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1.
Dialyzed sonicates from Chinese hamster ovary (CHO) and V-79 lung cells catalyze the addition of l-[U-14C]glutamate to tetrahydrofolate (H4PteGlu). Catalysis is optimal between pH 8.5 and 10.2 and is dependent on Mg2+ and a purine nucleotide triphosphate. Cobalamins do not stimulate the system even when the cells are grown in the absence of cyanocobalamin (CN-Cbl). Incubations with dl-H4-[G-3H]PteGlu + l-[U-14C]glutamate show that the product routinely assayed by DEAE-cellulose chromatography is tetrahydropteroyldiglutamate (H4PteGluGlu). Higher reduced folylpolyglutamates are formed when the standard assay level of dl-H4PteGlu is decreased from 100 μm to 1–5 μm. Using either dialyzed extracts or a 25-fold purified enzyme fraction, dATP is 1.6 times more effective than ATP. The folyl specificity for diglutamate synthesis is H4PteGlu > H4-homofolate > 5-formyl-H4PteGlu > 5-MeH4 PteGlu. dl-5-MeH4PteGlu is only about 15% as active as dl-H4PteGlu. Extracts from a CHO mutant AUXB1 (requiring glycine + adenosine + thymidine) and a V-79 mutant ght-1 (requiring glycine + hypoxanthine + thymidine) have <3% of their respective parent cell amounts of H4PteGluGlu synthetase activity. CHO AUXB1 and V-79 ght-1 extracts are also inactive with the other three reduced folyl compounds cited above and PteGlu. Twelve out of 16 revertant clones that were isolated from CHO AUXB1 in media lacking glycine + adenosine + thymidine contained 44–66% of the wild-type level of H4PteGluGlu synthetase activity. Both parent CHO and V-79 extracts catalyzed the conversion of H4PteGluGlu and tetrahydropteroyl triglutamate to higher glutamyl conjugates. The AUXB1 and ght-1 mutant extracts again lacked these catalytic properties. In contrast, revertants of AUXB1 with about 50% of the wild-type H4PteGluGlu synthetase activity displayed a proportionate ability to synthesize higher polyglutamyl conjugates. From our findings and published genetic data, we conclude that in cultured hamster cells a single synthetase can successively add at least three glutamates to H4PteGlu. Loss of its function in certain mutants is responsible for their triple auxotrophy. 相似文献
2.
Relationship between the cobalamin-dependent methyltransferase activities in Escherichia coli B and an E. coli B (Met H - ) mutant 总被引:1,自引:0,他引:1
The methylcobalamin (MeB12) homocysteine transmethylase activity and the B12-dependent 5-methyltetrahydrofolate (5-MeH4-folate) homocysteine transmethylase activity in cell-free extracts of E. coli B are catalytic functions of separate sites on a single enzyme-protein. Whether these two transmethylases exactly co-purify from extracts, and are protected against p-chloromercuribenzoate (pCMB), however, depends on whether or not the cells were previously cultured in the presence of approximately 1 × 10?8 m cyanocobalamin (CNB12). E. coli B (met H?) contains a defective 5-MeH4-folate apoenzyme which does not tightly bind B12 as a prosthetic group. While the folate-inactive apoenzyme from the mutant strain still catalyzes MeB12 homocysteine transmethylation, this second site on the defective protein is not protected by media CNB12 against pCMB inactivation. Both transmethylase activities are repressed 50% by growth in the presence of 10 m l-methionine. 相似文献
3.
