromatography using detergent elution from a novel column that mimics phospholipid membranes. This preparation exhibits a specific content of 2 nm P-450/mg protein and a turnover number of 1,500pm DMBA/nm P-450/minutes. A polyclonal antisera raised against this preparation provides a single western blot corresponding to the 57Kd ACTH sensitive protein. This antibody did not blot microsomal P-450 c21, nor did selected antibodies from known families react with this adrenal P-450 protein, suggesting substantial sequence differences from known P-450's.

  19. Transcriptional regulation of decreased protein synthesis during skeletal muscle unloading

    NASA Technical Reports Server (NTRS)

    Howard, G.; Steffen, J. M.; Geoghegan, T. E.

    1989-01-01

    The regulatory role of transcriptional alterations in unloaded skeletal muscles was investigated by determining levels of total muscle RNA and mRNA fractions in soleus, gastrocnemius, and extensor digitorum longus (EDL) of rats subjected to whole-body suspension for up to 7 days. After 7 days, total RNA and mRNA contents were lower in soleus and gastrocnemius, compared with controls, but the concentrations of both RNAs per g muscle were unaltered. Alpha-actin mRNA (assessed by dot hybridization) was significantly reduced in soleus after 1, 3, and 7 days of suspension and in gastrocnemius after 3 and 7 days, but was unchanged in EDL. Protein synthesis directed by RNA extracted from soleus and EDL indicated marked alteration in mRNAs coding for several small proteins. Results suggest that altered transcription and availability of specific mRNAs contribute significantly to the regulation of protein synthesis during skeletal muscle unloading.

  20. Microsomal metabolism of NDMA and analogs

    SciTech Connect

    Wade, D.; Yang, C.S.

    1987-05-01

    The metabolism of N-nitrosodimethylamine (NDMA), dimethylamine (DMA), N-nitro-DMA (N x NO/sub 2/ x DMA), N-nitrosodiethylamine (NDEA), and diethylamine (DEA) was studied using control, acetone (Ac)-, butylated hydroxytoluene (BHT)-, pregnenolone 16- ..cap alpha..-carbonitrile (PCN)-, and phenobarbital (PB)-induced rat liver microsomes. At low substrate concentrations, the NDMA demethylase activity of Ac-induced microsomes was 5-fold greater than that of control, BHT-, and PCN-induced microsomes. The rate of NDMA denitrosation was ca. 10% that of demethylation. N x NO/sub 2/ x DMA was metabolized to HCHO, but not to NO/sub 2//sup -/, and the rate of metabolism was greatest with Ac-induced microsomes; the K/sub m/ and V/sub max/ of Ac-induced microsomes were similar to those of NDMA. For the dealkylation of NDEA, Ac- and BHT-induced microsomes were twice as active as the control. Ratios of dealkylation/denitrosation for NDEA remained constant over a broad range of low substrate concentrations. BHT- or Ac-treatment appeared to cause a selective increase in the ability of microsomes to denitrosate NDEA. The activity of all microsome preparations with the amines, DMA and DEA was less than that with the nitrosamine or nitramine substrates. The results suggest that both the N-nitroso and N-nitro compounds are good substrates for microsomal P-450; the amines, which bear positive charges, are not. Denitrosation appeared to be a more important pathway with NDEA than with NDMA.

  1. The biosynthesis of crustacean chitin. Isolation and characterization of polyprenol-linked intermediates from brine shrimp microsomes.

    PubMed

    Horst, M N

    1983-05-01

    The biosynthesis of crustacean chitin appears to involve the participation of a lipid-linked intermediate. A microsomal preparation from larval stages of the brine shrimp Artemia salina was found to catalyze the glycosylation of exogenous [3H]dolichol phosphate, yielding a product which was insoluble in chloroform:methanol (2:1) but soluble in chloroform:methanol:water (10:10:3). Artemia microsomes catalyze the transfer of N-acetylglucosamine from UDP-N-acetylglucosamine to a lipid acceptor. After extraction of labeled lipids with either chloroform:methanol (2:1) or chloroform:methanol:water (10:10:3), labeled compounds could be purified by ion-exchange chromatography on DEAE-Sephacel. Mild acid hydrolysis of 3H-N-acetylglucosamine labeled material soluble in chloroform:methanol:water (10:10:3) yielded a series of oligosaccharides ranging from 2 to about 8 glycosyl units in size. The larger components were shown to be sensitive to chitinase digestion but resistant to treatment with alpha-mannosidase. Such 3H-N-acetylglucosamine containing compounds, prepared by both in vivo and in vitro procedures, appear to be chitin oligosaccharides. Brine shrimp microsomes also catalyze the transfer of mannose from GDP-mannose to a lipid acceptor. Mild acid hydrolysis of mannosyl lipids soluble in chloroform:methanol:water (10:10:3) yielded oligosaccharides which were sensitive to alpha-mannosidase digestion and resistant to treatment with endochitinase. The results suggest 3H-N-acetylglucosamine-labeled oligosaccharide-lipids are distinct from the mannose-labeled fraction and may participate in the formation of an endogenous primer for chitin synthesis after their transfer to a protein acceptor. PMID:6859859

  2. Ribosomal History Reveals Origins of Modern Protein Synthesis

    PubMed Central

    Harish, Ajith; Caetano-Anollés, Gustavo

    2012-01-01

    The origin and evolution of the ribosome is central to our understanding of the cellular world. Most hypotheses posit that the ribosome originated in the peptidyl transferase center of the large ribosomal subunit. However, these proposals do not link protein synthesis to RNA recognition and do not use a phylogenetic comparative framework to study ribosomal evolution. Here we infer evolution of the structural components of the ribosome. Phylogenetic methods widely used in morphometrics are applied directly to RNA structures of thousands of molecules and to a census of protein structures in hundreds of genomes. We find that components of the small subunit involved in ribosomal processivity evolved earlier than the catalytic peptidyl transferase center responsible for protein synthesis. Remarkably, subunit RNA and proteins coevolved, starting with interactions between the oldest proteins (S12 and S17) and the oldest substructure (the ribosomal ratchet) in the small subunit and ending with the rise of a modern multi-subunit ribosome. Ancestral ribonucleoprotein components show similarities to in vitro evolved RNA replicase ribozymes and protein structures in extant replication machinery. Our study therefore provides important clues about the chicken-or-egg dilemma associated with the central dogma of molecular biology by showing that ribosomal history is driven by the gradual structural accretion of protein and RNA structures. Most importantly, results suggest that functionally important and conserved regions of the ribosome were recruited and could be relics of an ancient ribonucleoprotein world. PMID:22427882

  3. Accelerated chemical synthesis of peptides and small proteins

    PubMed Central

    Miranda, Les P.; Alewood, Paul F.

    1999-01-01

    The chemical synthesis of peptides and small proteins is a powerful complementary strategy to recombinant protein overexpression and is widely used in structural biology, immunology, protein engineering, and biomedical research. Despite considerable improvements in the fidelity of peptide chain assembly, side-chain protection, and postsynthesis analysis, a limiting factor in accessing polypeptides containing greater than 50 residues remains the time taken for chain assembly. The ultimate goal of this work is to establish highly efficient chemical procedures that achieve chain-assembly rates of approximately 10–15 residues per hour, thus underpinning the rapid chemical synthesis of long polypeptides and proteins, including cytokines, growth factors, protein domains, and small enzymes. Here we report Boc chemistry that employs O-(7-azabenzotriazol-1-yl)-N,N,N′,N′-tetramethyluronium hexafluorophosphate (HATU)/dimethyl sulfoxide in situ neutralization as the coupling agent and incorporates a protected amino acid residue every 5 min to produce peptides of good quality. This rapid coupling chemistry was successfully demonstrated by synthesizing several small to medium peptides, including the “difficult” C-terminal sequence of HIV-1 proteinase (residues 81–99); fragment 65–74 of the acyl carrier protein; conotoxin PnIA(A10L), a potent neuronal nicotinic receptor antagonist; and the pro-inflammatory chemotactic protein CP10, an 88-residue protein, by means of native chemical ligation. The benefits of this approach include enhanced ability to identify and characterize “difficult couplings,” rapid access to peptides for biological and structure–activity studies, and accelerated synthesis of tailored large peptide segments (<50 residues) for use in chemoselective ligation methods. PMID:9989998

  4. Directed Evolution of Proteins through In Vitro Protein Synthesis in Liposomes

    PubMed Central

    Nishikawa, Takehiro; Sunami, Takeshi; Matsuura, Tomoaki; Yomo, Tetsuya

    2012-01-01

    Directed evolution of proteins is a technique used to modify protein functions through “Darwinian selection.” In vitro compartmentalization (IVC) is an in vitro gene screening system for directed evolution of proteins. IVC establishes the link between genetic information (genotype) and the protein translated from the information (phenotype), which is essential for all directed evolution methods, by encapsulating both in a nonliving microcompartment. Herein, we introduce a new liposome-based IVC system consisting of a liposome, the protein synthesis using recombinant elements (PURE) system and a fluorescence-activated cell sorter (FACS) used as a microcompartment, in vitro protein synthesis system, and high-throughput screen, respectively. Liposome-based IVC is characterized by in vitro protein synthesis from a single copy of a gene in a cell-sized unilamellar liposome and quantitative functional evaluation of the synthesized proteins. Examples of liposome-based IVC for screening proteins such as GFP and β-glucuronidase are described. We discuss the future directions for this method and its applications. PMID:22957209

  5. Determination of microsomal lauric acid hydroxylase activity by HPLC with flow-through radiochemical quantitation

    SciTech Connect

    Romano, M.C.; Straub, K.M.; Yodis, L.A.P.; Eckardt, R.D.; Newton, J.F.

    1988-04-01

    An assay for the microsomal hydroxylation of lauric acid (LA), based on HPLC with flow-through radiochemical detection, has been developed. Conditions were optimized for resolution and quantitation of three microsomal metabolites of /sup 14/C-LA, one of which has not been reported previously as a metabolite of LA in mammalian microsomal incubations. These products, 12-(omega)-hydroxy-LA, 11-(omega-1)-hydroxy-LA, and a novel metabolite, 10-(omega-2)-hydroxy-LA, were isolated by HPLC and identified by gas chromatography/mass spectrometry. In the presence of NADPH, the formation of all three metabolites was linear with time and microsomal protein concentration. Hydrogen peroxide also supported the microsomal metabolism of LA, although the ratio of metabolites was substantially different than that produced by NADPH-supported microsomes. Several biochemical probes (metyrapone, ..cap alpha..-naphthoflavone, 2-diethylaminoethyl-2,2-diphenylvalerate hydrochloride, and 10-undecynoic acid) were used to dissociate the three LA hydroxylase activities. These experiments suggest that the site-specific hydroxylation (omega-, (omega-1), (omega-2)-) of LA may be catalyzed by different isozymes of cytochrome P-450.

  6. Reduced protein synthesis in schizophrenia patient-derived olfactory cells

    PubMed Central

    English, J A; Fan, Y; Föcking, M; Lopez, L M; Hryniewiecka, M; Wynne, K; Dicker, P; Matigian, N; Cagney, G; Mackay-Sim, A; Cotter, D R

    2015-01-01

    Human olfactory neurosphere-derived (ONS) cells have the potential to provide novel insights into the cellular pathology of schizophrenia. We used discovery-based proteomics and targeted functional analyses to reveal reductions in 17 ribosomal proteins, with an 18% decrease in the total ribosomal signal intensity in schizophrenia-patient-derived ONS cells. We quantified the rates of global protein synthesis in vitro and found a significant reduction in the rate of protein synthesis in schizophrenia patient-derived ONS cells compared with control-derived cells. Protein synthesis rates in fibroblast cell lines from the same patients did not differ, suggesting cell type-specific effects. Pathway analysis of dysregulated proteomic and transcriptomic data sets from these ONS cells converged to highlight perturbation of the eIF2α, eIF4 and mammalian target of rapamycin (mTOR) translational control pathways, and these pathways were also implicated in an independent induced pluripotent stem cell-derived neural stem model, and cohort, of schizophrenia patients. Analysis in schizophrenia genome-wide association data from the Psychiatric Genetics Consortium specifically implicated eIF2α regulatory kinase EIF2AK2, and confirmed the importance of the eIF2α, eIF4 and mTOR translational control pathways at the level of the genome. Thus, we integrated data from proteomic, transcriptomic, and functional assays from schizophrenia patient-derived ONS cells with genomics data to implicate dysregulated protein synthesis for the first time in schizophrenia. PMID:26485547

  7. Synthesis of Hydrogen-Bond Surrogate α-helices as Inhibitors of Protein-Protein Interactions

    PubMed Central

    Miller, Stephen E.; Thomson, Paul F.; Arora, Paramjit S.

