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1.
The ability of a microsomal enzyme, glucose dehydrogenase (hexose 6-phosphate dehydrogenease) to supply NADPH to the microsomal electron transport system, was investigated. Microsomes could perform oxidative demethylation of aminopyrine using microsomal glucose dehydrogenase in situ as an NADPH generator. This demethylation reaction had apparent Km values of 2.61 X 10(-5) M for NADP+, 4.93 X 10(-5) m for glucose 6-phosphate, and 2.14 X 10(-4) m for 2-deoxyglucose 6-phosphate, a synthetic substrate for glucose dehydrogenase. Phenobarbital treatment enhanced this demethylation activity more markedly than glucose dehydrogenase activity itself. Latent activity of glucose dehydrogenase in intact microsomes could be detected by using inhibitors of microsomal electron transport, i.e. carbon monoxide and p-chloromercuribenzoate (PCMB), and under anaerobic conditions. These observations indicate that in microsomes the NADPH generated by glucose dehydrogenase is immediately oxidized by NADPH-cytochrome c reductase, and that glucose dehydrogenase may be functioning to supply NADPH. 相似文献
2.
Our previous work has shown that phenyl phosphate acts as an exogenous substrate for GDP-mannose:dolichyl phosphate mannosyltransferase in rat liver microsomal fractions to give rise to phenyl phosphate beta-D-mannose, a compound which, unlike Dol-P-Man (dolichyl phosphate beta-D-mannose), cannot act as mannose donor for further mannose-adding reactions in microsomal fractions. The study has now been extended to the action of various aryl phosphates and structurally related compounds on several other glycosyltransferase systems in the microsomal fractions. (1) Examination of the ability of these compounds to accept sugars from various sugar nucleotides indicated that the individual compounds have specificity as sugar acceptors. Thus phenyl phosphate acted as an effective acceptor for both mannose and glucose, whereas benzenephosphonic acid was active only in accepting mannose. p-Nitrophenyl phosphate was a more effective glucose acceptor than phenyl phosphate, but had only 8% of the mannose-accepting activity of phenyl phosphate. (2) Phenyl phosphate had an inhibitory effect on the transfer of mannose form GDP-mannose to lipid-linked oligosaccharides and to glycoproteins in rat liver microsomal fractions. The inhibition depended on the concentration of phenyl phosphate and on the extent of inhibition of Dol-P-Man synthesis. It is proposed that phenyl phosphate has a direct effect on the synthesis of Dol-P-Man and that its inhibition of synthesis of lipid-linked oligosaccharides and glycoproteins could be a consequence of this effect. 相似文献
3.
An oxidative metabolism of glucose 6-phosphate was studied in rat liver microsomal fraction. Although radioactive 14CO2 was formed from [1-14C]glucose 6-phosphate in the microsomal fraction (Hino, Y., and Minakami, S. (1982) J. Biochem. (Tokyo) 92, 547-557), the formation was negligible when [2-14C]glucose 6-phosphate was used as a starting substrate. These results indicated an inability of the microsomal fraction to rearrange [2-14C]glucose 6-phosphate to form [1-14C] glucose 6-phosphate, and it was expected that a certain compound derived from glucose 6-phosphate accumulated as an end-product of the reaction. We, therefore, have tried to identify the product by high performance liquid chromatography, and found that ribose accumulated as the end-product. The formation of ribose was inhibited in the same manner as that of 14CO2 by antibodies against rat liver microsomal hexose-6-phosphate dehydrogenase, and the ratios of ribose to 14CO2 formed in the reaction were 0.5-0.8 on a molar basis. The finding of ribose formation further suggested the involvement of ribose phosphate isomerase and phosphatase activities in the reaction. 相似文献
4.
