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1.
Hypoxanthine phosphoribosyltransferase (EC 2.4.2.8) of a strain of Streptomyces cyanogenus was purified 1,900-fold to an apparent homogenity from cell-free extracts. The enzyme had a molecular weight of 150,000 and consisted of eight identical subunits with a molecular weight of 18,000. The isoelectric point was at pH 4.4. The enzyme required Mg2+ or Ma2+ for activity and had a pH optimum at 8.5. Hypoxanthine and guanine were good substrates for the enzyme. Xanthine was a very poor substrate and adenine was not a substrate. Apparent Km values of the enzyme for hypoxanthine, guanine and 5-phosphoribose-1-pyro-phosphate were 1.6 × 10?8, 2.7 × 10?6 and 6.3 × 10?5 m, respectively. All purine nucleotides tested inhibited the activity significantly, apparently by competing with 5-phosphoribose-1-pyrophosphate.  相似文献   

2.
15-Hydroxyprostaglandin dehydrogenase from bovine lung was purified 7.4 times to a specific activity of 1.4 mU/mg of protein. The isoelectric point was estimated to 5.4 and the molecular weight by gelfiltration to 40,000. Km for prostaglandin E1 and for NAD+ were found to be 3.4 μM and 1.1 × 10?4M respectively. The enzyme was inhibited by indomethacin and aspirin. The indomethacin inhibition was found to be non-competitive to prostaglandin E1 having a Ki=1.4 × 10?4M and a Ki=1.6 × 10?5M.  相似文献   

3.
Heparin fractions of different molecular weights and anticoagulant activities were prepared by chromatography on protamine-Sepharose, and the association constants and stoichiometry for binding to antithrombin III were determined by measurement of enhancement of tryptophan fluorescence. A 7,900 molecular weight heparin preparation bound to antithrombin III with a stoichiometry of close to 2:1, whereas 14,300 and 21,600 molecular weight fractions bound at approximately 1:1 with the protein. Apparent association constants were 0.66 × 106 M?1 for the low molecular weight preparation and 2.89 × 106 M?1 for the high molecular weight material. Maximal fluorescence enhancement was greater with the higher molecular weight heparin. These results suggest a model of heparin-antithrombin III binding in which two sites on antithrombin III can accommodate one large heparin molecule with high affinity or two smaller molecules with low affinity.  相似文献   

4.
Abstract

Biliverdin reductase was purified from cow spleen. The specific activity of the final enzyme preparation was 24.01 u/mg, representing 686-fold purification as measured with NADPH. The yield was 3 grams of enzyme per 100 grams of cow spleen. The purified enzyme was a monomeric protein with an apparent molecular weight of about 34,000 and an isoelectric point of about 6.2. The biliverdin reductase was specific for biliverdin and reduced IXα faster than the biliverdin isomers IXβ, IXr, or IXδ. The purified enzyme could utilize both NADH and NADPH, but the kinectic properties of the NADH-dependent and the NADPH-dependent enzyme activities were different: the time course of the NADPH-dependent reaction displayed a sigmoidal curve, whereas that of the NADH-dependent reaction did not. Km for biliverdin IXα was 4 × 10?4 mM in the NADPH system, while it was 1.5 × 10?3 mM in the NADH system. Both enzyme activities were inhibited by excess biliverdin, but the inhibition of the NADPH-dependent enzyme activity was more pronounced. The pH optimum was 7.0 with NADH, and 6.8 with NADPH.  相似文献   

5.
Further support for hypotheses proposed earlier for the genetic control and subunit composition of the alcohol dehydrogenase of Triticum has been obtained through the purification and partial characterization of the enzyme. The alcohol dehydrogenase of the wheat T. monococcum was purified 103-fold to a specific activity of 55,900 units/mg. Purification was achieved using streptomycin sulfate precipitation, gel filtration chromatography, DEAE-cellulose anion-exchange chromatography, and preparative isoelectric focusing. The native enzyme has a molecular weight of 116,000 and a dimeric subunit structure. The apparent Michaelis constants are 1.2 × 10?2m for ethanol and 1 × 10?4m for NAD. The substrate specificity of wheat alcohol dehydrogenase differs significantly from the substrate specificities of the enzymes of horse and yeast.  相似文献   