    2014-01-01

    The α-helix is a prevalent secondary structure in proteins and critical in mediating protein-protein interactions (PPIs). Peptide mimetics that adopt stable helices have become powerful tools for the modulation of PPIs in vitro and in vivo. Hydrogen-bond surrogate (HBS) α-helices utilize a covalent bond in place of an N-terminal i to i+4 hydrogen bond and have been used to target and disrupt PPIs that become dysregulated in disease states. These compounds have improved conformational stability and cellular uptake as compared to their linear peptide counterparts. The protocol presented here describes current methodology for the synthesis of HBS α-helical mimetics. The solid phase synthesis of HBS helices involves solid phase peptide synthesis with three key steps involving incorporation of N-allyl functionality within the backbone of the peptide, coupling of a secondary amine, and a ring-closing metathesis step. PMID:24903885

  8. Modulation by estrogen of synthesis of specific uterine proteins.

    PubMed

    Skipper, J K; Eakle, S D; Hamilton, T H

    1980-11-01

    The contemporary procedure for high resolution two dimensional gel electrophoresis was extended to include an initial nondenaturing dimension of electrophoresis. Use of the resulting three dimensional procedure revealed that the previously described single peak of estrogen-induced protein in the uterus of the rat contains at least three distinct proteins whose rates of synthesis are regulated by estrogen. These proteins were localized within partial protein maps, thereby providing definitive operational definitions for the detection and identification of each. It was unambiguously demonstrated that each of the three proteins is continuously synthesized in control uteri. These findings cast doubt on the simplistic hypothesis that estrogen induces a single key protein that triggers a "cascade" of sequential transcriptional events in the uterus. Our finding that the major uterine protein induced by estrogen is also synthesized in liver and muscle cells is significant in that it points to a more general cellular function for the protein, rather than a unique role within uterine cells. Finally, our procedure for three dimensional gel electrophoresis opens new avenues for the detection of minor proteins in heterogeneous protein mixtures, such as those from the tissues of higher animals. PMID:7428041

  9. Antibiotics in development targeting protein synthesis.

    PubMed

    Sutcliffe, Joyce A

    2011-12-01

    The resolution of antibiotic-ribosomal subunit complexes and antibacterial-protein complexes at the atomic level has provided new insights into modifications of clinically relevant antimicrobials and provided new classes that target the protein cellular apparatus. New chemistry platforms that use fragment-based drug design or allow novel modifications in known structural classes are being used to design new antibiotics that overcome known resistance mechanisms and extend spectrum and potency by circumventing ubiquitous efflux pumps. This review provides details on seven antibiotics in development for treatment of moderate-to-severe community-acquired bacterial pneumonia and/or acute bacterial skin and skin structure infections: solithromycin, cethromycin, omadacycline, CEM-102, GSK1322322, radezolid, and tedizolid. Two antibiotics of the oxazolidinone class, PF-02341272 and AZD5847, are being developed as antituberculosis agents. Only three antibiotics that target the protein cellular machinery, TP-434, GSK2251052, and plazomicin, have a spectrum that encompasses multidrug-resistant Gram-negative pathogens. These compounds provide hope for treating key pathogens that cause serious disease in both the community and the hospital. PMID:22191530

  10. Polyaromatic compounds alter placental protein synthesis in pregnant rats

    SciTech Connect

    Shiverick, K.T.; Ogilvie, S.; Medrano, T. )

    1991-03-15

    The administration of the polyaromatic compounds {beta}-naphthoflavone ({beta}NF) and 3-methylcholanthrene (3MC) to pregnant rats during mid-gestation has been shown to produce marked feto-placental growth retardation. This study examined secretory protein synthesis in placental tissue from rats following administration of {beta}NF on gestation days (gd) 11-14 or 3MC on gd 12-14. Explants of placental basal zone tissue were cultured for 24 hours in serum-free medium in the presence of ({sup 3}H)leucine. Secreted proteins were analyzed by two-dimensional SDS-polyacrylamide gel electrophoresis followed by either fluorography or immunostaining. Total incorporation of ({sup 3}H)leucine into secreted proteins was not altered in BZ explants from {beta}NF or 3MC-treated animals. However a selective decrease was observed in ({sup 3}H)leucine incorporation into a major complex of proteins with apparent molecular weight of 25-30,000 and isoelectric point between 5.3 to 5.7. This group of proteins has been further identified as being related to rat pituitary growth hormone (GH) using N-terminal amino acid microsequencing of individual spots from 2-D SDS-PA gels. This is the first report that synthesis of GH-related proteins by rat placenta is decreased following {beta}NF and 3MC administration, a change which may underlie the feto-placental growth retardation associated with these polyaromatic compounds.

  11. Impaired rate of microsomal fatty acid elongation in undernourished neonatal rat brain

    SciTech Connect

    Yeh, Y.Y.

    1986-05-01

    Hypomyelination caused by undernourishment in characterized by low concentrations of myelin lipids and marked reduction in lignocerate (C/sub 24:0/) and nervonate (C/sub 24:1/) moiety of cerebroside and sulfatide. Since microsomal elongation is the major source of long chain (22 to 24 carbons) fatty acids in the brain, the effect of neonatal undernourishment on acyl elongation was investigated. Undernourishment of suckling rats were induced after birth by restricting maternal dietary intake to 40% of that consumed by dams fed ad libitum. Neonates suckled by the normally fed dams served as controls. Microsomal elongation was measured as nmol from (2-/sup 14/C) malonyl CoA incorporated/h per mg of protein. At 19 days of age, rates of behenoyl CoA (C/sub 22:0/) and erucoyl CoA (C/sub 22:1/) elongation in whole brain of undernourished neonates were 30-40% lower than that of the control, whereas the elongation rates of acyl CoA 16, 18 and 20 carbons in length either saturated or monounsaturated were similar in both groups. Undernourishment had no effect on cytoplasmic de novo fatty acid synthesis from acetyl CoA. If there are multiple elongation factors, the results indicate that the depressed activity of elongating enzyme(s) for C/sub 22:0/ and C/sub 22:1/ is an important contributing factor in lowering S/sub 24:0/ and C/sub 24:1/ content in cerebroside and sulfatide. This impairment may be a specific lesion leading to hypomyelination in undernourished rats.

  12. Question 7: Optimized Energy Consumption for Protein Synthesis

    NASA Astrophysics Data System (ADS)

    Szaflarski, Witold; Nierhaus, Knud H.

    2007-10-01

    In our previous contribution (Nierhaus, Orig Life Evol Biosph, this volume, 2007) we mentioned that life had solved the problem of energy supply in three major steps, and that these steps also mark major stages during the development of life. We further outlined a possible scenario concerning a minimal translational apparatus focusing on the essential components necessary for protein synthesis. Here we continue that consideration by addressing on one of the main problems of early life, namely avoiding wasteful energy loss. With regard to the limiting energy supply of early living systems, i.e. those of say more than 3,000 Ma, a carefully controlled and product oriented energy consumption was in demand. In recent years we learned how a bacterial cell avoids energy drain, thus being able to pump most of the energy into protein synthesis. These lessons must be followed by the design of a minimal living system, which is surveyed in this short article.

  13. Global protein synthesis in human trophoblast is resistant to inhibition by hypoxia

    PubMed Central

    Williams, S.F.; Fik, E.; Zamudio, S.; Illsley, N.P.

    2012-01-01

    Placental growth and function depend on syncytial cell processes which require the continuing synthesis of cellular proteins. The substantial energy demands of protein synthesis are met primarily from oxidative metabolism. Although the responses of individual proteins produced by the syncytiotrophoblast to oxygen deprivation have been investigated previously, there is no information available on global protein synthesis in syncytiotrophoblast under conditions of hypoxia. These studies were designed to test the hypothesis that syncytial protein synthesis is decreased in a dose-dependent manner by hypoxia. Experiments were performed to measure amino acid incorporation into proteins in primary syncytiotrophoblast cells exposed to oxygen concentrations ranging from 0 to 10%. Compared to cells exposed to normoxia (10% O2), no changes were observed following exposure to 5% or 3% O2, but after exposure to 1% O2, protein synthesis after 24 and 48 h decreased by 24% and 23% and with exposure to 0% O2, by 65% and 50%. As a consequence of these results, we hypothesized that global protein synthesis in conditions of severe hypoxia was being supported by glucose metabolism. Additional experiments were performed therefore to examine the role of glucose in supporting protein synthesis. These demonstrated that at each oxygen concentration there was a significant, decreasing linear trend in protein synthesis as glucose concentration was reduced. Under conditions of near-anoxia and in the absence of glucose, protein synthesis was reduced by >85%. Even under normoxic conditions (defined as 10% O2) and in the presence of oxidative substrates, reductions in glucose were accompanied by decreases in protein synthesis. These experiments demonstrate that syncytiotrophoblast cells are resistant to reductions in protein synthesis at O2 concentrations greater than 1%. This could be explained by our finding that a significant fraction of protein synthesis in the syncytiotrophoblast is

  14. Concurrent protein synthesis is required for in vivo chitin synthesis in postmolt blue crabs

    SciTech Connect

    Horst, M.N. )

    1990-12-01

    Chitin synthesis in crustaceans involves the deposition of a protein-polysaccharide complex at the apical surface of epithelial cells which secrete the cuticle or exoskeleton. The present study involves an examination of in vivo incorporation of radiolabeled amino acids and amino sugars into the cuticle of postmolt blue crabs, Callinectes sapidus. Rates of incorporation of both 3H leucine and 3H threonine were linear with respect to time of incubation. Incorporation of 3H threonine into the endocuticle was inhibited greater than 90% in the presence of the protein synthesis inhibitor, puromycin. Linear incorporation of 14C glucosamine into the cuticle was also demonstrated; a significant improvement of radiolabeling was achieved by using 14C-N-acetylglucosamine as the labeled precursor. Incorporation of 3H-N-acetylglucosamine into the cuticle of postmolt blue crabs was inhibited 89% by puromycin, indicating that concurrent protein synthesis is required for the deposition of chitin in the blue crab. Autoradiographic analysis of control vs. puromycin-treated crabs indicates that puromycin totally blocks labeling of the new endocuticle with 3H glucosamine. These results are consistent with the notion that crustacean chitin is synthesized as a protein-polysaccharide complex. Analysis of the postmolt and intermolt blue crab cuticle indicates that the exoskeleton contains about 60% protein and 40% chitin. The predominant amino acids are arginine, glutamic acid, alanine, aspartic acid, and threonine.