Differences in distribution of esterase between cell fractions of rat liver homogenates prepared in various media. Relevance to the lysosomal location of the enzyme in the intact cell 总被引:1,自引:0,他引:1
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The distribution of esterase in subcellular fractions of rat liver homogenates was compared with that of the lysosomal enzyme acid phosphatase and the microsomal enzyme glucose 6-phosphatase. Most of the esterase from sucrose homogenate sediments with glucose 6-phosphatase and about 8% is recovered in the supernatant. However, up to 53% of the esterase can be washed from microtome sections of unfixed liver, in which less cellular damage would be expected than that caused by homogenization. About 40% of both esterase and acid phosphatase are recovered in the soluble fraction after homogenization in aqueous glycerol or in a two-phase system (Arcton 113-0.25m-sucrose), although glucose 6-phosphatase is still recovered in the microsomal fraction of such homogenates. The esterase of the microsomal fraction prepared from a sucrose homogenate is much more readily released by treatment with 0.26% deoxycholate than are other constituents of this fraction. The release of esterase from the microsomal fraction by the detergent and its concomitant release with acid phosphatase after homogenization in glycerol or the two-phase system suggests that a greater proportion of esterase may be present in lysosomes of the intact cell than is indicated by the results of standard fractionation procedures. 相似文献
5.
The cholic acid CoA ligase activity of rat liver was quantitatively inactivated by proteolysis with pronase, chymotrypsin, subtilisin, or proteinase K in intact microsomal vesicles. Under the conditions employed, less than 14% of the lumenal mannose-6-phosphate phosphatase activity was lost, and the mannose-6-phosphate phosphatase activity remained highly latent. After microsomal integrity was disrupted with sodium deoxycholate, protease treatment resulted in a loss of greater than 74% of the mannose-6-phosphate phosphatase activity. Cholic acid CoA ligase activity was unaffected by preincubation of microsomes with sodium taurocholate under conditions that led to the complete expression of latent mannose-6-phosphate phosphatase activity. The data suggest that cholic acid CoA ligase activity is located on the cytoplasmic surface of hepatic microsomal vesicles. 相似文献
6.
Studies on the fractionation of mucosal homogenates from the small intestine 总被引:50,自引:41,他引:9
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1. Homogenates of the mucosa of the small intestine of the guinea pig were separated by fractional sedimentation into seven different fractions. The enzymic properties of some of these subcellular fractions were compared with those obtained from the mucosa of the small intestine of the rabbit and cat. 2. The enzymic properties of the low-speed sediment (15000g-min.) were investigated and it was shown that invertase and alkaline ribonuclease were predominantly located in this subcellular fraction, whereas alkaline phosphatase, aryl-amidase, acid phosphatase, acid ribonuclease and phosphoprotein phosphatase, though true constituents of this fraction, occurred to varying degrees in other subcellular structures also. 3. It was shown that the most probable source of the enzymic activities observed in the low-speed sediment was the brush border. Electron micrographs of the purified brush-border fraction indicated vesicles derived from the brush-border membrane. 4. A method is described for the fractionation of mucosal homogenates into a brush border-plus-nuclei fraction, a mitochondrial fraction, a microsomal fraction and a particle-free supernatant. The fractions were shown to be relatively pure, as indicated by the distribution of invertase, DNA, succinate dehydrogenase, glucose 6-phosphatase and 6-phosphogluconate dehydrogenase. 5. Most of the activity of four lysosomal enzymes present in the nuclei-free homogenate was sedimented at 375000g-min., suggesting the occurrence of lysosomal particles in mucosal homogenates. 6. Further fractionation of the microsomal membranes into three fractions is described. The enzymic composition of the membrane fractions is given and discussed in relation to their structure as seen in electron micrographs. 相似文献
7.
C J Fernandez M Lacort J M Gandarias B Ochoa 《Biochemical and biophysical research communications》1987,146(3):1212-1217
Lateral and transversal distribution of cholesterol ester hydrolase activity in rat liver microsomal membranes has been studied. Total cholesterol ester hydrolase activity was found predominantly (75%) in rough microsomes though specific esterase activities were similar in rough and smooth microsomal fractions. The transversal asymmetry of the enzyme was examined using the criteria of protease sensitivity and latency of mannose-6-phosphate phosphatase. Cholesterol ester hydrolase resulted drastically inhibited by proteolysis with trypsin when microsomal integrity had been previously disrupted with sodium deoxycholate or sodium taurocholate. Under these conditions, most lumenal mannose-6-phosphate phosphatase activity was destroyed. However, cholesterol esterase was unaffected by preincubating microsomes with the detergent alone, which led to the complete expression of latent mannose-6-phosphate phosphatase or by preincubating them with trypsin, where less than a 15% of the lumenal mannose-6-phosphate phosphatase was lost. These findings suggest that cholesterol ester hydrolase activity is located on the lumenal surface of the hepatic microsomal vesicles. 相似文献
8.