6.
The two most common variants of placental alkaline phosphatase, the F and S variants, were purified to homogeneity and characterized. Their molecular weights were determined by equilibrium ultracentrifugation and sodium dodecylsulfate polyacrylamide gel electrophoresis, which gave almost identical values for the two variants, 118,000 (F) and 119,000 (S). The amino acid compositions of the F and S variants presented here are found to be very similar. Differences between the two variants were found in specific activity (160 U/mg for F and 250 U/mg for S), isoelectric point (IP=4.5 for F and 4.7 for S), sedimentation coefficient (6.5×10?13 sec for F and 6.4×10?13 sec for S). Thus the structural differences observed for these enzyme variants seem to affect both the active site and the protein conformation.  相似文献   

7.
The significant betaine aldehyde dehydrogenase activity was found in the cells of Pseudomonas aeruginosa A-16. The enzyme was inducibly formed and accumulated in the presence of choline, acetylcholine or betaine in the medium. The enzyme was purified approximately 620-fold with an overall recovery of 2.6% and proved to be homogeneous by ultracentrifugation. The molecular weight of the enzyme was determined as approximately 145,000 by gel filtration method. The enzyme had an isoelectric point around pH 5.1. The enzyme was quite specific for its substrate, betaine aldehyde. Both NADP and NAD functioned as coenzyme. The estimated values of Km at pH 7.4 and 25°C were 3.8 × 10?4 m for betaine aldehyde, 8.9 × 10?5 m for NADP and 2.2 × 10?4 m for NAD.  相似文献   

8.
Insulin and glucagon degradation by rat kidney homogenates and subcellular fractions was examined under a variety of conditions including high and low substrate concentrations, at pH 4 and pH 7, with and without glutathione. At high insulin concentration (4.1 · 10?5 M) insulin degradation by the homogenate was greatest at pH 4 but at low insulin concentration (1 · 10?10 M) insulin degradation was greatest at pH 7. At either high or low glucagon concentration glucagon degradation by the homogenate was greatest at pH 7. Glutathione at pH 7 stimulated insulin degradation at high insulin concentrations and inhibited insulin degradation at low concentrations. Glucagon degradation at pH 7 was inhibited at both high and low concentrations of glucagon by glutathione.Separation of kidney into cortex and medulla prior to homogenation produced a pattern of insulin and glucagon degradation identical to the whole homogenate but glucagon degradation by the medulla was greater than by the cortex.Examination of degradation by subcellular fractions revealed that at high concentration at neutral pH most insulin was degraded by the 100 000 × g pellet but at low insulin concentrations over 90% of the activity was in the 100 000 × g supernatant. At pH 7, at both high and low concentrations, most glucagon-degrading activity was in the 100 000 × g pellet, although the cytosol also had activity. At pH 4 most degradation occurred in the lysosomal fractions.Separation into cortex and medulla again showed similar distribution of activity as the whole gland with the medulla having more glucagon-degrading activity than the cortex. With low insulin concentrations the cortex 100 000 × g supernatant had higher relative specific activities than the medulla supernatant.Examination of recoveries of enzyme activity revealed that the subcellular fractions consistently had markedly less insulin-degrading activity than the original homogenate. This loss of activity was only discernible when insulin degradation was performed at pH 7 at low substrate concentrations. Comparable losses of glucagon-degrading activity were not seen.  相似文献   