The effects of media vitamin B12(CNB12), l-methionine, folic acid, dl-5-methyltetrahydrofolate (5-MeH4folate), homocysteine, and other nutrients on four one-carbon enzymes in cultured Chinese hamster ovary (CHO) cells were examined. Excess 10 mm methionine elevates the amount of B12 methyltransferase 1.8 – 2.3-fold at media folate concentrations of 0.2 – 2.0 μm. Conversely, excess 100 μm folic acid increases the amount of B12 holoenzyme by 2.4 – 3.0-fold when the medium contains 0.01 – 0.1 mm methionine. These increases in B12 methyltransferase promoted by 100 μm media folate and 10 mm methionine are inhibited by cycloheximide. 5-MeH4folate will support growth and induce methyltransferase synthesis more efficiently than folic acid.Upon transfer to methionine-free media, wild-type CHO cells will survive and can be repeatedly subcultured in the absence of exogenous methionine, provided it is supplemented with 1.0 μm CNB12, 0.1 mm homocysteine, and 100 μm folic acid or 10 μm dl-5-MeH4folate. No growth occurs if homocysteine is omitted, but a requirement for added CNB12 does not become evident until the cells have undergone at least two or three divisions. Survival upon transfer from 0.1 mm methionine-containing to methionine-free media is dependent upon the B12 holomethyltransferase content of the cells used as an inoculum. Inoculum cells must have been previously grown in media supplemented with 1.0 μm CNB12 to stabilize and convert apo- to holomethyltransferase, and 100 μm folate (or 10 μm dl-5-MeH4folate) to induce maximal enzyme-protein synthesis. Transfer to methionine-deficient medium does not result in more than a 20–25% increase in the cellular B12 enzyme content over the level already induced by 100 μm folate in 0.1 mm methionine-supplemented media. A mutant auxotroph CHO AUXB1 with a triple growth requirement for glycine + adenosine + thymidine (McBurney, M. W., and Whitmore, G. F. (1974) Cell, 2, 173) cannot survive in media lacking exogenous methionine. High concentrations of media folic acid or dl-5-MeH4folate fail to induce elevated amounts of B12 methyltransferase in this mutant. Excess 10 mm medium methionine does, however, elevate its B12 enzyme as in the parent CHO cells. An additional mutant AUXB3 that requires glycine + adenosine (McBurney, M. W., and Whitmore, G. F. (1974) Cell, 2, 173) barely survives in methionine-deficient media. It has a folate-induced B12 enzyme level intermediate between wild-type CHO cells and AUXB1. The level of B12 methyltransferase induced by high media folate concentrations is a critical determinant of CHO cell survival in methionine-free media. 相似文献
4.
Leon Milewich Terry S. Hendricks Bettie Sue Masters Rene A. Frenkel Paul C. Mac Donald 《Archives of biochemistry and biophysics》1981,211(2):530-536
The estrogen synthetase present in human placental microsomes appears to be dependent on the cooperative interaction of the reduced cofactors NADPH and NADH for optimal activity. Using steady-state concentrations of either cofactor, it was found that while the estrogen synthetase activity followed hyperbolic saturation kinetics with NADPH (Kmapp = 14 μM), the enzyme followed sigmoidal saturation kinetics when the cofactor was NADH, with the half-maximum velocity attained at a cofactor concentration of 1.1 mm. The maximum velocity obtained with NADPH as the cofactor was greater than with corresponding concentrations of NADH. Estrogen synthetase activity in the presence of NADH was not due to NADPH contamination. NADH, in the presence of small concentrations of NADPH (0.5 to 5 μm), stimulated significantly the rate of estrogen formation from androstenedione by placental microsomes and, in addition, the enzyme saturation kinetics changed from sigmoidal to hyperbolic, thus mimicking the effect of NADPH. Estrogen synthetase activity, measured in the presence of 1 mm NADH, was stimulated in a dose-dependent manner by NADPH (Kmapp = 0.4 μM NADPH) and, when the enzyme was measured in the presence of 5 μm NADPH, the activity was stimulated in a dose-dependent manner by NADH (Kmapp = 45 μM NADH). Estrogen synthetase activity measured in the presence of NADH, without and with NADPH (1 μm) remained linear both with time of incubation for approximately 15 min and with microsomal protein concentration up to 3 mg/ml. The apparent Km of estrogen synthetase for androstenedione, when measured in the presence of NADH, was 1 μm. The synergistic interaction between NADH and NADPH in stimulating placental estrogen synthetase activity observed in vitro may, conceivably, take place in vivo in the intact placenta. 相似文献
5.
