Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: T02G6 .1
 572,118 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

A cellulase [EC 3.2.1.4] component was purified from a crude cellulase preparation of Trichoderma viride (Meicelase) by consecutive column chromatography procedures, and was designated as cellulase III. The enzyme was homogeneous on polyacrylamide gel disc electrophoresis. The molecular weight of the enzyme was estimated to be about 45,000 by gel filtration. The optimum pH and temperature of the enzyme were pH 4.5-5.0 and 50 degrees, respectively. The enzyme was stable over the range of pH 4.5-7.5 at 4 degrees for 24 hr, and retained 40% of the original carboxymethylcellulose-saccharifying activity after heating at 100 degrees for 10 min. The enzyme was completely inactivated by 1 mM Hg2+, and partially by 1 mM Ag+ and Cu2+. The enzyme was characterized as a less-random type cellulase on the basis of its action on carboxymethylcellulose. The enzyme split cellohexaose, retaining the beta-configuration of the anomeric carbon atoms in the hydrolysis products. The Km values of cellulase III for cellooligosaccharides decreased in parallel with increase of the chain length of the substrates, while Vmax values showed a tendency to increase. The enzyme produced predominantly cellobiose and glucose from various cellulosic substrates as well as from higher cellooligosaccharides. Cellulase III preferentially attacked the aglycone linkage of p-nitrophenyl beta-D-cellobioside. The enzyme was found to catalyze the rapid synthesis of cellotetraose from cellobiose (condensation action).
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PMID:Enzymatic studies on a cellulase system of Trichoderma viride. IV. Purification and properties of a less-random type cellulase. 1 53

A 37,000 X g supernatant fraction prepared from fat lung homogenate demonstrated a 2- to 3-fold increase in guanylate cyclase activity after incubation at 30 degrees for 30 min (preincubation). Treatment of the supernatant fraction with Triton X-100 increased activity to approximately the same extent as preincubation, but would not increase the activity after preincubation. By chromatography on Sepharose 2B, before and after preincubation, it was demonstrated that the increase in activity was only associated with the soluble guanylate cyclase, and not the particulate enzyme. Activation by preincubation required O2. It was completely inhibited by thiols such as 2-mercaptoethanol, and by bovine serum albumin, KCN, and sodium diethyldithiocarbamate. These inhibitors suggested a copper requirement for activation, and this was confirmed by demonstrating that 20 to 60 muM CuCl2 could relieve the inhibition by 0.1 mM sodium diethyldithiocarbamate. 2-Mercaptoethanol inhibition could also be reversed by removal of the thiol on a Sephadex G-25 column, however, this treatment partially activated the enzyme. Addition of 2-mercaptoethanol to a preincubated preparation would not reverse the activation. H2O2 was found to activate guanylate cyclase, either by its generation in the lung supernatant with glucose oxidase and glucose, or by its addition to a preparation in which the catalase was inhibited with KCN. KCN or bovine serum albumin was able to partially inhibit activation by glucose oxidase plus glucose, however, larger amounts of glucose oxidase could overcome that inhibition, indicating a catalytic role for Cu2+ at low H2O2 concentrations. No direct evidence for H2O2 formation during preincubation could be found, however, indirect evidence was obtained by the spectrophotometric detection of choleglobin formation from hemoglobin present in the lung supernatant fluid. The H2O2 is believed to result from the reaction of oxyhemoglobin with ascorbate.
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PMID:Activation of soluble guanylate cyclase from rat lung by incubation or by hydrogen peroxide. 1 60

Placental aldose reductase (EC 1.1.1.21) was incubated with glucose in the presence of [4A-2H] NADPH prepared in the oxidation of [2-2H] isocitrate by isocitrate dehydrogenase (EC 1.1.1.42) or [4B-2H] NADPH prepared in the oxidation of [1-2H] glucose-6-phosphate dehydrogenase (EC 1.1.1.49). The sorbitol formed from [4A-2H] NADPH contained deuterium and from [4B-2H] NADPH it did not. Therefore, aldose reductase in an A-type enzyme.
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PMID:Stereospecificity of the hydrogen transfer catalyzed by human placental aldose reductase. 1 22

A method for serum or plasma cholesterol assay involving amperometric measurement of the rate of oxygen depletion in the cholesterol oxidase-catalyzed oxidation of cholesterol is described. The hydrolysis of the serum cholesterol esters is accomplished by saponification of 50 mul of sample with 0.2 ml of ethanolic KOH (1.0 mol/1) containing 0.5% Triton X-100 for 5 min at 75 degrees C. The rate of oxygen consumption in a 25-mul aliquot of this is measured with a Clark electrode in a Beckman Glucose Analyzer and the assay takes about one minute after incubation; results are read digitally on the instrument. The analyzer cell contains 1 ml of 1 M phosphate buffer, pH 7.4, with 100 mg sodium cholate/100 ml and 0.1-0.2 U cholesterol oxidase.
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PMID:Enzymatic assay of total cholesterol in serum or plasma by amperometric measurement of rate of oxygen depletion following saponification. 1 91