  15. Impact of protein coingestion on muscle protein synthesis during continuous endurance type exercise.

    PubMed

    Beelen, Milou; Zorenc, Antoine; Pennings, Bart; Senden, Joan M; Kuipers, Harm; van Loon, Luc J C

    2011-06-01

    This study investigates the impact of protein coingestion with carbohydrate on muscle protein synthesis during endurance type exercise. Twelve healthy male cyclists were studied during 2 h of fasted rest followed by 2 h of continuous cycling at 55% W(max). During exercise, subjects received either 1.0 g·kg(-1)·h(-1) carbohydrate (CHO) or 0.8 g·kg(-1)·h(-1) carbohydrate with 0.2 g·kg(-1)·h(-1) protein hydrolysate (CHO+PRO). Continuous intravenous infusions with l-[ring-(13)C(6)]phenylalanine and l-[ring-(2)H(2)]tyrosine were applied, and blood and muscle biopsies were collected to assess whole body protein turnover and muscle protein synthesis rates at rest and during exercise conditions. Protein coingestion stimulated whole body protein synthesis and oxidation rates during exercise by 22 ± 3 and 70 ± 17%, respectively (P < 0.01). Whole body protein breakdown rates did not differ between experiments. As a consequence, whole body net protein balance was slightly negative in CHO and positive in the CHO+PRO treatment (-4.9 ± 0.3 vs. 8.0 ± 0.3 μmol Phe·kg(-1)·h(-1), respectively, P < 0.01). Mixed muscle protein fractional synthetic rates (FSR) were higher during exercise compared with resting conditions (0.058 ± 0.006 vs. 0.035 ± 0.006%/h in CHO and 0.070 ± 0.011 vs. 0.038 ± 0.005%/h in the CHO+PRO treatment, respectively, P < 0.05). FSR during exercise did not differ between experiments (P = 0.46). We conclude that muscle protein synthesis is stimulated during continuous endurance type exercise activities when carbohydrate with or without protein is ingested. Protein coingestion does not further increase muscle protein synthesis rates during continuous endurance type exercise. PMID:21364122

  16. Synthesis of Nanogel-Protein Conjugates

    PubMed Central

    Chacko, Reuben T.; Maynard, Heather D.; Thayumanavan, S.

    2014-01-01

    The covalent conjugation of bovine serum albumin (BSA) to disulfide cross-linked polymeric nanogels is reported. Polymeric nanogel precursors were synthesized via a reversible addition-fragmentation chain transfer (RAFT) random copolymerization of poly(ethylene glycol) methyl ether methacrylate (PEGMA) and pyridyl disulfide methacrylate (PDSMA). Reaction of the p(PEGMA-co-PDSMA) with dithiothreitol resulted in the formation of nanogels. PDSMA serves as both a crosslinking agent and a reactive handle for the surface modification of the nanogels. Lipophilic dye, DiI, was sequestered within the nanogels by performing the crosslinking reaction in the presence of the hydrophobic molecule. Thiol-enriched BSA was conjugated to nanogels loaded with DiI via a disulfide reaction between the BSA and the surface exposed nanogel pyridyl disulfides. Conjugation was confirmed by fast protein liquid chromatography, dynamic light scattering, and agarose and polyacrylamide gel electrophoresis. We expect that this methodology is generally applicable to the preparation of nanogel-protein therapeutics. PMID:24761162

  17. Synthesis of Nanogel-Protein Conjugates.

    PubMed

    Matsumoto, Nicholas M; González-Toro, Daniella C; Chacko, Reuben T; Maynard, Heather D; Thayumanavan, S

    2013-04-21

    The covalent conjugation of bovine serum albumin (BSA) to disulfide cross-linked polymeric nanogels is reported. Polymeric nanogel precursors were synthesized via a reversible addition-fragmentation chain transfer (RAFT) random copolymerization of poly(ethylene glycol) methyl ether methacrylate (PEGMA) and pyridyl disulfide methacrylate (PDSMA). Reaction of the p(PEGMA-co-PDSMA) with dithiothreitol resulted in the formation of nanogels. PDSMA serves as both a crosslinking agent and a reactive handle for the surface modification of the nanogels. Lipophilic dye, DiI, was sequestered within the nanogels by performing the crosslinking reaction in the presence of the hydrophobic molecule. Thiol-enriched BSA was conjugated to nanogels loaded with DiI via a disulfide reaction between the BSA and the surface exposed nanogel pyridyl disulfides. Conjugation was confirmed by fast protein liquid chromatography, dynamic light scattering, and agarose and polyacrylamide gel electrophoresis. We expect that this methodology is generally applicable to the preparation of nanogel-protein therapeutics. PMID:24761162

  18. Protein synthesis in liposomes with a minimal set of enzymes.

    PubMed

    Murtas, Giovanni; Kuruma, Yutetsu; Bianchini, Paolo; Diaspro, Alberto; Luisi, Pier Luigi

    2007-11-01

    In a significant step towards the construction of the semi-synthetic minimal cell, a protein expression system with a minimal set of pure and specific enzymes is required. A novel cell-free transcription and translation system named PURESYSTEM (PS), consisting of a specified set of 36 enzymes and ribosomes, has been entrapped in POPC liposomes for protein synthesis. The PS has been used to transcribe and translate an Enhanced Green Fluorescent Protein (EGFP) gene from plasmid DNA. The synthesis is confirmed by the EGFP fluorescence emitting liposomes on fluorometric analysis and on confocal microscopy analysis. Furthermore the PS encapsulated into POPC liposomes can drive the expression of the plsB and plsC genes encoding for the sn-glycerol-3-phosphate acyltransferase (GPAT) and 1-acyl-sn-glycerol-3-phosphate acyltransferase (LPAAT) involved in the first step of the "salvage pathway" for synthesis of POPC. The expression of GPAT and LPAAT in liposomes would in principle allow the production of the cell boundary from within. PMID:17850764

  19. Voluntary Exercise Regionally Augments Rates of Cerebral Protein Synthesis

    PubMed Central

    Nadel, Jeffrey; Huang, Tianjian; Xia, Zengyan; Burlin, Thomas; Zametkin, Alan; Smith, Carolyn Beebe

    2016-01-01

    Exercise is a natural form of neurophysiologic stimulation that has known benefits for mental health, maintenance of cerebral function, and stress reduction. Exercise is known to induce an upregulation of brain-derived neurotrophic factor and this is thought to be involved in associated increases in neural plasticity. Protein synthesis is also an essential component of adaptive plasticity. We hypothesized that exercise may stimulate changes in brain protein synthesis as part of its effects on plasticity. Here, we applied the quantitative autoradiographic L-[1-14C] leucine method to the in vivo determination of regional rates of cerebral protein synthesis (rCPS) in adult rats following a seven day period of voluntary wheel-running and their sedentary counterparts. In four of 21 brain regions examined, the mean values of rCPS in the exercised rats were statistically significantly higher than in sedentary controls; regions affected were paraventricular hypothalamic nucleus, ventral hippocampus as a whole, CA1 pyramidal cell layer in ventral hippocampus, and frontal cortex. Increases in rCPS approached statistical significance in dentate gyrus of the ventral hippocampus. Our results affirm the value of exercise in encouraging hippocampal and possibly cortical neuroplasticity, and also suggest that exercise may modulate stimulation of stress-response pathways. Ultimately, our study indicates that measurement of rCPS with PET might be used as a marker of brain response to exercise in human subjects. PMID:24016692

  20. Eukaryotic protein synthesis inhibitors identified by comparison of cytotoxicity profiles

    PubMed Central

    CHAN, JENNY; KHAN, SHAKILA N.; HARVEY, ISABELLE; MERRICK, WILLIAM; PELLETIER, JERRY

    2004-01-01

    The National Cancer Institute (NCI) Human Tumor Cell Line Anti-Cancer Drug Screen has evaluated the cytotoxicity profiles of a large number of synthetic compounds, natural products, and plant extracts on 60 different cell lines. The data for each compound/extract can be assessed for similarity of cytotoxicity pattern, relative to a given test compound, using an algorithm called COMPARE. In applying a chemical biology approach to better understand the mechanism of eukaryotic protein synthesis, we used these resources to search for novel inhibitors of translation. The cytotoxicity profiles of 31 known protein synthesis inhibitors were used to identify compounds from the NCI database with similar activity profiles. Using this approach, two natural products, phyllanthoside and nagilactone C, were identified and characterized as novel protein synthesis inhibitors. Both compounds are specific for the eukaryotic translation apparatus, function in vivo and in vitro, and interfere with translation elongation. Our results demonstrate the feasibility of utilizing cytotoxicity profiles to identify new inhibitors of translation. PMID:14970397

  1. Ultrafast sonochemical synthesis of protein-inorganic nanoflowers

    PubMed Central

    Batule, Bhagwan S; Park, Ki Soo; Kim, Moon Il; Park, Hyun Gyu

    2015-01-01

    We developed a simple but efficient method to synthesize protein-inorganic hybrid nanostructures with a flower-like shape (nanoflowers), which relies on sonication to facilitate the synthesis of the nanoflowers. With this technique, we synthesized nanoflowers containing laccase as a model protein and copper phosphate within 5 minutes at room temperature. The resulting laccase nanoflowers yielded greatly enhanced activity, stability, and reusability, and their usefulness was successfully demonstrated by applying them in the colorimetric detection of epinephrine. The strategy developed could be used to rapidly synthesize nanoflowers for various applications in biosensor and enzyme catalysis and would expand the utilization of nanoflowers in diverse fields of biotechnology. PMID:26346235

  2. Quantitating protein synthesis, degradation, and endogenous antigen processing.

    PubMed

    Princiotta, Michael F; Finzi, Diana; Qian, Shu-Bing; Gibbs, James; Schuchmann, Sebastian; Buttgereit, Frank; Bennink, Jack R; Yewdell, Jonathan W

    2003-03-01

    Using L929 cells, we quantitated the macroeconomics of protein synthesis and degradation and the microeconomics of producing MHC class I associated peptides from viral translation products. To maintain a content of 2.6 x 10(9) proteins, each cell's 6 x 10(6) ribosomes produce 4 x 10(6) proteins min(-1). Each of the cell's 8 x 10(5) proteasomes degrades 2.5 substrates min(-1), creating one MHC class I-peptide complex for each 500-3000 viral translation products degraded. The efficiency of complex formation is similar in dendritic cells and macrophages, which play a critical role in activating T cells in vivo. Proteasomes create antigenic peptides at different efficiencies from two distinct substrate pools: rapidly degraded newly synthesized proteins that clearly represent defective ribosomal products (DRiPs) and a less rapidly degraded pool in which DRiPs may also predominate. PMID:12648452

  3. Fluorinated proteins: from design and synthesis to structure and stability.

    PubMed

    Marsh, E Neil G

    2014-10-21

    Fluorine is all but absent from biology; however, it has proved to be a remarkably useful element with which to modulate the activity of biological molecules and to study their mechanism of action. Our laboratory's interest in incorporating fluorine into proteins was stimulated by the unusual physicochemical properties exhibited by perfluorinated small molecules. These include extreme chemical inertness and thermal stability, properties that have made them valuable as nonstick coatings and fire retardants. Fluorocarbons also exhibit an unusual propensity to phase segregation. This phenomenon, which has been termed the "fluorous effect", has been effectively exploited in organic synthesis to purify compounds from reaction mixtures by extracting fluorocarbon-tagged molecules into fluorocarbon solvents. As biochemists, we were curious to explore whether the unusual physicochemical properties of perfluorocarbons could be engineered into proteins. To do this, we developed a synthesis of a highly fluorinated amino acid, hexafluoroleucine, and designed a model 4-helix bundle protein, α4H, in which the hydrophobic core was packed exclusively with leucine. We then investigated the effects of repacking the hydrophobic core of α4H with various combinations of leucine and hexafluoroleucine. These initial studies demonstrated that fluorination is a general and effective strategy for enhancing the stability of proteins against chemical and thermal denaturation and proteolytic degradation. We had originally envisaged that the "fluorous interactions", postulated from the self-segregating properties of fluorous solvents, might be used to mediate specific protein-protein interactions orthogonal to those of natural proteins. However, various lines of evidence indicate that no special, favorable fluorine-fluorine interactions occur in the core of the fluorinated α4 protein. This makes it unlikely that fluorinated amino acids can be used to direct protein-protein interactions. More

  4. Protein synthesis in chloroplasts. Characteristics and products of protein synthesis in vitro in etioplasts and developing chloroplasts from pea leaves.