A previously reported stimulation of brain 5-methyltetrahydrofolate (5-MeH4-folate) N-methyltransferase by FAD and methylcobalamin (MeB12 is attributed to their roles as nonspecific electron acceptors. Evidence is presented that the catalyst involved is not an aromatic alkylamine methyltransferase, but the widely distributed enzyme, 5,10-methyleneH4-folate reductase. In the presence of an electron acceptor it catalyzes the oxidation of [5-14C]MeH4-folate to [5,10-14C]methyleneH4-folate which equilibrates to yield dimedone reactive H14CHO. The material being measured when incubation systems containing β-phenylethylamine or tryptamine are extracted with tolueneisoamyl alcohol is a condensation product of the H14CHO and the aromatic alkylamine. The aromatic alkylamine is not a co-substrate in the enzymic oxidation mechanism. It is required to react nonenzymically with reductase formed H14CHO and render it extractable. Our failure and that recently of others to detect significant N-methylation using [5-14C]MeH4-folate as a Me group donor make the existence of a folate-biogenic amine methyltransferase seem highly improbable. 相似文献
6.
Amino ketone formation and aminopropanol-dehydrogenase activity in rat-liver preparations 总被引:2,自引:2,他引:0
1. Rat tissue homogenates convert dl-1-aminopropan-2-ol into aminoacetone. Liver homogenates have relatively high aminopropanol-dehydrogenase activity compared with kidney, heart, spleen and muscle preparations. 2. Maximum activity of liver homogenates is exhibited at pH9·8. The Km for aminopropanol is approx. 15mm, calculated for a single enantiomorph, and the maximum activity is approx. 9mμmoles of aminoacetone formed/mg. wet wt. of liver/hr.at 37°. Aminoacetone is also formed from l-threonine, but less rapidly. An unidentified amino ketone is formed from dl-4-amino-3-hydroxybutyrate, the Km for which is approx. 200mm at pH9·8. 3. Aminopropanol-dehydrogenase activity in homogenates is inhibited non-competitively by dl-3-hydroxybutyrate, the Ki being approx. 200mm. EDTA and other chelating agents are weakly inhibitory, and whereas potassium chloride activates slightly at low concentrations, inhibition occurs at 50–100mm. 4. It is concluded that aminopropanol-dehydrogenase is located in mitochondria, and in contrast with l-threonine dehydrogenase can be readily solubilized from mitochondrial preparations by ultrasonic treatment. 5. Soluble extracts of disintegrated mitochondria exhibit maximum aminopropanol-dehydrogenase activity at pH9·1 At this pH, Km values for the amino alcohol and NAD+ are approx. 200 and 1·3mm respectively. Under optimum conditions the maximum velocity is approx. 70mμmoles of aminoacetone formed/mg. of protein/hr. at 37°. Chelating agents and thiol reagents appear to have little effect on enzyme activity, but potassium chloride inhibits at all concentrations tested up to 80mm. dl-3-Hydroxybutyrate is only slightly inhibitory. 6. Dehydrogenase activities for l-threonine and dl-4-amino-3-hydroxybutyrate appear to be distinct from that for aminopropanol. 7. Intraperitoneal injection of aminopropanol into rats leads to excretion of aminoacetone in the urine. Aminoacetone excretion proportional to the amount of the amino alcohol administered, is complete within 24hr., but represents less than 0·1% of the dose given. 8. The possible metabolic role of amino alcohol dehydrogenases is discussed. 相似文献
7.
Alfonso L. Pogolotti Kathryn M. Ivanetich Hans Sommer Daniel V. Santi 《Biochemical and biophysical research communications》1976,70(3):972-978
Studies are reported on the FdUMP-CH2-H4 folate-peptide obtained upon proteolysis of the complex formed from thymidylate synthetase, FdUMP and 5,10-CH2-H4folate. Contrary to a previous report from this laboratory, the peptide does contain a cysteine residue. The sequence of the largest peptide obtained is Ala-Leu-Pro-Pro-(His,Cys)-Thr. Quantitative modification of the histidine residue with the Pauly reagent indicates that imidazole is not directly linked to the nucleotide. The stability of the peptide indicates the covalent bond to the cofactor involves its 5-nitrogen; from this, it may be concluded that the reactive form of the cofactor is the 5-iminium ion. 相似文献
8.