Physiological function of periplasmic hexose phosphatase in Salmonella typhimurium. 总被引:3,自引:3,他引:0
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Hydrolysis of sugar phosphates by crude and purified preparations of periplasmic hexose phosphatase from Salmonella typhimurium followed Michaelis-Menten kinetics. The enzyme bound glucose 1-phosphate with high affinity (Km = 10 microM) but bound glucose 6-phosphate with low affinity (Km = 2,000 microM). The order of substrate affinities was glucose 1-phosphate greater than mannose 1-phosphate = galactose 1-phosphate greater than fructose 1-phosphate greater than glucose 6-phosphate. These results and others suggest that the physiological function of the enzyme is the periplasmic hydrolysis of hexose 1-phosphates. 相似文献
9.
The levels of nicotinamide nucleotides in liver microsomes and their possible significance to the function of hexose phosphate dehydrogenase. 总被引:1,自引:0,他引:1
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The concentrations of NAD and NADP have been determined in detergent extracts of washed rat liver microsomes. Precautions were taken during the preparation of the microsomes to remove nicotinamide nucleotides from their external surface both by hydrolysis by nucleotide pyrophosphatase (EC 3.6.1.9) and by washing them three times in 0.15 M-Tris/HCl, pH 8.0, to remove soluble proteins which bind these nucleotides. The mannose phosphatase was essentially completely latent, indicating that the microsomes were intact. Assuming these nucleotides are in the cisternae of the microsomes, the concentrations in the cisternae are 240 +/- 25 microM-NAD and 55 +/- 12 microM-NADP. These levels of nucleotides are compatible with both the glucose:NAD+ and the glucose 6-phosphate:NADP+ oxidoreductase activities of hexose phosphate dehydrogenase (EC 1.1.1.47). Since the organ and subcellular distributions of this dehydrogenase and glucose-6-phosphatase are similar, and Pi stimulates the glucose:NAD+ oxidoreductase activity, it is proposed that the combined action of these two enzymes leads to the reduction of both coenzymes in the lumen of the endoplasmic reticulum. A modification of the colorimetric method of Nisselbaum & Green [(1969) Anal. Biochem. 27, 212-217] for the determination of NADP+ is described. Colour formation is linear with the concentration of NADP+ and is sensitive to less than 0.3 nmol of NADP+. 相似文献
10.
E R Lax E Kreuzfelder H Schriefers 《Hoppe-Seyler's Zeitschrift für physiologische Chemie》1979,360(12):1807-1817
This investigation was undertaken to elucidate the amount of oestradiol and duration of its administration necessary to cause complete feminization of the activities of cytoplasmic 3 alpha- and 17 beta-hydroxysteroid dehydrogenase, microsomal 3 alpha- and 3 beta-hydroxysteroid dehydrogenase and microsomal 5 alpha-reductase in male rat liver. With the exception of cytoplasmic 3 alpha-hydroxysteroid dehydrogenase, 5 microgram oestradiol/d for 8 days and less was sufficient to cause complete feminization. The order of oestrogen sensitivity was cytoplasmic 3 alpha-hydroxysteroid dehydrogenase greater than microsomal 3 beta-hydroxysteroid dehydrogenase greater than microsomal 3 alpha-hydroxysteroid dehydrogenase greater than microsomal 5 alpha-reductase greater than cytoplasmic 17 beta-hydroxysteroid dehydrogenase. Although the changes occurring after oestradiol administration are qualitatively the same as after testectomy, they occur more rapidly. This rules out the possibility that oestradiol exerts its effect via androgen deprivation. Diethylstilboestrol administration causes the same changes in cytoplasmic 17 beta- and microsomal 3 beta-hydroxysteroid dehydrogenase activity as oestradiol, although the dosage must be increased 100 fold. The effect of diethylstilboestrol on 5 alpha-reductase activity changes with the dose applied. Doses up to 100 microgram/d partially feminize the activity, but at higher doses the enzyme activity is repressed. 相似文献
11.