9.
Intact adrenocortical cells possess cyclic nucleotide-independent protein kinase activity which is capable of phosphorylating endogenous proteins and casein when incubated in the presence of [γ-32P]ATP. The cyclic nucleotide-independent enzyme was dependent on cell number and temperature and had an apparent Km for ATP of 6.5 × 10?5 M and a Vmax of 12.5 pmol/3 min/2 × 105 cells at 37°C. Phosphorylation of endogenous proteins by this kinase was increased by treatment of intact cells with corticotropin (2.2 nM) for 24 h. In control cells, two endogenous proteins of apparent molecular weights of 39,000 and 76,000 were phosphorylated. In corticotropin-treated cells, another protein of apparent molecular weight of 87,000 was also phosphorylated. Thus, this protein kinase activity, which appears to be located on the plasma membrane, may be involved in mediating longer term actions of corticotropin on the adrenal cortex.  相似文献   

10.
Abstract— –Enzymic transformation of [4-14C]dehydroepiandrosterone or [4-14C]dehydro-epiandrosterone sulphate to androstenediol or its sulphate occurred when incubated with a microsomal preparation of rat brain or a whole rat blood homogenate. The brain enzyme which appeared to cause this transformation had a pH optimum at 60, was NADPH2-dependent, and had an apparent Km of 4·6 × 10?6m . When the subcellular fractions of rat brain were compared for transformation, microsomes had the highest specific activity, followed by the cytosol. The crude nuclear and mitochondrial fractions had no significant activity. The level of enzymic activity in the brain microsomes increased from that for rats sacrificed at 7 days of postnatal age to a maximum for rats sacrificed at 1 month of age; then the activity appeared to level off in rats older than 1 month. Microsomes obtained from the cerebellum had the highest specific activity in comparison to that obtained from the cerebral cortex, the diencephalon, and the brain stem. The incubated preparations of rat brain also converted dehydroepiandrosterone sulphate to androstenediol sulphate without hydrolysis. The enzyme in rat blood which was similar to that in the brain was also partially characterized. The blood enzyme had a pH optimum at 6–5, was nearly exclusively present in erythrocytes, was also NADPH2-dependent, and had an apparent Km of 2·7 × 10?4m . The developmental pattern of the blood enzyme specific activity was similar to that of the rat brain enzyme. Upon haemolysis, most activity was recovered in the haemolysate.  相似文献   

11.
Hexose 1-phosphate uridylyltransferase (EC 2.7.7.12) was present constitutively in Bifidobacterium bifidum. The enzyme was purified to a homogeneous state from B. bifidum grown on a glucose medium and characterized. The molecular weight of the enzyme is about 110,000.The pH optimum of the enzyme was 7.5. The enzyme was very labile on the acidic side below pH 4.5. Thymidine diphosphate glucose could serve as a substrate with about 60% efficiency of UDP-glucose. The Km values for UDP-gtucose, galactose 1-phosphate (Gal-l-P), UDP-galactose and glucose 1-phosphate (Glc-1-P) were estimated to be 2.3×10?5M, 5.0 × 10?4M, 3.1 × 10?5 M and 1.4 × 10?4M, respectively. From these results the physiological roles of the enzyme were considered in relation to galactose metabolism in B. bifidum.  相似文献   

12.
Prostaglandin A isomerase has been purified 120-fold from rabbit serum by the use of ammonium sulfate fractionation, isoelectric focusing, and Sephadex G-200 chromatography. The molecular weight of the enzyme was estimated to be 110,000 from the elution volume on Sephadex G-200. Prostaglandin A isomerase is a heterogeneous protein with respect to charge. This has been concluded from the spread of enzymatic activity over 1 pH unit after isoelectric focusing. The enzymatic activity is inhibited by N-ethylmaleimide but not by other sulfhydryl blocking agents. The Km was determined to be 5 × 10?5m.  相似文献   

13.
Although spermatozoa possess a very active carnitine acetyltransferase, there is no satisfactory explanation for such a high activity. In order to help elucidate possible roles for carnitine acetyltransferase in spermatozoa, we examined the intracellular location and properties of carnitine acetyltransferase from ejaculated ram spermatozoa. The spermatozoa were disrupted by hypotonic treatment with 10 mm phosphate buffer (pH 7.4), followed by mild sonication. The resulting homogenate was separated by sucrose step-gradient centrifugation into soluble, plasma membrane, acrosomal membrane, and mitochondrial fractions. These fractions were characterized by electron microscopy and marker enzyme assays. The particulate fractions were made soluble by treatment with 0.1% deoxycholate and then were assayed for carnitine acetyltransferase activity. Carnitine acetyltransferase activity was found exclusively in the mitochondrial fraction with a specific activity of 0.151 μmol CoASH · min?1 · mg?1. The apparent Km values for acetyl-CoA and l-carnitine were 1.1 × 10?5 and 1.3 × 10?4m respectively.  相似文献   