《Biochimica et Biophysica Acta (BBA)/General Subjects》1986,881(2):175-184
5′-Bromoacetamido-5′-deoxythymidine (BAT), 5′-iodoacetamido-5′-deoxythymidine (IAT), 5′-chloroacetamido-5′-deoxythymidine (CAT) and [14C]BAT were synthesized and their interactions with thymidylate synthase purified from L1210 cells were invesatigated. The inhibitory effects of these compounds on thymidylate synthase were in the order BAT > IAT > CAT, which is in agreement with their cytotoxic effects in L1210 cells. In the presence of substrate during preincubation, the concentration required for 50% inhibition of the enzyme activity by these inhibitors was 4–8 fold higher than it was in the absence of dUMP. The I50 values for BAT were 1·10−5 M and 1.2·10−6 M in the presence and absence, respectively, of dUMP during preincubation. These results were in agreement with the observed inhibition of thynmidylate synthase by BAT in intact L1210 cells. A Lineweaver-Burk plot revealed that BAT behaved as a competitive inhibitor. The Km for the enzyme was 9.2 μM, and the Ki determined for competitive inhibition by BAT was 5.4 μM. Formation of a tight, irreversible compledx is referred from the finding that BAT-inactivation of thymidylate synthase was not reversible on prolonged dialysis and that the enzyme-BAT complex was nondissociable by gel filtration through a Sephadex G-25 column or by TSK-125 column chromatography. Incubation of thymidylate synthase with BAT resulted in time-dependent, irreversible loss of enzyme activity by first-order kinetics. The rate constant for inactivation was 0.4 min−1, and the steady-state constant of inactivation, Ki, was estimated to be 6.6 μM. The 5′-haloacetamido-5′-deoxythymidines provide specific inhibitors of thymidylate synthase that may also serve as reagents for studying the enzyme mechanism. 相似文献
9.
Alteration in properties of thymidylate synthetase from pyrimethamine-resistant smodium chabaudi. International Journal for Parasitology16: 483–490. Thymidylate synthetase from cloned strains of pyrimethamine-sensitive and resistant P. chubaudi were partially purified and characterized. The enzyme from both strains have equal mol. wt of 120,000 as estimated by Sephadex G-200 column chromatography. The enzyme from drug-sensitive parasites has an optimum pH of 6.5–7.5 and is stable at pH 4–11 while that from drug-resistant strain has an pH optimum of 7.0–8.0 and is stable at pH 5–10. The Km for methylenetetrahydrofolate are 206 ± 6 and 495 ± 5 μm for the enzyme from drug-resistant and sensitive parasites, respectively. The Km for dUMP of the enzyme from drug-resistant and sensitive parasites are 42 ± 1 and 49 ± 6 μm, respectively. Inhibition of the enzyme from both strains by FdUMP are competitive with dUMP; however,the Kis for the enzyme from drug-resistant strain (0.043 ± 0.005 μm) is less than that from drug-sensitive strain (0.11 ± 0.007 μm) by a factor of 2.5. The Kii for methotrexate with respect to methylenetetrahydrofolate of the enzyme from drug-resistant parasites (58 ± 3 μm) is 3 times larger than that from drug-sensitive parasites (17 ± 1 μm). 相似文献
10.