产甘油假丝酵母甘油代谢关键酶的研究 总被引:15,自引:2,他引:15
本文对产甘油假丝酵母的甘油代谢关键酶进行了研究,发现产甘油假丝酵母同化甘油能力极弱,少量葡萄糖明显改善其同化甘油的能力;线粒体3磷酸甘油脱氢酶受3磷酸甘油的强烈诱导,受葡萄糖代谢的阻遏。在甘油发酵过程中,产甘油假丝酵母胞浆3磷酸甘油脱氢酶酶活处于较高水平并在36h和60h时出现两次酶活高峰,其中第一次酶活峰值水平决定产甘油假丝酵母的甘油合成和积累水平,成为甘油高速积累期(18~48h)甘油合成的关键性的限速酶。在甘油发酵18~48h内,3磷酸甘油酯酶的酶活处于高水平,并在36h时出现酶活峰值;处于缓慢甘油积累阶段的48~72h间,3磷酸甘油酯酶已处于低水平表达,此时,3磷酸甘油酯酶则成为甘油合成的限速酶。产甘油假丝酵母稳定并高表达其胞浆3磷酸甘油脱氢酶基因并且其所表达的3磷酸甘油酯酶酶活远高于胞浆3磷酸甘油脱氢酶这一特征是其高产甘油根本所在。 相似文献
12.
The transfer of mannose to dolichol diphosphate oligosaccharides in pig liver endoplasmic reticulum 总被引:4,自引:0,他引:4
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The transfer, catalysed by pig liver microsomal preparations, of mannose, from GDP-mannose, to lipid-linked oligosaccharides and the properties of the products are described. Solubility, hydrolytic and chromatographic data suggest that they are dolichol diphosphate derivatives. The presence of two N-acetyl groups in at least part of the heterogenous oligosaccharide portion was tentatively deduced. Reduction with borohydride of the oligosaccharide showed that the newly added mannose residues were not at its reducing end. Periodate oxidation suggested that 60% of these were at the non-reducing terminus and that 40% were positioned internally. T.l.c. showed the presence of seven oligosaccharide fractions with chromatographic mobilities corresponding to glucose oligomers with 7-13 residues. The molar proportions of the oligosaccharide fractions in the mixture were determined by borotritiide reduction and the number of mannose residues added to each oligosaccharide fraction during the incubation was calculated. Two of the oligosaccharide fractions had received on average one, or slightly more than one, mannose residue per chain during the incubation; four of the other fractions were each shown to be a mixture, 20-25% of which had received one mannose residue during the incubation and 75-80% of which had not been mannosylated during the incubation. This supported other evidence for the presence of endogenous lipid-linked oligosaccharides in the microsomal preparation which had been formed before the incubation in vitro. Evidence for the possibility of two pools of dolichol monophosphate mannose, one being more closely associated with mannosyl transfer to dolichol diphosphate oligosaccharides than the other, is also discussed. 相似文献
13.
Eric G. Brunngraber Leonard G. Davis Chandrasiri W. Somawardhana 《Neurochemical research》1982,7(10):1243-1256
Glycoproteins that contain phosphohexosyl groups were found to be present in the myelin- and synaptosomal-enriched fractions as well as in the microsomes of rat brain. The kinetics of flow of intraperitoneally injected [32P]phosphate suggests that the phosphate is enzymatically added in structures found in the microsomal fraction. The newly synthesized phosphoglycoproteins then appear in the soluble fraction of the synaptosomes and in the cytosol, prior to incorporation into the membranes of the synaptosomes and myelin. Phosphoglycopeptides recovered from the phosphoglycoprotein contain 3 Mannose units per N-acetylglucosamine residue; one of the mannose residues is phosphorylated. [13C]NMR studies indicate that the phosphoglycopeptides contain a chitobiose group and more than four sugar residues. Thus, the phosphomannoglycopeptides from rat brain contain an average of 2 N-acetylglucosamine, 6 mannose, and two phosphate moieties per oligosaccharide chain. Enzymatic treatment with -mannosidase failed to remove the phosphomannose, although some mannose residues were released. Thus, the phosphorylated mannose is not removed by the glycosidase and terminal nonphosphorylated mannose residues are present in the oligosaccharide. The phosphate residues are removed by treatment with alkaline phosphatase. 相似文献
14.