14.
A major carcinogenic aminoazo dye-binding protein having Ip of 9.7 (isoelectric focusing) was isolated from the liver cytosol of rats given 40 mg 3'MeDAB. The protein has the molecular weight of 6.8 × 104 (gel-filtration) and two subunits of about 3.9 × 104 molecular weight (SDS-polyacrylamide gel electrophoresis). The amino acid composition was similar to that reported for liver alcohol dehydrogenase of animals. The enzymatic activity was shown to be associated consistently with the dye-binding protein fractions throughout the purification steps suggesting identity of the dye-binding protein as liver alcohol dehydrogenase.  相似文献   

15.
Bacillus macerans enzyme (BME)-derived high molecular weight dextrins, which are by-products in the course of the industrial production of cylodextrins, were isolated and their chemical structures were characterized.Dextrin I was obtained in a yield of about 24% from BME-hydrolyzate (a mixture of dextrin and cylodextrins, 50% each) of potato starch by fractionation with an ultrafiltrator having a membrane of cut-off molecular weight 2.0 × 104. Dextrin II was obtained in a yield of about 15% from BME-hydrolyzate (a mixture of dextrins and cyclodextrins, 70 : 30) of Dextrin I by the same method.Dextrin I and II consisted of dextrin having molecular weights over 20 × 106 and dextrins having molecular weights 4 × 103−1 × 105 in the ratio of 80 : 12 and 66: 15, respectively.The results of hydrolysis by β-amylase and methylation analysis indicated that the average, exterior and interior chain lenghts of the dextrins having molecular weights over 20 × 106 and 4 × 103−1 × 105 from Dextrin I were 16.5, 8.2 and 7.3, and 11.5, 6.9 and 3.6, respectively, than those from Dextrin II were 13.6, 4.7 and 9.9, and 10.4, 5.1 and 4.3, respectively.  相似文献   

16.
Euglena gracilis chloroplast valyl-tRNA synthetase was purified 990 fold to a specific activity of about 1100 units/mg protein, by a series of steps including ammonium sulfate precipitation and chromatography on hydroxyapatite, DEAE-cellulose, Blue Dextran — Sepharose and Sephadex G200. The enzyme gives a single band upon polyacrylamide gel electrophoresis, appears to be a monomer with a molecular weight of 126,000 daltons and has Km values of 1.5 × 10?5 M for L-valine, 5 × 10?5 M for ATP, and 6 × 10?8 for tRNAVal.  相似文献   

17.
A method for analysis of elution data of proteins, obtained from Sephadex gel filtration experiments, is described. The relevant elution data from seven different proteins, with known molecular weights and Stoke's radii, were fitted into various equations relating elution parameters and molecular size parameters. It was observed that polynomial relationships represented elution data for proteins with a much greater degree of precision than linear equations. The validity of this procedure was also checked by analysing gel filtration data available in the literature and it was concluded that a better fit was obtained using polynomial relationships, provided a sufficiently large number of experimental points were available for numerical analysis. Using this method, values of 320,000 ± 7000 for the molecular weight, and (60 ± 0.4) × 10?8 cm for the Stoke's radius of Neurospora NAD-specific glutamate dehydrogenase were calculated.  相似文献   