    PubMed Central

    Siddell, S G; Ellis, R J

    1975-01-01

    The function of plastid ribosomes in pea (Pisum sativum L.) was investigated by characterizing the products of protein synthesis in vitro in plastids isolated at different stages during the transition from etioplast to chloroplast. Etioplasts and plastids isolated after 24, 48 and 96h of greening in continuous white light, use added ATP to incorporate labelled amino acids into protein. Plastids isolated from greening leaves can also use light as the source of energy for protein synthesis. The labelled polypeptides synthesized in isolated plastids were analysed by electrophoresis in sodium dodecyl sulphate-ureapolyacrylamide gels. Six polypeptides are synthesized in etioplasts with ATP as energy source. Only one of these polypeptides is present in a 150 000g supernatant fraction. This polypeptide has been identified as the large subunit of Fraction I protein (3-phospho-D-glycerate carboxylyase EC 4.1.1.39) by comparing the tryptic 'map' of its L-(35S)methionine-labelled peptides with the tryptic 'map' of large subunit peptides from Fraction I labelled with L-(35S)methionine in vivo. The same gel pattern of six polypeptides is seen when plastids isolated from greening leaves are incubated with either added ATP or light as the energy source. However, the rates of synthesis of particular polypeptides are different in plastids isolated at different stages of the etioplast to chloroplast transition. The results support the idea that plastid ribosomes synthesize only a small number of proteins, and that the number and molecular weight of these proteins does not alter during the formation of chloroplasts from etioplasts. Images PLATE 1 PMID:1147911

  5. VCP and ATL1 regulate endoplasmic reticulum and protein synthesis for dendritic spine formation

    PubMed Central

    Shih, Yu-Tzu; Hsueh, Yi-Ping

    2016-01-01

    Imbalanced protein homeostasis, such as excessive protein synthesis and protein aggregation, is a pathogenic hallmark of a range of neurological disorders. Here, using expression of mutant proteins, a knockdown approach and disease mutation knockin mice, we show that VCP (valosin-containing protein), together with its cofactor P47 and the endoplasmic reticulum (ER) morphology regulator ATL1 (Atlastin-1), regulates tubular ER formation and influences the efficiency of protein synthesis to control dendritic spine formation in neurons. Strengthening the significance of protein synthesis in dendritic spinogenesis, the translation blocker cyclohexamide and the mTOR inhibitor rapamycin reduce dendritic spine density, while a leucine supplement that increases protein synthesis ameliorates the dendritic spine defects caused by Vcp and Atl1 deficiencies. Because VCP and ATL1 are the causative genes of several neurodegenerative and neurodevelopmental disorders, we suggest that impaired ER formation and inefficient protein synthesis are significant in the pathogenesis of multiple neurological disorders. PMID:26984393

  6. Lipid-mediated Protein-protein Interactions Modulate Respiration-driven ATP Synthesis

    PubMed Central

    Nilsson, Tobias; Lundin, Camilla Rydström; Nordlund, Gustav; Ädelroth, Pia; von Ballmoos, Christoph; Brzezinski, Peter

    2016-01-01

    Energy conversion in biological systems is underpinned by membrane-bound proton transporters that generate and maintain a proton electrochemical gradient across the membrane which used, e.g. for generation of ATP by the ATP synthase. Here, we have co-reconstituted the proton pump cytochrome bo3 (ubiquinol oxidase) together with ATP synthase in liposomes and studied the effect of changing the lipid composition on the ATP synthesis activity driven by proton pumping. We found that for 100 nm liposomes, containing 5 of each proteins, the ATP synthesis rates decreased significantly with increasing fractions of DOPA, DOPE, DOPG or cardiolipin added to liposomes made of DOPC; with e.g. 5% DOPG, we observed an almost 50% decrease in the ATP synthesis rate. However, upon increasing the average distance between the proton pumps and ATP synthases, the ATP synthesis rate dropped and the lipid dependence of this activity vanished. The data indicate that protons are transferred along the membrane, between cytochrome bo3 and the ATP synthase, but only at sufficiently high protein densities. We also argue that the local protein density may be modulated by lipid-dependent changes in interactions between the two proteins complexes, which points to a mechanism by which the cell may regulate the overall activity of the respiratory chain. PMID:27063297

  7. Protein synthesis directly from PCR: progress and applications of cell-free protein synthesis with linear DNA.

    PubMed

    Schinn, Song-Min; Broadbent, Andrew; Bradley, William T; Bundy, Bradley C

    2016-06-25

    A rapid, versatile method of protein expression and screening can greatly facilitate the future development of therapeutic biologics, proteomic drug targets and biocatalysts. An attractive candidate is cell-free protein synthesis (CFPS), a cell-lysate-based in vitro expression system, which can utilize linear DNA as expression templates, bypassing time-consuming cloning steps of plasmid-based methods. Traditionally, such linear DNA expression templates (LET) have been vulnerable to degradation by nucleases present in the cell lysate, leading to lower yields. This challenge has been significantly addressed in the recent past, propelling LET-based CFPS as a useful tool for studying, screening and engineering proteins in a high-throughput manner. Currently, LET-based CFPS has promise in fields such as functional proteomics, protein microarrays, and the optimization of complex biological systems. PMID:27085957

  8. Isolation of a cDNA clone for spinach lipid transfer protein and evidence that the protein is synthesized by the secretory pathway

    SciTech Connect

    Bernhard, W.R.; Thoma, S.; Botella, J.; Somerville, C.R. )

    1991-01-01

    A cDNA clone encoding a nonspecific lipid transfer protein from spinach (Spinacia oleracea) was isolated by probing a library with synthetic oligonucleotides based on the amino acid sequence of the protein. Determination of the DNA sequence indicated a 354-nucleotide open reading frame which encodes a 118-amino acid residue polypeptide. The first 26 amino acids of the open reading frame, which are not present in the mature protein, have all the characteristics of a signal sequence which is normally associated with the synthesis of membrane proteins or secreted proteins. In vitro transcription of the cDNA and translation in the presence of canine pancreatic microsomes or microsomes from cultured maize endosperm cells indicated that proteolytic processing of the preprotein to the mature form was associated with cotranslational insertion into the microsomal membranes. Because there is no known mechanism by which the polypeptide could be transferred from the microsomal membranes to the cytoplasm, the proposed role of this protein in catalyzing lipid transfer between intracellular membranes is in doubt. Although the lipid transfer protein is one of the most abundant proteins in leaf cells, the results of genomic Southern analysis were consistent with the presence of only one gene. Analysis of the level of mRNA by Northern blotting indicated that the transcript was several-fold more abundant than an actin transcript in leaf and petiole tissue, but was present in roots at less than 1% of the level in petioles.

  9. Respective influences of age and weaning on skeletal and visceral muscle protein synthesis in the lamb.

    PubMed Central

    Attaix, D; Aurousseau, E; Bayle, G; Rosolowska-Huszcz, D; Arnal, M

    1988-01-01

    1. The influences of age and weaning on muscle protein synthesis were studied in vivo, by injecting a large dose of [3H]valine into 1-, 5- and 8-week-old suckling or 8-week-old weaned lambs. 2. The fractional rates of protein synthesis, in red- and white-fibre-type skeletal muscles or striated and smooth visceral muscles, were in 8-week-old suckling animals 24-37% of their values at 1 week of age. This developmental decline was related to decreased capacities for protein synthesis, i.e. RNA/protein ratios. 3. At 8 weeks of age, suckling and weaned lambs had similar fractional synthesis rates, capacities for protein synthesis and efficiencies of protein synthesis (i.e. rates of protein synthesis relative to RNA) in skeletal muscles. 4. In contrast, visceral-muscle fractional synthesis rates were lower in 8-week-old suckling lambs than in weaned animals, owing to decreased efficiencies of protein synthesis. It was concluded that developmental factors and the change to a solid diet, or weaning in itself, or both, affect differently skeletal and visceral muscle protein synthesis in the immature lamb. PMID:3223952

  10. Lil3 Assembles with Proteins Regulating Chlorophyll Synthesis in Barley

    PubMed Central

    Gargano, Daniela; Furnes, Clemens; Reisinger, Veronika; Arnold, Janine; Kmiec, Karol; Eichacker, Lutz Andreas

    2015-01-01

    The light-harvesting-like (LIL) proteins are a family of membrane proteins that share a chlorophyll a/b-binding motif with the major light-harvesting antenna proteins of oxygenic photoautotrophs. LIL proteins have been associated with the regulation of tetrapyrrol biosynthesis, and plant responses to light-stress. Here, it was found in a native PAGE approach that chlorophyllide, and chlorophyllide plus geranylgeraniolpyrophosphate trigger assembly of Lil3 in three chlorine binding fluorescent protein bands, termed F1, F2, and F3. It is shown that light and chlorophyllide trigger accumulation of protochlorophyllide-oxidoreductase, and chlorophyll synthase in band F3. Chlorophyllide and chlorophyll esterified to geranylgeraniol were identified as basis of fluorescence recorded from band F3. A direct interaction between Lil3, CHS and POR was confirmed in a split ubiquitin assay. In the presence of light or chlorophyllide, geranylgeraniolpyrophosphate was shown to trigger a loss of the F3 band and accumulation of Lil3 and geranylgeranyl reductase in F1 and F2. No direct interaction between Lil3 and geranylgeraniolreductase was identified in a split ubiquitin assay; however, accumulation of chlorophyll esterified to phytol in F1 and F2 corroborated the enzymes assembly. Chlorophyll esterified to phytol and the reaction center protein psbD of photosystem II were identified to accumulate together with psb29, and APX in the fluorescent band F2. Data show that Lil3 assembles with proteins regulating chlorophyll synthesis in etioplasts from barley (Hordeum vulgare L.). PMID:26172838

  11. Biochemical heterogeneity of skeletal-muscle microsomal membranes. Membrane origin, membrane specificity and fibre types

    PubMed Central

    Salviati, Giovanni; Volpe, Pompeo; Salvatori, Sergio; Betto, Romeo; Damiani, Ernesto; Margreth, Alfredo; Pasquali-Ronchetti, Ivonne

    1982-01-01

    1. Microsomes were isolated from rabbit fast-twitch and slow-twitch muscle and were separated into heavy and light fractions by centrifugation in a linear (0.3–2m) sucrose density gradient. The membrane origin of microsomal vesicles was investigated by studying biochemical markers of the sarcoplasmic-reticulum membranes and of surface and T-tubular membranes, as well as their freeze-fracture properties. 2. Polyacrylamide-gel electrophoresis showed differences in the Ca2+-dependent ATPase/calsequestrin ratio between heavy and light fractions, which were apparently consistent with their respective origin from cisternal and longitudinal sarcoplasmic reticulum, as well as unrelated differences, such as peptides specific to slow-muscle microsomes (mol.wts. 76000, 60000, 56000 and 45000). 3. Freeze-fracture electron microscopy of muscle microsomes demonstrated that vesicles truly derived from the sarcoplasmic reticulum, with an average density of 9nm particles on the concave face of about 3000/μm2 for both fast and slow muscle, were admixed with vesicles with particle densities below 1000/μm2. 4. As determined in the light fractions, the sarcoplasmic-reticulum vesicles accounted for 84% and 57% of the total number of microsomal vesicles, for fast and slow muscle respectively. These values agreed closely with the percentage values of Ca2+-dependent ATPase protein obtained by gel densitometry. 5. The T-tubular origin of vesicles with a smooth concave fracture face in slow-muscle microsomes is supported by their relative high content in total phospholipid and cholesterol, compared with the microsomes of fast muscle, and by other correlative data, such as the presence of (Na++K+)-dependent ATPase activity and of low amounts of Na+-dependent membrane phosphorylation. 6. Among intrinsic sarcoplasmic-reticulum membrane proteins, a proteolipid of mol.wt. 12000 is shown to be identical in the microsomes of both fast and slow muscle and the Ca2+-dependent ATPase to be

  12. Marginal B-6 intake affects protein synthesis in rat tissues

    SciTech Connect

    Sampson, D.A.; Kretsch, M.J.; Young, L.A.; Jansen, G.R.