1. Microsomal fractions of lactating rabbit mammary gland incubated with UDP-glucose formed lipid-linked mono- and oligo-saccharides. The lipid-linked monosaccharide had chromatographic properties similar to those of dolichol phosphate mannose and yielded glucose on acid hydrolysis. 2. Incubation of the microsomal fraction with GDP-[U14C]-mannose yielded an oligosaccharide lipid of approximately seven monosaccharide units. Further incubation with UDP-glucose increased the size of the oligosaccharide by approximately two units. 3. Explants of lactating rabbit mammary gland incorporated [U-14C]glucose into both lipid-linked mono- and oligo-saccharides. The oligosaccharide lipid was of approx. 11 monosaccharide units. 4. Considerable redistribution of radioactive label occurred in the explant system, and radioactively labelled glucosamine and mannose, as well as glucose, were detected on acid hydrolysis of the oligosaccharide lipid. 5. Glucose was also detected in the acid hydrolysate of explant proteins. Radioactive glucosamine, galactosamine, galactose and mannose were also found in this fraction. 相似文献
15.
G M Stephenson 《Archives of biochemistry and biophysics》1991,289(2):324-328
Rat liver microsomes contain type-1 S6 phosphatase (acting on the serine residues phosphorylated by protein kinase A) and type-1 phosphorylase phosphatase activities. The main aim of this study has been to characterize the microsomal S6 phosphatase activity and to compare its properties with those of the phosphorylase phosphatase activity in the same microsomal preparation. The specific activities of both microsomal S6 phosphatase and phosphorylase phosphatase were 1.6- to 1.7-fold higher in the smooth endoplasmic reticulum than in the rough sarcoplasmic reticulum. Both phosphatase activities were inhibited to a similar extent by MgCl2 (10 mM) and NaF (22 mM), were completely suppressed by glycerophosphate (80 mM) and ZnCl2(10 mM), and were stimulated by MnCl2(1 mM). When analyzed by gel filtration on Sephadex G-100 superfine, both phosphatase activities eluted as broad peaks, stretching from the void volume to 45-60 kDa. The microsomal S6 phosphatase and phosphorylase phosphatase activities also displayed the following distinct characteristics: (a) Mn2+ stimulated the S6 phosphatase activity 2.9-fold more than the phosphorylase phosphatase activity, (b) limited trypsin digestion of microsomal preparations increased the phosphorylase phosphatase activity by 1.5- to 2-fold, but decreased the S6 phosphatase activity by 50%, (c) a synthetic peptide analog of S6 (S6229-239) (200 microM), which did not act as a substrate for the microsomal S6 phosphatase and did not affect its activity, inhibited the microsomal phosphorylase phosphatase activity by about 50%, and (d) the elution profile of the phosphorylase phosphatase activity was markedly broader than that of the S6 phosphatase activity. A series of in vivo studies showed that streptozotocin-diabetes and insulin replacement therapy as well as ip injection of insulin or vanadate, which modified the microsomal S6 phosphatase activity, had no statistically significant effects on the microsomal phosphorylase phosphatase activity. Taken together, these results suggest that the microsomal S6 phosphatase and phosphorylase phosphatase activities are due to two distinct enzyme populations. 相似文献
16.
Studies on glycogen synthesis in pigeon liver homogenates. Incorporation of hexose into glycogen 总被引:2,自引:1,他引:1
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Liver homogenates of avian species, but not of mammals, form glycogen from glucose, mannose, fructose and galactose. Incorporation of labelled glucose, fructose and mannose, but not of labelled galactose, into glycogen is diluted isotopically by unlabelled glucose. Except for fructose, glycogen formation from other substrates by pigeon liver homogenates compares favourably with that from the same substrates in pigeon liver slices. Optimum conditions for glycogen synthesis from glucose by pigeon liver homogenate are: medium of incubation, 0.175m-sucrose-45mm-potassium chloride-15mm-glycylglycine buffer, pH7.5; concentration of substrate, 15mm; concentration of tissue, less than 120mg./ml.; temperature of incubation, 37-43 degrees ; atmosphere, oxygen. Uncouplers of oxidative phosphorylation, Ca(2+), EDTA, PP(i), 2-deoxyglucose 6-phosphate and microsomal fraction of rat liver are inhibitory to glycogen synthesis from glucose. Starvation of pigeons for 24 and 48hr. leads to a slight stimulation of glycogen synthesis in their liver homogenates as compared with fed controls. Pigeon liver homogenates can be separated into subcellular fractions that on reconstitution can synthesize glycogen. All the enzymes of the glycogen pathway except soluble high-K(m) glucokinase are present in pigeon liver. 相似文献
17.