18.
Hamster liver glutathione peroxidase was purified to homogeneity in three chromatographic steps and with 30% yield. The purified enzyme had a specific activity of approximately 500 μmol cumene hydroperoxide reduced/min/mg of protein at 37 °C, pH 7.6, and 0.25 mm GSH. The enzyme was shown to be a tetramer of indistinguishable subunits, the molecular weight of which was approximately 23,000 as estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. A single isoelectric point of 5.0 was attributed to the active enzyme. Amino acid analysis determined that selenocysteine, identified as its carboxymethyl derivative, was the only form of selenium. One residue of cysteine was found to be present in each glutathione peroxidase subunit. The presence of tryptophan was colorimetrically determined. Pseudo-first-order kinetics of inactivation of the enzyme by iodoacetate was observed at neutral pH with GSH as the only reducing agent. An optimal pH of 8.0 at 37 °C and an activation energy of 3 kcal/mol at pH 7.6 were found. A ter-uni-ping-pong mechanism was shown by the use of an integrated-rate equation. At pH 7.6, the apparent second-order rate constants for reaction of glutathione peroxidase with hydroperoxides were as follows: k1 (t-butyl hydroperoxide), 7.06 × 105 mm min?1; k1 (cumene hydroperoxide), 1.04 × 106 mm?1 min?1; k1 (p-menthane hydroperoxide), 1.2 × 106 mm?1 min?1; k1 (diisopropylbenzene hydroperoxide), 1.7 × 106 mm?1 min?1; k1 (linoleic acid hydroperoxide), 2.36 × 106 mm?1 min?1; k1 (ethyl hydroperoxide), 2.5 × 106 mm?1 min?1; and k1 (hydrogen peroxide), 2.98 × 106 mm?1 min?1. It is concluded that for bulky hydroperoxides, the more hydrophobic the substrate, the faster its reduction by glutathione peroxidase.  相似文献   

19.
Phospholipase A2 was isolated from Trypanosoma congolense and purified to electrophoretic homogeneity. The enzyme appeared to exist in a dimeric form with subunit molecular weights of 16 500 and 18 000. It had a pH optimum of 6·8. Kinetic analysis with different substrates, showed that the enzyme had exceptional specificity for 1,2,dimyristoyl-sn-phosphatidylcholine and 1,2,dioleoyl-sn-phosphatidylcholine with Km values of 1·85 × 10?3 M and 2·12 × 10?3 M respectively. The Arrhenius plot was linear with an activation energy of 5·8 kcal mol?1. Inhibition studies with parahydroxymercuribenzoate and tri-butyltinoxide were positive thus implicating a thiol group at the catalytic site of the enzyme. The enzyme was stable to heat treatment and possessed haemolytic and anticoagulating properties.  相似文献   

20.
Chlorophyllase from a diatom alga (Phaeodactylum tricornutum) was obtained and the partially purified extract has been further purified using preparative isoelectric focusing on a Rotofor cell. Three fractions, FI, FII, and FIII, were separated from the Rotofor cell and salt and ampholytes were removed to give fractions FI′, FII′, and FIII′, respectively. Enzyme fractions FI′, FII′, and FIII′, respectively. Enzyme fractions FI′, FII′, and FIII′ showed specific activities of 15.2 × 10?4, 226.7 ×10?4 and 33.8 × 10?4 µmol/mg protein/min, respectively. Most of the enzyme activity (84%) was in fraction FII′. The optimum pH for chlorophyllase activity was 8.0 for FI′ and 8.5 for both FII′ and FIII′. Apparent Km values for enzyme fractions FI′, FII′, and FIII′ were 2.1nM, 2.3nM, and 2.0 nM, respectively. Enzyme fractions FII′ and FIII′ showed higher chlorophyllase activity towards the partially purified chlorophyll when it was compared to that with the crude chlorophyll as well as with both chlorophylls a and b. However, the enzyme fraction FI′ had higher activity towards the crude chlorophyll when it was compared to that with both chlorophylls a and b, but with a preference for chlorophyll a over chlorophyll b. The inhibitory effect of diisopropyl flurophosphate (DIFP) on chlorophyllase activity demonstrates a noncompetitive inhibitor kinetics with Ki values of 1.29mM, 2.14mM, and 0.71mM for FI′. FII′, and FIII′, respectively.  相似文献   

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