    1986-03-05

    The role of vitamin B-6 in amino acid metabolism suggests that inadequate B-6 intake may impair protein synthesis. To test this hypothesis, 30 male rats (initially 227 g) were fed AIN76A diets that contained control, marginal or devoid levels of B-6 (5.8, 1.2 or 0.1 mg B-6/kg diet, by analysis) ad libitum for 9 weeks. Protein synthesis rates (PSRs) were measured in liver, kidney and calf muscle using a flooding dose of /sup 3/H-phenylalanine. Marginal and control groups ate and gained weight at similar rates. The marginal diet did not elevate xanthurenic acid (XA) excretion following a tryptophan load. However, marginal B-6 intake did depress liver PSR by 29% (2182 vs 1549 mg/day, P<.05), liver wet weight by 15% (19.0 vs 16.1 g, P<.05) and muscle PSR by 23% (3.0 vs 2.3%/day, P<.10). Unexpectedly, marginal B-6 intake increased PSR in kidney 47% (90 vs 132 mg/day, P<.05). The devoid diet, which increased XA excretion following a tryptophan load by more than 3-fold, depressed PSRs 56% in liver and 31% in muscle. However, the devoid diet decreased food intake by 40% (25.0 vs 15.0 g/day); therefore effects of devoid B-6 intake on PSRs may have been confounded by deficits in protein-energy intake in devoid vs control groups. These data demonstrate that marginal B-6 intake alters protein synthesis in tissues of the rat.

  13. Chloroplast protein synthesis: thylakoid bound polysomes synthesize thylakoid proteins

    SciTech Connect

    Hurewitz, J.; Jagendorf, A.T.

    1986-04-01

    Previous work indicated more polysomes bound to pea thylakoids in light than in the dark, in vivo. With isolated intact chloroplasts incubated in darkness, 24 to 74% more RNA was thylakoid-bound at pH 8.3 than at pH 7. Thus the major effect of light in vivo may be due to higher stroma pH. In isolated pea chloroplasts, initiation inhibitors (pactamycin and kanamycin) decreased the extent of RNA binding, and elongation inhibitors (lincomycin and streptomycin) increased it. Thus translation initiation and termination probably control the cycling of bound ribosomes. While only 3 to 6% of total RNA is in bound polysomes the incorporation of /sup 3/H-Leu into thylakoids was proportional to the amount of this bound RNA. When Micrococcal nuclease-treated thylakoids were added to labeled runoff translation products of stroma ribosomes, less than 1% of the label adhered to the added membranes; but 37% of the labeled products made by thylakoid polysomes were bound. These data support the concept that stroma ribosomes are recruited into thylakoid proteins.

  14. Amino acid metabolism and protein synthesis in malarial parasites*

    PubMed Central

    Sherman, I. W.

    1977-01-01

    Malaria-infected red cells and free parasites have limited capabilities for the biosynthesis of amino acids. Therefore, the principal amino acid sources for parasite protein synthesis are the plasma free amino acids and host cell haemoglobin. Infected cells and plasmodia incorporate exogenously supplied amino acids into protein. However, the hypothesis that amino acid utilization (from an external source) is related to availability of that amino acid in haemoglobin is without universal support: it is true for isoleucine and for Plasmodium knowlesi and P. falciparum, but not for methionine, cysteine, and other amino acids, and it does not apply to P. lophurae. More by default than by direct evidence, haemoglobin is believed to be the main amino acid reservoir available to the intraerythrocytic plasmodium. Haemoglobin, ingested via the cytostome, is held in food vacuoles where auto-oxidation takes place. As a consequence, haem is released and accumulates in the vacuole as particulate haemozoin (= malaria pigment). Current evidence favours the view that haemozoin is mainly haematin. Acid and alkaline proteases (identified in crude extracts from mammalian and avian malarias) are presumably secreted directly into the food vacuole. They then digest the denatured globin and the resulting amino acids are incorporated into parasite protein. Cell-free protein synthesizing systems have been developed using P. knowlesi and P. lophurae ribosomes. In the main these systems are typically eukaryotic. Studies of amino acid metabolism are exceedingly limited. Arginine, lysine, methionine, and proline are incorporated into protein, whereas glutamic acid is metabolized via an NADP-specific glutamic dehydrogenase. Glutamate oxidation generates NADPH and auxiliary energy (in the form of α-ketoglutarate). The role of red cell glutathione in the economy of the parasite remains obscure. Important goals for future research should be: quantitative assessment of the relative importance of

  15. Characterization of the microsomal steroid-8-ene isomerase of cholesterol biosynthesis.

    PubMed

    Yamaga, N; Gaylor, J L

    1978-03-01

    Rat liver microsomes contain an enzyme that catalyzes the isomerization of the nuclear double bond of steroids from the 8(9) position to the 7(8) position. The enzyme is most active with zymosterol, 5alpha-cholesta-8,24-dien-3beta-ol, which is a precursor of cholesterol. Properties of the microsomal isomerase have now been studied, and preliminary data are reported on both regulation of enzymic activity and first steps in the solubilization of the enzyme from membranes. After a brief lag period, the velocity of isomerase is relatively constant for about 5 min of incubation, and then isomerization subsides. The apparent Michaelis constant (52-70 micro M) is difficult to determine accurately, due to these complex kinetic changes. V(max) is 4.0-4.7 nmol/min per mg of microsomal protein. The apparent specific activity is more than ten times that of liver microsomal methyl sterol oxidase. The maximal specific activity of microsomal isomerase is approximately doubled when rats are fed an intestinal bile acid sequestrant, cholestyramine. Changes in specific activity of isomerase parallel changes in activities of other microsomal enzymes of cholesterol biosynthesis, such as 3-hydroxy-3-methylglutaryl-CoA reductase and 4-methyl sterol oxidase. Isomerase activity is destroyed by phospholipase A digestion, high concentration of bile salts, and solvent extraction, all of which are known either to remove phospholipid or to alter microsomal membrane integrity. On the other hand, isomerase remains active in the presence of a mild, nonionic detergent, Triton WR-1339; thus, solubilization with nonionic detergents is under study. PMID:650094

  16. Synthesis and trafficking of prion proteins in cultured cells.

    PubMed Central

    Taraboulos, A; Raeber, A J; Borchelt, D R; Serban, D; Prusiner, S B

    1992-01-01

    Scrapie prions are composed largely, if not entirely, of the scrapie prion protein (PrPSc) that is encoded by a chromosomal gene. Scrapie-infected mouse neuroblastoma (ScN2a) and hamster brain (ScHaB) cells synthesize PrPSc from the normal PrP isoform (PrPC) or a precursor through a posttranslational process. In pulse-chase radiolabeling experiments, we found that presence of brefeldin A (BFA) during both the pulse and the chase periods prevented the synthesis of PrPSc. Removal of BFA after the chase permitted synthesis of PrPSc to resume. BFA also blocked the export of nascent PrPC to the cell surface but did not alter the distribution of intracellular deposits of PrPSc. Under the same conditions, BFA caused the redistribution of the Golgi marker MG160 into the endoplasmic reticulum (ER). Using monensin as an inhibitor of mid-Golgi glycosylation, we determined that PrP traverses the mid-Golgi stack before acquiring protease resistance. About 1 h after the formation of PrPSc, its N-terminus was removed by a proteolytic process that was inhibited by ammonium chloride, chloroquine, and monensin, arguing that this is a lysosomal event. These results suggest that the ER is not competent for the synthesis of PrPSc and that the synthesis of PrPSc occurs during the transit of PrP between the mid-Golgi stack and lysosomes. Presumably, the endocytic pathway features in the synthesis of PrPSc. Images PMID:1356522

  17. Interferon Production and Protein Synthesis in Chick Cells

    PubMed Central

    Friedman, Robert M.

    1966-01-01

    Friedman, Robert M. (National Cancer Institute, Bethesda, Md.). Interferon production and protein synthesis in chick cells. J. Bacteriol. 91:1224–1229. 1966.—Overnight incubation of chick embryo fibroblasts (CEF) at 4 C before infection with live Semliki Forest virus (SFV) increased virus yields but decreased interferon production. The same findings were noted when CEF were incubated for 4 hr with p-fluorophenylalanine (FPA) before infection with live SFV or inactivated Chikungunya virus. In both systems incorporation of C14-leucine into protein appeared to be increased after pretreatment at 4 C or with FPA. Protein synthesis could be raised in CEF incubated in 0.5% serum after trypsinization by increasing the concentration of serum. CEF in 10% serum had higher rates of C14-leucine incorporation than did cells in 1.5% serum, but again the cells with the apparently high rate of incorporation produced less interferon. These findings may be related to the mechanism of cellular control over interferon production. PMID:5929753

  18. Lewis lung carcinoma regulation of mechanical stretch-induced protein synthesis in cultured myotubes.

    PubMed

    Gao, Song; Carson, James A

    2016-01-01

    Mechanical stretch can activate muscle and myotube protein synthesis through mammalian target of rapamycin complex 1 (mTORC1) signaling. While it has been established that tumor-derived cachectic factors can induce myotube wasting, the effect of this catabolic environment on myotube mechanical signaling has not been determined. We investigated whether media containing cachectic factors derived from Lewis lung carcinoma (LLC) can regulate the stretch induction of myotube protein synthesis. C2C12 myotubes preincubated in control or LLC-derived media were chronically stretched. Protein synthesis regulation by anabolic and catabolic signaling was then examined. In the control condition, stretch increased mTORC1 activity and protein synthesis. The LLC treatment decreased basal mTORC1 activity and protein synthesis and attenuated the stretch induction of protein synthesis. LLC media increased STAT3 and AMP-activated protein kinase phosphorylation in myotubes, independent of stretch. Both stretch and LLC independently increased ERK1/2, p38, and NF-κB phosphorylation. In LLC-treated myotubes, the inhibition of ERK1/2 and p38 rescued the stretch induction of protein synthesis. Interestingly, either leukemia inhibitory factor or glycoprotein 130 antibody administration caused further inhibition of mTORC1 signaling and protein synthesis in stretched myotubes. AMP-activated protein kinase inhibition increased basal mTORC1 signaling activity and protein synthesis in LLC-treated myotubes, but did not restore the stretch induction of protein synthesis. These results demonstrate that LLC-derived cachectic factors can dissociate stretch-induced signaling from protein synthesis through ERK1/2 and p38 signaling, and that glycoprotein 130 signaling is associated with the basal stretch response in myotubes. PMID:26491045

  19. Combining in Vitro Folding with Cell Free Protein Synthesis for Membrane Protein Expression.

    PubMed

    Focke, Paul J; Hein, Christopher; Hoffmann, Beate; Matulef, Kimberly; Bernhard, Frank; Dötsch, Volker; Valiyaveetil, Francis I

    2016-08-01

    Cell free protein synthesis (CFPS) has emerged as a promising methodology for protein expression. While polypeptide production is very reliable and efficient using CFPS, the correct cotranslational folding of membrane proteins during CFPS is still a challenge. In this contribution, we describe a two-step protocol in which the integral membrane protein is initially expressed by CFPS as a precipitate followed by an in vitro folding procedure using lipid vesicles for converting the protein precipitate to the correctly folded protein. We demonstrate the feasibility of using this approach for the K(+) channels KcsA and MVP and the amino acid transporter LeuT. We determine the crystal structure of the KcsA channel obtained by CFPS and in vitro folding to show the structural similarity to the cellular expressed KcsA channel and to establish the feasibility of using this two-step approach for membrane protein production for structural studies. Our studies show that the correct folding of these membrane proteins with complex topologies can take place in vitro without the involvement of the cellular machinery for membrane protein biogenesis. This indicates that the folding instructions for these complex membrane proteins are contained entirely within the protein sequence. PMID:27384110

  20. Kluyveromyces marxianus as a host for heterologous protein synthesis.

    PubMed

    Gombert, Andreas K; Madeira, José Valdo; Cerdán, María-Esperanza; González-Siso, María-Isabel