1. Glutamate dehydrogenase and malate dehydrogenase solubilized from liver microsomes were able to rebind to microsomal vesicles while the corresponding dehydrogenases extracted from mitochondria showed no affinity for microsomes. 2. Competition was noticed between microsomal glutamate dehydrogenase and microsomal malate dehydrogenase in the binding to microsomal membranes. Mitochondrial malate dehydrogenase or bovine serum albumin did not inhibit the binding of microsomal glutamate dehydrogenase to microsomes. 3. Binding of microsomal glutamate dehydrogenase to microsomal membranes decreased when microsomes was preincubated with trypsin. 4. Rough microsomal glutamate dehydrogenase was more efficiently bound to rough microsomes than smooth microsomes. Conversely, smooth microsomal glutamate dehydrogenase had higher affinity for smooth microsomes than for rough microsomes. 5. A difference was noticed among the glutamate dehydrogenase isolated from rough and smooth microsomes, and from mitochondria, which suggested the possibility of minor post-translational modification of enzyme molecules in the transport from the site of synthesis to mitochondria. 相似文献
18.
Summary P-Chloromercuribenzoate alters various reactions of rat liver glucose (hexose phosphate) dehydrogenase differently. The reagent has little effect on the glucose: NAD or the glucose: NADP oxidoreductases, doubles the rates of oxidations of galactose-6-phosphate and glucose-6-phosphate by NADP and greatly stimulates the oxidations of glucose-6-phosphate and galactose-6-phosphate by NAD. The reagent appears to react with a sulfhydryl group of the enzyme since activation is reversed and prevented by mercaptoethanol. The direct reaction of the reagent with the enzyme is indicated by its lower thermal stability in the presence of the p-chloromercuribenzoate. The size of the enzyme appears to be the same when determined by sucrose gradient centrifugation in the presence or absence of p-chloromercuribenzoate. In microsomes, the oxidation of NADH or NADPH hampers measurements of glucose dehydrogenase. Since p-chloromercuribenzoate inhibits microsomal oxidation of reduced nicontinamide nucleotides, it is possible to assay for glucose dehydrogenase accurately in the presence of the mercurial in microsomes and microsomal extracts and thus measure the effectiveness of a detergent in extracting the enzyme from microsomes.Abbreviation pcMB
p-chloromercuribenzoic acid 相似文献
19.
Male mice were castrated at 2 months of age and a pellet of testosterone propionate was implanted subcutaneously ten days later. After 14 days, normal, castrated and androgen treated mice were autopsied simultaneously. The kidneys of each group were homogenized, pooled and fractionated by centrifugation into the various subcellular components. The alkaline ribonuclease and phosphatase activities were localized in the microsomal fraction and changed in inverse relationship to the previously observed changes in the rate of protein biosynthesis and the concentration of microsomal and polysomal RNA induced by castration and androgen administration. Esterase activity also was localized in the microsomal fraction and its specific activity decreased after castration and was restored to normal by testosterone. The activities of acid ribonuclease, acid phosphatase and the several marker enzymes (glucose-6-phosphatase, nucleotidase, succinic dehydrogenase and glucose-6-phosphate dehydrogenase) changed in direct proportion to the changes in weight of the kidney after castration and androgen stimulation. 相似文献
20.
Glucose dehydrogenase of bovine heart: activity, purification and properties of the enzyme 总被引:2,自引:0,他引:2
K Brzek 《Acta biochimica Polonica》1987,34(4):431-440
The activity of glucose dehydrogenase (EC 1.1.1.47; GDH; glucose NAD(P) oxidoreductase) was demonstrated in the supernatant obtained after centrifugation (1500 g) of bovine heart homogenate. More than 50% of GDH activity was found in the microsomal fraction. The optimum pH for the microsomal enzyme was 8.9. About 200-fold purification of GDH was achieved by successive application of ultracentrifugation in 0.25 M mannitol, treatment with solid ammonium sulphate, and CM-32 cellulose chromatography. The purified preparation oxidized not only glucose but also D-glucosamine, N-acetylglucosamine and xylose in 87, 63 and 23%, respectively. Purified GDH was inhibited by p-chloromercuribenzoate and glucose 6-phosphate. 相似文献