The alpha-L-fucosidase from rat liver lysosomes was purified approximately 27,000-fold (from cytoplasmic extract) by a rapid procedure requiring only 7 h anf providing enzyme in a 20 per cent yield. The procedure is based upon affinity chromatography with agarose-epsilon-aminocaproyl-fucosamine. The isolated enzyme was found to be pure by a number of different analytical gel techniques and is essentially free of other lysosomal gylcosidases. The purified enzyme exhibits a positive periodic acid-Schiff stain, suggesting that it is a glycoprotein. The purified enzyme has a pH optimum of 5.7 to 5.9, a Vmax of 27 mumol/min/mg of protein, and a Km of 0.19 mM with p-nitrophenyl alpha-L-fucopyranoside as substrate. L-Fucose was the only possibly physiological effector of the enzyme which was identified; it exhibited a Ki of 1.6 mM, with p-nitrophenyl alpha-L-fucopyranoside as substrate. The enzyme has a subunit molecular weight of approximately 55,000 by Na dodecyl-SO4 electrophoresis in a variety of gel systems. The molecular weight of the native enzyme was indicated to be approximately 160,000 by sucrose density centrifugation, 300,000 by molecular sieve chromatography on Sephadex G-200, and 217,000 by sedimentation equilibrium centrifugation. The weight of evidence suggests that the enzyme is a tetramer. Incubation on the absence of sulfhydryl reagents under appropriate conditions generates a second alpha-L-fucosidase activity band on gels corresponding to a molecular weight of approximately 40,000 to 50,000. This result suggests that the subunit is relatively stable and may reassociate to form active enzyme. Alpha-L-Fucosidase requires a high concentration of protein and the presence of a sulfhydryl reagent for stabilization. It is rapidly inactivated by p-chloromercuriphenyl sulfonic acid, this inactivation being rapidly reversible by the addition of 10 mM 2-mercaptoethanol. The enzyme catalyzed the hydrolysis of 1 leads to 2, 1 leads to 3, and 1 leads to 4 fucosyl linkages and was found to be active on glycopeptides but not on native glycoproteins. The amino acid and carbohydrate composition of the enzyme was determined. The native enzyme contains the following sugars (residues per tetramer): fucose (3.5), mannose (32), galactose (8), glucose (9), glucosamine (32), and sialic acid (8). Rat liver lysosomal alpha-glucosidase, also produced in the rapid isolation procedure described herein, contained less than 0.1 residue of sialic acid per subunit.
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PMID:The purification and characterization of rat liver lysosomal alpha-L-fucosidase. 1 77

Fatty acid synthesis was studied in testes of rats fed a fat-free or fat-supplemented diet. Testes of fat-deficient rats incorporated nearly twice as much intratesticularly injected [1-14C]acetate into total fatty acids (primarily into palmitic acid) as did supplemented rats. To determine the mechanism for the increased synthesis, the activities of the following enzymes were determined in the cytoplasmic fraction of testicular homogenates: fatty acid synthetase, acetyl CoA carboxylase [EC 6.4.1.2], citrate-cleavage [EC 4.1.3.8], malic [EC 1.1.1.38], and the glucose-l-phosphate dehydrogenase [EC 1.1.1.49]: 6-phosphogluconate dehydrogenase pair [EC 1.1.1.44]. Although the activity of fatty acid synthetase did increase in livers from fat-deficient rats, no change was observed in corresponding testes. No difference between the two groups could be demonstrated in testicular activity of citrate-cleavage enzyme, malic enzyme, or the glucose-6-phosphate dehydrogenase: 6-phosphogluconate dehydrogenase pair. However, the activity of cytoplasmic acetyl CoA carboxylase in testes of rats fed the fat-deficient diet was 1.4 times higher than the activity in testes of rats fed the supplemented diet. Fat deficiency did not affect the specific activity of the testicular microsomal elongation system, assayed by incubation with 14C-malonyl CoA. The concentration of unesterified fatty acids was lower in testes of the fat-deficient compared to supplemented rats, indicating that decreased inhibition of acetyl CoA carboxylase in the fat-deficient rats testes might have been responsible for the observed increased de novo synthesis of palmitic acid.
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PMID:Fatty acid synthesis in testes of fat-deficient and fat-supplemented rats. 1 68