    2016-07-01

    The preferentially respiring and thermotolerant yeast Kluyveromyces marxianus is an emerging host for heterologous protein synthesis, surpassing the traditional preferentially fermenting yeast Saccharomyces cerevisiae in some important aspects: K . marxianus can grow at temperatures 10 °C higher than S. cerevisiae, which may result in decreased costs for cooling bioreactors and reduced contamination risk; has ability to metabolize a wider variety of sugars, such as lactose and xylose; is the fastest growing eukaryote described so far; and does not require special cultivation techniques (such as fed-batch) to avoid fermentative metabolism. All these advantages exist together with a high secretory capacity, performance of eukaryotic post-translational modifications, and with a generally regarded as safe (GRAS) status. In the last years, replication origins from several Kluyveromyces spp. have been used for the construction of episomal vectors, and also integrative strategies have been developed based on the tendency for non-homologous recombination displayed by K. marxianus. The recessive URA3 auxotrophic marker and the dominant Kan(R) are mostly used for selection of transformed cells, but other markers have been made available. Homologous and heterologous promoters and secretion signals have been characterized, with the K. marxianus INU1 expression and secretion system being of remarkable functionality. The efficient synthesis of roughly 50 heterologous proteins has been demonstrated, including one thermophilic enzyme. In this mini-review, we summarize the physiological characteristics of K. marxianus relevant for its use in the efficient synthesis of heterologous proteins, the efforts performed hitherto in the development of a molecular toolbox for this purpose, and some successful examples. PMID:27260286

  1. Responses of insect cells to baculovirus infection: protein synthesis shutdown and apoptosis.

    PubMed Central

    Du, X; Thiem, S M

    1997-01-01

    Protein synthesis is globally shut down at late times postinfection in the baculovirus Autographa californica M nuclear polyhedrosis virus (AcMNPV)-infected gypsy moth cell line Ld652Y. A single gene, hrf-1, from another baculovirus, Lymantria dispar M nucleopolyhedrovirus, is able to preclude protein synthesis shutdown and ensure production of AcMNPV progeny in Ld652Y cells (S. M. Thiem, X. Du, M. E. Quentin, and M. M. Berner, J. Virol. 70:2221-2229, 1996; X. Du and S. M. Thiem, Virology 227:420-430, 1997). AcMNPV contains a potent antiapoptotic gene, p35, and protein synthesis arrest was reported in apoptotic insect cells induced by infection with AcMNPV lacking p35. In exploring the function of host range factor 1 (HRF-1) and the possible connection between protein synthesis shutdown and apoptosis, a series of recombinant AcMNPVs with different complements of p35 and hrf-1 were employed in apoptosis and protein synthesis assays. We found that the apoptotic suppressor AcMNPV P35 was translated prior to protein synthesis shutdown and functioned to prevent apoptosis. HRF-1 prevented protein synthesis shutdown even when the cells were undergoing apoptosis, but HRF-1 could not functionally substitute for P35. The DNA synthesis inhibitor aphidicolin could block both apoptosis and protein synthesis shutdown in Ld652Y cells infected with p35 mutant AcMNPVs but not the protein synthesis shutdown in wild-type AcMNPV-infected Ld652Y cells. These data suggest that protein synthesis shutdown and apoptosis are separate responses of Ld652Y cells to AcMNPV infection and that P35 is involved in inducing a protein synthesis shutdown response in the absence of late viral gene expression in Ld652Y cells. A model was developed for these responses of Ld652Y cells to AcMNPV infection. PMID:9311875

  2. Electrochemical template synthesis of multisegment nanowires: fabrication and protein functionalization.

    PubMed

    Wildt, Bridget; Mali, Prashant; Searson, Peter C

    2006-12-01

    Multisegment nanowires represent a unique platform for engineering multifunctional nanoparticles for a wide range of applications. For example, the optical and magnetic properties of nanowires can be tailored by modifying the size, shape, and composition of each segment. Similarly, surface modification can be used to tailor chemical and biological properties. In this article, we report on recent work on electrochemical template synthesis of nanogap electrodes, the fabrication of multisegment nanowires with embedded catalysts, and the selective functionalization of multisegment nanowires with proteins. PMID:17129026

  3. Truly Absorbed Microbial Protein Synthesis, Rumen Bypass Protein, Endogenous Protein, and Total Metabolizable Protein from Starchy and Protein-Rich Raw Materials: Model Comparison and Predictions.

    PubMed

    Parand, Ehsan; Vakili, Alireza; Mesgaran, Mohsen Danesh; van Duinkerken, Gert; Yu, Peiqiang

    2015-07-29

    This study was carried out to measure truly absorbed microbial protein synthesis, rumen bypass protein, and endogenous protein loss, as well as total metabolizable protein, from starchy and protein-rich raw feed materials with model comparisons. Predictions by the DVE2010 system as a more mechanistic model were compared with those of two other models, DVE1994 and NRC-2001, that are frequently used in common international feeding practice. DVE1994 predictions for intestinally digestible rumen undegradable protein (ARUP) for starchy concentrates were higher (27 vs 18 g/kg DM, p < 0.05, SEM = 1.2) than predictions by the NRC-2001, whereas there was no difference in predictions for ARUP from protein concentrates among the three models. DVE2010 and NRC-2001 had highest estimations of intestinally digestible microbial protein for starchy (92 g/kg DM in DVE2010 vs 46 g/kg DM in NRC-2001 and 67 g/kg DM in DVE1994, p < 0.05 SEM = 4) and protein concentrates (69 g/kg DM in NRC-2001 vs 31 g/kg DM in DVE1994 and 49 g/kg DM in DVE2010, p < 0.05 SEM = 4), respectively. Potential protein supplies predicted by tested models from starchy and protein concentrates are widely different, and comparable direct measurements are needed to evaluate the actual ability of different models to predict the potential protein supply to dairy cows from different feedstuffs. PMID:26118653

  4. Involvement of protein kinase C activation in L-leucine-induced stimulation of protein synthesis in l6 myotubes.

    PubMed

    Yagasaki, Kazumi; Morisaki, Naoko; Kitahara, Yoshiro; Miura, Atsuhito; Funabiki, Ryuhei

    2003-11-01

    Effects of leucine and related compounds on protein synthesis were studied in L6 myotubes. The incorporation of [(3)H]tyrosine into cellular protein was measured as an index of protein synthesis. In leucine-depleted L6 myotubes, leucine and its keto acid, alpha-ketoisocaproic acid (KIC), stimulated protein synthesis, while D-leucine did not. Mepacrine, an inhibitor of both phospholipases A(2) and C, canceled stimulatory actions of L-leucine and KIC on protein synthesis. Neither indomethacin, an inhibitor of cyclooxygenase, nor caffeic acid, an inhibitor of lipoxygenase, diminished their stimulatory actions, suggesting no involvement of arachidonic acid metabolism. Conversely, 1-O-hexadecyl-2-O-methylglycerol, an inhibitor of proteinkinase C, significantly canceled the stimulatory actions of L-leucine and KIC on protein synthesis, suggesting an involvement of phosphatidylinositol degradation and activation of protein kinase C. L-Leucine caused a rapid activation of protein kinase C in both cytosol and membrane fractions of the cells. These results strongly suggest that both L-leucine and KIC stimulate protein synthesis in L6 myotubes through activation of phospholipase C and protein kinase C. PMID:19003213

  5. Expanding the chemical toolbox for the synthesis of large and uniquely modified proteins

    NASA Astrophysics Data System (ADS)

    Bondalapati, Somasekhar; Jbara, Muhammad; Brik, Ashraf

    2016-05-01

    Methods to prepare proteins that include a specific modification at a desired position are essential for understanding their cellular functions and physical properties in living systems. Chemical protein synthesis, which relies on the chemoselective ligation of unprotected peptides, enables the preparation of modified proteins that are not easily fabricated by other methods. In contrast to recombinant approaches, chemical synthesis can be used to prepare protein analogues such as D-proteins, which are useful in protein structure determination and the discovery of novel therapeutics. Post-translationally modifying proteins is another example where chemical protein synthesis proved itself as a powerful approach for preparing samples with high homogeneity and in workable quantities. In this Review, we discuss the basic principles of the field, focusing on novel chemoselective peptide ligation approaches such as native chemical ligation and the recent advances based on this method with a proven record of success in the synthesis of highly important protein targets.

  6. Amyloid Precursor Protein (APP) Affects Global Protein Synthesis in Dividing Human Cells

    PubMed Central

    Liang, Shuang; Rambo, Brittany; Skucha, Sylvia; Weber, Megan J.; Alani, Sara; Bocchetta, Maurizio

    2015-01-01

    Hypoxic non-small cell lung cancer (NSCLC) is dependent on Notch-1 signaling for survival. Targeting Notch-1 by means of γ-secretase inhibitors (GSI) proved effective in killing hypoxic NSCLC. Post-mortem analysis of GSI-treated, NSCLC-burdened mice suggested enhanced phosphorylation of 4E-BP1 at threonines 37/46 in hypoxic tumor tissues. In vitro dissection of this phenomenon revealed that Amyloid Precursor Protein (APP) inhibition was responsible for a non-canonical 4E-BP1 phosphorylation pattern rearrangement—a process, in part, mediated by APP regulation of the pseudophosphatase Styx. Upon APP depletion we observed modifications of eIF-4F composition indicating increased recruitment of eIF-4A to the mRNA cap. This phenomenon was supported by the observation that cells with depleted APP were partially resistant to silvestrol, an antibiotic that interferes with eIF-4A assembly into eIF-4F complexes. APP downregulation in dividing human cells increased the rate of global protein synthesis, both cap- and IRES-dependent. Such an increase seemed independent of mTOR inhibition. After administration of Torin-1, APP downregulation and Mechanistic Target of Rapamycin Complex 1 (mTORC-1) inhibition affected 4E-BP1 phosphorylation and global protein synthesis in opposite fashions. Additional investigations indicated that APP operates independently of mTORC-1. Key phenomena described in this study were reversed by overexpression of the APP C-terminal domain. The presented data suggest that APP may be a novel regulator of protein synthesis in dividing human cells, both cancerous and primary. Furthermore, APP appears to affect translation initiation using mechanisms seemingly dissimilar to mTORC-1 regulation of cap-dependent protein synthesis. PMID:25283437

  7. Amyloid precursor protein (APP) affects global protein synthesis in dividing human cells.

    PubMed

    Sobol, Anna; Galluzzo, Paola; Liang, Shuang; Rambo, Brittany; Skucha, Sylvia; Weber, Megan J; Alani, Sara; Bocchetta, Maurizio

    2015-05-01

    Hypoxic non-small cell lung cancer (NSCLC) is dependent on Notch-1 signaling for survival. Targeting Notch-1 by means of γ-secretase inhibitors (GSI) proved effective in killing hypoxic NSCLC. Post-mortem analysis of GSI-treated, NSCLC-burdened mice suggested enhanced phosphorylation of 4E-BP1 at threonines 37/46 in hypoxic tumor tissues. In vitro dissection of this phenomenon revealed that Amyloid Precursor Protein (APP) inhibition was responsible for a non-canonical 4E-BP1 phosphorylation pattern rearrangement-a process, in part, mediated by APP regulation of the pseudophosphatase Styx. Upon APP depletion we observed modifications of eIF-4F composition indicating increased recruitment of eIF-4A to the mRNA cap. This phenomenon was supported by the observation that cells with depleted APP were partially resistant to silvestrol, an antibiotic that interferes with eIF-4A assembly into eIF-4F complexes. APP downregulation in dividing human cells increased the rate of global protein synthesis, both cap- and IRES-dependent. Such an increase seemed independent of mTOR inhibition. After administration of Torin-1, APP downregulation and Mechanistic Target of Rapamycin Complex 1 (mTORC-1) inhibition affected 4E-BP1 phosphorylation and global protein synthesis in opposite fashions. Additional investigations indicated that APP operates independently of mTORC-1. Key phenomena described in this study were reversed by overexpression of the APP C-terminal domain. The presented data suggest that APP may be a novel regulator of protein synthesis in dividing human cells, both cancerous and primary. Furthermore, APP appears to affect translation initiation using mechanisms seemingly dissimilar to mTORC-1 regulation of cap-dependent protein synthesis. PMID:25283437

  8. Cell-free synthesis of enzymically active tissue-type plasminogen activator. Protein folding determines the extent of N-linked glycosylation.

    PubMed Central

    Bulleid, N J; Bassel-Duby, R S; Freedman, R B; Sambrook, J F; Gething, M J

    1992-01-01

    Tissue-type plasminogen activator (t-PA) is synthesized in mammalian cells as a mixture of two forms that differ in their extent of N-linked glycosylation. We have investigated the mechanism underlying this variation in glycosylation, using a cell-free system that consists of a rabbit reticulocyte lysate optimized for the formation of disulphide bonds and supplemented with dog pancreas microsomal membranes. Molecules of human t-PA synthesized in vitro are enzymically active and responsive to natural activators and inhibitors, and are glycosylated in a pattern identical with that of the protein produced in vivo. This demonstrates that t-PA synthesized in vitro folds into the same conformation as the protein synthesized in vivo. We show that the extent of glycosylation of individual t-PA molecules is dependent on the state of folding of the polypeptide chain, since the probability of addition of an oligosaccharide side chain at Asn-184 is decreased under conditions that promote the formation of enzymically active molecules. This variation in glycosylation is independent of the rate of protein synthesis. Images Fig. 3. Fig. 4. Fig. 5. Fig. 6. PMID:1520279

  9. Evidence for microsomal variation of opioid sites not coupled to N/sub i/

    SciTech Connect

    Spain, J.W.; Coscia, C.J.

    1986-03-05

    Previously, the authors demonstrated a multi-step association of variation of opioid agonists to bovine hippocampal synaptic membranes (SPM). A slowly formed high affinity state is most sensitive to the GTP analog, Gpp(NH)p. Multi-step association can be inferred from the assoc.-time dependent rate of dissociation seen for agonists, i.e., following a brief incubation period the rate of dissociation is more rapid than following a longer association. When 1 nM /sup 3/H-DADL (in the presence of 20 nM unlabeled DAGO) was incubated with microsomal membranes for 7, 12, 20, or 60 min, the off-rate remained constant, suggesting a bimolecular interaction. The monophasic rate of association to microsomes contrasted with the observed biphasic association to SPM. Computer simulation of binding models indicated that, while binding kinetics for SPM can best be simulated by a three-step sequential equilibrium, the pattern for microsomes represents a bimolecular interaction. In contrast to the profound increase in rate of dissociation from SPM's in the presence of Gpp(NH)p, microsome kinetics are not affected. The authors proposed that for SPM's the slowly formed state with a high sensitivity to Gpp(NH)p is a form of the ternary complex consisting of receptor, ligand and GTP binding protein. These results suggest that microsomal membranes, which they believe are primarily internal sites, do not form the ternary complex and are not coupled to N/sub i/.

  10. Evolution, structure, and synthesis of vertebrate egg-coat proteins

    PubMed Central

    Litscher, Eveline S.; Wassarman, Paul M.

    2015-01-01

    All vertebrate eggs are surrounded by an extracellular coat that supports growth of oocytes, protects oocytes, eggs, and early embryos, and participates in the process of fertilization. In mammals (platypus to human beings) the coat is called a zona pellucida (ZP) and in non-mammals (molluscs to birds), a vitelline envelope (VE). The ZP and VE are composed of just a few proteins that are related to one another and possess a common motif, called the zona pellucida domain (ZPD). The ZPD arose more than ~600 million years ago, consists of ~260 amino acids, and has 8 conserved Cys residues that participate in 4 intramolecular disulfides. It is likely that egg-coat proteins are derived from a common ancestral gene. This gene duplicated several times during evolution and gave rise to 3–4 genes in fish, 5 genes in amphibians, 6 genes in birds, and 3–4 genes in mammals. Some highly divergent sequences, N- and C-terminal to the ZPD, have been identified in egg-coat proteins and some of these sequences may be under positive Darwinian selection that drives evolution of the proteins. These and other aspects of egg-coat proteins, including their structure and synthesis, are addressed in this review. PMID:26504367

  11. A Network Synthesis Model for Generating Protein Interaction Network Families

    PubMed Central

    Sahraeian, Sayed Mohammad Ebrahim; Yoon, Byung-Jun

    2012-01-01

    In this work, we introduce a novel network synthesis model that can generate families of evolutionarily related synthetic protein–protein interaction (PPI) networks. Given an ancestral network, the proposed model generates the network family according to a hypothetical phylogenetic tree, where the descendant networks are obtained through duplication and divergence of their ancestors, followed by network growth using network evolution models. We demonstrate that this network synthesis model can effectively create synthetic networks whose internal and cross-network properties closely resemble those of real PPI networks. The proposed model can serve as an effective framework for generating comprehensive benchmark datasets that can be used for reliable performance assessment of comparative network analysis algorithms. Using this model, we constructed a large-scale network alignment benchmark, called NAPAbench, and evaluated the performance of several representative network alignment algorithms. Our analysis clearly shows the relative performance of the leading network algorithms, with their respective advantages and disadvantages. The algorithm and source code of the network synthesis model and the network alignment benchmark NAPAbench are publicly available at http://www.ece.tamu.edu/bjyoon/NAPAbench/. PMID:22912671

  12. Microsomal quercetin glucuronidation in rat small intestine depends on age and segment

    Technology Transfer Automated Retrieval System (TEKTRAN)

    UDP-glucuronosyltransferase (UGT) activity toward the flavonoid quercetin and UGT protein were characterized in 3 equidistant small intestine (SI) segments from 4, 12, 18, and 28 mo male F344 rats, n=8/age using villin to control for enterocyte content. SI microsomal intrinsic clearance of quercetin...

  13. Prion protein interaction with stress-inducible protein 1 enhances neuronal protein synthesis via mTOR

    PubMed Central

    Roffé, Martín; Beraldo, Flávio Henrique; Bester, Romina; Nunziante, Max; Bach, Christian; Mancini, Gabriel; Gilch, Sabine; Vorberg, Ina; Castilho, Beatriz A.; Martins, Vilma Regina; Hajj, Glaucia Noeli Maroso

    2010-01-01

    Transmissible spongiform encephalopathies are fatal neurodegenerative diseases caused by the conversion of prion protein (PrPC) into an infectious isoform (PrPSc). How this event leads to pathology is not fully understood. Here we demonstrate that protein synthesis in neurons is enhanced via PrPC interaction with stress-inducible protein 1 (STI1). We also show that neuroprotection and neuritogenesis mediated by PrPC–STI1 engagement are dependent upon the increased protein synthesis mediated by PI3K-mTOR signaling. Strikingly, the translational stimulation mediated by PrPC–STI1 binding is corrupted in neuronal cell lines persistently infected with PrPSc, as well as in primary cultured hippocampal neurons acutely exposed to PrPSc. Consistent with this, high levels of eukaryotic translation initiation factor 2α (eIF2α) phosphorylation were found in PrPSc-infected cells and in neurons acutely exposed to PrPSc. These data indicate that modulation of protein synthesis is critical for PrPC–STI1 neurotrophic functions, and point to the impairment of this process during PrPSc infection as a possible contributor to neurodegeneration. PMID:20615969

  14. Consumption of Milk Protein or Whey Protein Results in a Similar Increase in Muscle Protein Synthesis in Middle Aged Men

    PubMed Central

    Mitchell, Cameron J.; McGregor, Robin A.; D’Souza, Randall F.; Thorstensen, Eric B.; Markworth, James F.; Fanning, Aaron C.; Poppitt, Sally D.; Cameron-Smith, David

    2015-01-01

    The differential ability of various milk protein fractions to stimulate muscle protein synthesis (MPS) has been previously described, with whey protein generally considered to be superior to other fractions. However, the relative ability of a whole milk protein to stimulate MPS has not been compared to whey. Sixteen healthy middle-aged males ingested either 20 g of milk protein (n = 8) or whey protein (n = 8) while undergoing a primed constant infusion of ring 13C6 phenylalanine. Muscle biopsies were obtained 120 min prior to consumption of the protein and 90 and 210 min afterwards. Resting myofibrillar fractional synthetic rates (FSR) were 0.019% ± 0.009% and 0.021% ± 0.018% h−1 in the milk and whey groups respectively. For the first 90 min after protein ingestion the FSR increased (p < 0.001) to 0.057% ± 0.018% and 0.052% ± 0.024% h−1 in the milk and whey groups respectively with no difference between groups (p = 0.810). FSR returned to baseline in both groups between 90 and 210 min after protein ingestion. Despite evidence of increased rate of digestion and leucine availability following the ingestion of whey protein, there was similar activation of MPS in middle-aged men with either 20 g of milk protein or whey protein. PMID:26506377

  15. Evolution of Protein Synthesis from an RNA World

    PubMed Central

    Noller, Harry F.

    2012-01-01

    SUMMARY Because of the molecular complexity of the ribosome and protein synthesis, it is a challenge to imagine how translation could have evolved from a primitive RNA World. Two specific suggestions are made here to help to address this, involving separate evolution of the peptidyl transferase and decoding functions. First, it is proposed that translation originally arose not to synthesize functional proteins, but to provide simple (perhaps random) peptides that bound to RNA, increasing its available structure space, and therefore its functional capabilities. Second, it is proposed that the decoding site of the ribosome evolved from a mechanism for duplication of RNA. This process involved homodimeric “duplicator RNAs,” resembling the anticodon arms of tRNAs, which directed ligation of trinucleotides in response to an RNA template. PMID:20610545

  16. Metabolism of N-methylcarbazole by rat lung microsomes.

    PubMed

    Ibe, B O; Raj, J U

    1994-01-01

    N-methylcarbazole (NMC) is a procarcinogenic component of tobacco smoke particulate matter. It is metabolized by liver microsomes into some hydroxylated metabolites such as the potent mutagen N-hydroxymethylcarbazole (NHMC). Lung metabolism and toxicity of NMC is not known. Since the lung is the primary organ of inhalation of tobacco smoke, NMC metabolism by lung microsomes was studied in comparison with the metabolism by liver microsomes. Liver or lung microsomes (1 mg/mL) were incubated with 0.5 mM NMC for 30 min at 37 degrees C. NMC metabolites were extracted with ethyl acetate and analyzed by reversed-phase high-performance liquid chromatography. Rat lung microsomes metabolized NMC with a similar profile to liver microsomes, although lung microsomes produced greater number of metabolites. The potent mutagen NHMC was also the major NMC metabolite produced by lung microsomes, as confirmed by particle beam mass spectrometry. However, lung microsomes produced only 10% of NHMC produced by liver microsomes. Metabolism of NMC by lung microsomes also led to depletion of the endogenous antioxidant glutathione by 34% compared to controls, indicating a significant generation of some reactive intermediates during NMC metabolism by lung microsomes. The data show that the lung participates directly in producing the potent mutagen NHMC from NMC present in tobacco smoke. PMID:7925139

  17. Physiological Content and Intrinsic Activities of 10 Cytochrome P450 Isoforms in Human Normal Liver Microsomes.

    PubMed

    Zhang, Hai-Feng; Wang, Huan-Huan; Gao, Na; Wei, Jun-Ying; Tian, Xin; Zhao, Yan; Fang, Yan; Zhou, Jun; Wen, Qiang; Gao, Jie; Zhang, Yang-Jun; Qian, Xiao-Hong; Qiao, Hai-Ling