By using D-glucose, D-xylose, D-galactose and D-fructose in the strictly aerobic yeast Rhodotorula glutinis and by comparing the half-saturation constants with inhibition constants the yeast was shown to possess a single common system for D-xylose and D-galactose (Km's and Ki's all between 0.5 and 1.1 mM) but another distinct transport system for D-fructose. The transport of D-glucose has a special position in that glucose blocks apparently allotopically all the other systems observed although it uses at least one of them for its own transport. The different character of D-glucose uptake is underlined by its relative independence of pH (its "Km" is completely pH-insensitive) in contrast with all other sugars. At low concentrations, all sugars show mutual positive cooperativity in uptake, suggesting at least two transport sites plus possibly a modifier site on the carrier.
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PMID:Monosaccharide transport systems in the yeast Rhodotorula glutinis. 1 56

Molar growth yields for anaerobic growth of Aerobacter aerogenes in complex medium were much higher than for growth in minimal medium. In batch cultures the molar growth yield for glucose varied from 44 to 50 and YATP from 17.1 to 18.8. For glucose-limited chemostat cultures a value of 17.5 g/mole was found for Y max ATP and a value of 2.3 mmoles ATP/g dry weight h for the maintenance coeficient. Growth-dependent pH changes were used to control the addition of fresh medium, containing excess of glucose to a continuous culture. The specific growth rate and the population density were dependent on the pH difference between the inflowing medium and the culture. At a mu value of 1.44 h-1 the molar growth yield for glucose was about 70 and Y ATP about 28.5. An equation is presented, which gives the relation between theoretical and experimental Y max ATP values.
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PMID:Energetic aspects of anaerobic growth of Aerobacter aerogenes in complex medium. 1 57

Glycogen debranching enzyme (4-alpha-glucanotransferase amylo-1,6-glucosidase, EC 2.4.1.25 + 3.2.1.33) was purified 140-fold from dogfish muscle in a rapid, high-yield procedure that takes advantage of a strong binding of the enzyme to glycogen, and its quantitative adsorption to concanavalin A-Sepharose only when the polysaccharide is present. The final product was hrophoresis in the presence and absence of dodecyl sulfate. A molecular weight of 162,000 +/- 5000 was determined by sedimentation equilibrium analysis in good agreement with the value of 160,000 estimated by gel electrophoresis, but a low-sedimentation constant of 6.5 S suggests that the enzyme is asymmetric. The molecule appears to be made up of a single polypeptide chain with no evidence for multiple repeating sequences: it could not be dissociated into smaller fragments by dodecyl sulfate even after complete carboxymethylation; tryptic cleavage of the native protein yielded only two fragments of molecular weight 20,000 and 140,000 without loss of enzymatic activity. The amino acid composition of the enzyme is reported; no covalently bound phosphate or carbohydrate could be detected. All 32 sulfhydryl groups present were titrated with 5,5'-dithiobis(2-nitrobenzoic acid) under denaturing conditions; eight reacted readily in the native enzyme without loss of catalytic activity, while substitution of eight additional ones lowered the activity by 50%. Inactivation was greatly reduced by glycogen; the polysaccharide also influenced markedly the electrophoretic behavior of the enzyme and large filamentous aggregates were formed when solutions of both were mixed. Purified debranching enzyme releases 3 mumol of glucose min-1 mg-1 at 19 degrees C, pH 6.0, from a glycogen limit dextrin and one-tenth this amount when the native polysaccharide is used as substrate; glycogen is quantitatively degraded in the presence of phosphorylase. None of the usual sugar phosphates or nucleotide effectors of glycolysis affected enzymatic activity. No phosphorylation by either dogfish or rabbit skeletal muscle protein kinase or phosphorylase kinase could be demonstrated, nor any direct interaction with phosphorylase as measured by SH-group reactivity, enzymatic activity, or rate of phosphorylase b to a conversion. Purification of the 160,000 molecular weight M-line protein of skeletal muscle resulted in the quantitative removal of debranching enzyme, indicating that the two proteins are different.
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PMID:Purification and properties of debranching enzyme from dogfish muscle. 1 9

1. Glucokinase (ATP : D-glucose 6-phosphotransferase, EC 2.7.1.2) was extracted from pea seeds and purified by fractionation with (NH4)2SO4 and chromatography on DEAE-cellulose and Sephadex. 2. The relative rates of phosphorylation of glucose, mannose and fructose (final concentration 5 mM) were 100, 64 and 11. 3. The Km for glucose of pea-seed glucokinase was 70 muM and the Km for mannose was 0.5 mM. The Km for fructose was much higher (30 mM). 4. Mg2+ ions were essential for activity. Mn2+ could partially replace Mg2+. 5. Enzyme activity was not inhibited by glucose 6-phosphate. A number of other metabolites had no effect on glucokinase activity. 6. Pea-seed glucokinase was inhibited by relatively low concentrations of ADP.
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PMID:Glucokinase of pea seeds. 1 40


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