    2016-07-01

    Due to a lack of physiologic cytochrome P450 (P450) isoform content, P450 activity is typically only determined at the microsomal level (per milligram of microsomal protein) and not at the isoform level (per picomole of P450 isoform), which could result in the misunderstanding of variations in P450 activity between individuals and further hinder development of personalized medicine. We found that there were large variations in protein content, mRNA levels, and intrinsic activities of the 10 P450s in 100 human liver samples, in which CYP2E1 and CYP2C9 showed the highest expression levels. P450 gene polymorphisms had different effects on activity at two levels: CYP3A5*3 and CYP2A6*9 alleles conferred increased activity at the isoform level but decreased activity at the microsomal level; CYP2C9*3 had no effect at the isoform level but decreased activity at the microsomal level. The different effects at each level stem from the different effects of each polymorphism on the resulting P450 protein. Individuals with CYP2A6*1/*4, CYP2A6*1/*9, CYP2C9*1/*3, CYP2D6 100C>T TT, CYP2E1 7632T>A AA, CYP3A5*1*3, and CYP3A5*3*3 genotypes had significantly lower protein content, whereas CYP2D6 1661G>C mutants had a higher protein content. In conclusion, we first offered the physiologic data of 10 P450 isoform contents and found that some single nucleotide polymorphisms had obvious effects on P450 expression in human normal livers. The effects of gene polymorphisms on intrinsic P450 activity at the isoform level were quite different from those at the microsomal level, which might be due to changes in P450 protein content. PMID:27189963

  18. Synthesis of several membrane proteins during developmental aggregation in Myxococcus xanthus.

    PubMed

    Orndorff, P E; Dworkin, M

    1982-01-01

    We have examined the pattern of synthesis of several membrane proteins during the aggregation phase of development in Myxococcus xanthus. Development was initiated by plating vegetative cells on polycarbonate filters placed on top of an agar medium that supported fruiting body formation. At various times during aggregation a filter was removed, the cells were pulse-labeled with [35S]methionine, and the membrane proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The rate of synthesis of numerous individual proteins changed during aggregation; we concentrated on six whose pattern of synthesis was greatly altered during aggregation. The rate of synthesis of five of the six proteins increased considerably during aggregation; that of the remaining protein was curtailed and appeared to be regulated by nutrient conditions. Three of the five major membrane proteins that increased during aggregation had a unique pattern of synthesis that was displayed only under conditions that are are required for development - high cell density, nutrient depletion, and a solid (agar) surface. The remaining two proteins were not unique to development; the appearance of one protein could be induced under conditions of high cell density, whereas the other could be induced by placing the cells on a solid agar surface. All of the five major proteins that appeared during development did so during the preaggregation stage, and the synthesis of four of the five proteins appeared to be curtailed late in aggregation. The synthesis of the remaining protein continued throughout aggregation. PMID:6798022

  19. Application of electroimmunoassay to the study of plasma protein synthesis in cultured hepatocytes.

    PubMed

    Grieninger, G; Pindyck, J; Hertzberg, K M; Mosesson, M W

    1979-01-01

    Electroimmunoassay has been applied to the study of plasma protein synthesis and secretion in liver cell cultures. The assay is performed on unconcentrated samples of culture medium containing the secreted plasma proteins and yields results within 2 hours. The characteristics of plasma protein production by the cultured hepatocytes coupled with the sensitivity of this assay permit the study of plasma protein in synthesis and its regulation by hormones and other agents without the routine use of radioisotopes. PMID:518014

  20. The mitochondrial outer membrane protein MDI promotes local protein synthesis and mtDNA replication.

    PubMed

    Zhang, Yi; Chen, Yong; Gucek, Marjan; Xu, Hong

    2016-05-17

    Early embryonic development features rapid nuclear DNA replication cycles, but lacks mtDNA replication. To meet the high-energy demands of embryogenesis, mature oocytes are furnished with vast amounts of mitochondria and mtDNA However, the cellular machinery driving massive mtDNA replication in ovaries remains unknown. Here, we describe a Drosophila AKAP protein, MDI that recruits a translation stimulator, La-related protein (Larp), to the mitochondrial outer membrane in ovaries. The MDI-Larp complex promotes the synthesis of a subset of nuclear-encoded mitochondrial proteins by cytosolic ribosomes on the mitochondrial surface. MDI-Larp's targets include mtDNA replication factors, mitochondrial ribosomal proteins, and electron-transport chain subunits. Lack of MDI abolishes mtDNA replication in ovaries, which leads to mtDNA deficiency in mature eggs. Targeting Larp to the mitochondrial outer membrane independently of MDI restores local protein synthesis and rescues the phenotypes of mdi mutant flies. Our work suggests that a selective translational boost by the MDI-Larp complex on the outer mitochondrial membrane might be essential for mtDNA replication and mitochondrial biogenesis during oogenesis. PMID:27053724

  1. Integrating gene synthesis and microfluidic protein analysis for rapid protein engineering

    PubMed Central

    Blackburn, Matthew C.; Petrova, Ekaterina; Correia, Bruno E.; Maerkl, Sebastian J.

    2016-01-01

    The capability to rapidly design proteins with novel functions will have a significant impact on medicine, biotechnology and synthetic biology. Synthetic genes are becoming a commodity, but integrated approaches have yet to be developed that take full advantage of gene synthesis. We developed a solid-phase gene synthesis method based on asymmetric primer extension (APE) and coupled this process directly to high-throughput, on-chip protein expression, purification and characterization (via mechanically induced trapping of molecular interactions, MITOMI). By completely circumventing molecular cloning and cell-based steps, APE-MITOMI reduces the time between protein design and quantitative characterization to 3–4 days. With APE-MITOMI we synthesized and characterized over 400 zinc-finger (ZF) transcription factors (TF), showing that although ZF TFs can be readily engineered to recognize a particular DNA sequence, engineering the precise binding energy landscape remains challenging. We also found that it is possible to engineer ZF–DNA affinity precisely and independently of sequence specificity and that in silico modeling can explain some of the observed affinity differences. APE-MITOMI is a generic approach that should facilitate fundamental studies in protein biophysics, and protein design/engineering. PMID:26704969

  2. Non-standard amino acid incorporation into proteins using Escherichia coli cell-free protein synthesis

    NASA Astrophysics Data System (ADS)

    Hong, Seok Hoon; Kwon, Yong-Chan; Jewett, Michael

    2014-06-01

    Incorporating non-standard amino acids (NSAAs) into proteins enables new chemical properties, new structures, and new functions. In recent years, improvements in cell-free protein synthesis (CFPS) systems have opened the way to accurate and efficient incorporation of NSAAs into proteins. The driving force behind this development has been three-fold. First, a technical renaissance has enabled high-yielding (>1 g/L) and long-lasting (>10 h in batch operation) CFPS in systems derived from Escherichia coli. Second, the efficiency of orthogonal translation systems has improved. Third, the open nature of the CFPS platform has brought about an unprecedented level of control and freedom of design. Here, we review recent developments in CFPS platforms designed to precisely incorporate NSAAs. In the coming years, we anticipate that CFPS systems will impact efforts to elucidate structure/function relationships of proteins and to make biomaterials and sequence-defined biopolymers for medical and industrial applications.

  3. Effect of dietary protein quality and feeding level on milk secretion and mammary protein synthesis in the rat

    SciTech Connect

    Sampson, D.A.; Jansen, G.R.

    1985-04-01

    Protein synthesis was studied in mammary tissue of rats fed diets deficient in protein quality and/or restricted in food intake throughout gestation and lactation. Diets containing 25% wheat gluten (WG), wheat gluten plus lysine and threonine (WGLT), or casein (C) were pair-fed from conception until day 15 of lactation at 100% or 85% of WG ad libitum consumption (PF100 and PF85, respectively). A seventh group was fed C ad libitum. Rates of protein synthesis were measured in vivo at day 15 of lactation from incorporation of (3-/sup 3/H)phenylalanine. At both PF100 and PF85, fractional and absolute rates of mammary gland protein synthesis were two- to three-fold higher in rats fed C than in those fed WG. Pup weights showed similar treatment effects. Both mammary protein synthesis rates and pup weights were significantly higher in rats fed C at PF85 than rats fed WG ad libitum. Food restriction from PF100 to PF85 depressed pup weights and mammary protein synthesis rates in rats fed WGLT, but had no effect in rats fed WG. These results demonstrate that when food intake is restricted, improvement of protein quality of the maternal diet increases milk output in the rat in association with increased rates of mammary protein synthesis.

  4. Identification of a microsomal retinoic acid synthase as a microsomal cytochrome P-450-linked monooxygenase system.

    PubMed

    Tomita, S; Tsujita, M; Matsuo, Y; Yubisui, T; Ichikawa, Y

    1993-12-01

    1. To characterize an enzyme which metabolizes retinal in liver microsomes, several properties of the enzymatic reaction from retinal to retinoic acid were investigated using rabbit liver microsomes. 2. The maximum pH of the reaction in the liver microsomes was 7.6. 3. The Km and Vmax values for all-trans, 9-cis and 13-cis-retinals were determined. 4. The reaction proceeded in the presence of NADPH and molecular oxygen. 5. The incorporation of one atom of molecular oxygen into retinal was confirmed by using oxygen-18, showing that the reaction comprised monooxygenation, not dehydrogenation. 6. The monooxygenase activity was inhibited by carbon monoxide, phenylisocyanide and anti-NADPH-cytochrome P-450 reductase IgG, but not by anti-cytochrome b5 IgG. 7. The enzymatic activity inhibited by carbon monoxide was photoreversibly restored by light of a wavelength of around 450 nm. 8. The retinal-induced spectra of liver microsomes with three isomeric retinals were type I spectra. 9. The microsomal monooxygenase activity induced by phenobarbital or ethanol were more effective than that by 3-methylcholanthrene, clotrimazole or beta-naphthoflavone. 10. These results showed that the monooxygenase reaction from retinal to retinoic acid in liver microsomes is catalyzed by a cytochrome P-450-linked monooxygenase system. PMID:8138015

  5. Feeding rapidly stimulates protein synthesis in skeletal muscle of neonatal pigs by enhancing translation initiation

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Food consumption increases protein synthesis in most tissues by promoting translation initiation, and in the neonate, this increase is greatest in skeletal muscle. In this study, we aimed to identify the currently unknown time course of changes in the rate of protein synthesis and the activation of ...

  6. Long-term leucine induced stimulation of muscle protein synthesis is amino acid dependent

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Infusing leucine for 1 h increases skeletal muscle protein synthesis in the neonate, but this is not sustained for 2 h unless the corresponding fall in amino acids is prevented. This study aimed to determine whether a continuous leucine infusion can stimulate protein synthesis for a prolonged period...

  7. On the Role of Hippocampal Protein Synthesis in the Consolidation and Reconsolidation of Object Recognition Memory

    ERIC Educational Resources Information Center

    Rossato, Janine I.; Bevilaqua, Lia R. M.; Myskiw, Jociane C.; Medina, Jorge H.; Izquierdo, Ivan; Cammarota, Martin

    2007-01-01

    Upon retrieval, consolidated memories are again rendered vulnerable to the action of metabolic blockers, notably protein synthesis inhibitors. This has led to the hypothesis that memories are reconsolidated at the time of retrieval, and that this depends on protein synthesis. Ample evidence indicates that the hippocampus plays a key role both in…

  8. Differential effects of long-term leucine infusion on tissue protein synthesis in neonatal pigs

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Leucine is unique among the amino acids in its ability to promote protein synthesis by activating translation initiation via the mammalian target of rapamycin (mTOR) pathway. Previously, we showed that leucine infusion acutely stimulates protein synthesis in fast-twitch glycolytic muscle of neonatal...

  9. Recalling an Aversive Experience by Day-Old Chicks Is Not Dependent on Somatic Protein Synthesis

    ERIC Educational Resources Information Center

    Mileusnic, Radmila; Lancashire, Christine L.; Rose, Steven P. R.

    2005-01-01

    Long-term memory is dependent on protein synthesis and inhibiting such synthesis following training results in amnesia for the task. Proteins synthesized during training must be transported to the synapse and disrupting microtubules with Colchicines, and hence, blocking transport, results in transient amnesia. Reactivating memory for a previously…

  10. Social Recognition Memory Requires Two Stages of Protein Synthesis in Mice

    ERIC Educational Resources Information Center

    Wolf, Gerald; Engelmann, Mario; Richter, Karin

    2005-01-01

    Olfactory recognition memory was tested in adult male mice using a social discrimination task. The testing was conducted to begin to characterize the role of protein synthesis and the specific brain regions associated with activity in this task. Long-term olfactory recognition memory was blocked when the protein synthesis inhibitor anisomycin was…