A methyltransferase, which catalyzes the methylation of luteolin (Km, 16 μM) using S-adenosyl-l-methionine as the methyl donor, has been purified about 38-fold from cell suspension cultures of soybean (Glycine max L., var. Mandarin). The following 3,4-dihydroxy phenolic compounds were also methylated: luteolin 7-O-glucoside (Km, 28 μm), quercetin (Km, 35 μm), eriodictyol (Km, 75 μm), 5-hydroxyferulic acid (Km, 227 μm), dihydroquercetin (Km, 435 μm), and caffeic acid (Km, 770 μm). Rutin and quercetin 3-O-glucoside were poor substrates. Methylation proceeded only in the meta position. The enzyme was unable to catalyze the methylation of p-coumaric acid, m-coumaric acid, ferulic acid, isoferulic acid, sinapic acid, apigenin, or naringenin. While the isoflavones biochanin A and daidzein did not serve as substrates, texasin (6,7-dihydroxy-3′-methoxyisoflavone) was methylated (Km, 35 μm). The methylation of caffeic acid and quercetin showed a pH optimum of 8.6–8.9. The enzyme required Mg2+ ions for maximum activity (approximately 1 mm) and could be totally inhibited by EDTA (10 mm). The Km for S-adenosyl-l-methionine was 11 μm. S-Adenosyl-l-homocysteine inhibited the methylation of luteolin by S-adenosyl-l-methionine. 相似文献
11.
Previous work from this laboratory has shown that 5-thio-d-glucose is a competitive inhibitor for active transport of d-glucose. The present work indicates that the thiosugar analog and its 1-phosphate can also interfere with d-glucose 6-P formation.5-Thio-d-glucose serves as a substrate for yeast hexokinase with a Km of 4 mm, and V of 8.8 nmol/min/μg of protein. The analog competitively inhibits d-glucose phosphorylation with a Ki of 20 mm.5-Thio-d-glucose 1-P can act as a substrate for rabbit skeletal muscle phosphoglucomutase with a Km of 60 μm and V of 0.17 μmol/min/μg of protein. Thus, 5-thio-d-glucose 1-P behaves as a near metabolic analog of d-glucose 1-P. 5-Thio-d-glucose 1-P is a competitive inhibitor of d-glucose 1-P conversion to the 6-P with a Ki of 16.2 μm.5-Thio-d-glucose 6-P produced by phosphorylation of 5-thio-d-glucose and by conversion from 5-thio-d-glucose 1-P was identified by chromatographic mobility and by color reactions. 相似文献
12.
M Terai C Furihata T Matsushima T Sugimura 《Archives of biochemistry and biophysics》1976,176(2):621-629
(i) Three forms of cyclic AMP phosphodiesterases (3′,5′-cyclic AMP 5′-nucleotidohydrolase, EC 3.1.4.17), F1, F2-I and F2-II, were partially purified from the soluble fraction of rat pancreas in the presence of excess protease inhibitors by DEAE-cellulose column chromatography and gel filtration and were characterized. (ii) F2-II, which was purified 31-fold, exhibited a single peak of activity on both polyacrylamide-gel electrophoresis and isoelectric focusing. The enzyme had a molecular weight of about 70,000, an isoelectric point of 3.9, and an optimal pH around 8.5 and required Mg2+ or Mn2+ but not Ca2+ for activity. The Km values of this enzyme for cyclic AMP and cyclic GMP were 1 and 50 μm, respectively, while V values of this enzyme for cyclic AMP and cyclic GMP were 36.1 and 12.6 nmol min?1 (mg of protein)?1, respectively. Cyclic GMP competitively inhibited hydrolysis of cyclic AMP by this enzyme. Ro20-1724 [4-(3-butoxy-4-methoxybenzyl)-2-imidazolidinone] also inhibited hydrolysis of cyclic AMP competitively, with a Ki value of 1 μm. (iii) Fraction F1, which was purified 10-fold, had a molecular weight of more than 500,000 and required Mg2+ for activity. Its Km values for cyclic AMP were 1 and 5 μm. Its Km value for cyclic GMP was 45 μm. Fraction F2-I, which was purified 26-fold, had a molecular weight of about 70,000. The ratio of the initial velocity of hydrolysis of cyclic GMP to that of cyclic AMP was 0.5 at a substrate concentration of 1 μm. 相似文献
13.
Matthew P. Galloway Bryan L. Roth Carmine J. Coscia 《Archives of biochemistry and biophysics》1981,209(2):620-627
The effects of tetrahydroisoquinolinecarboxylic acids, derived from dopamine and various phenylpyruvates, on the enzyme tyrosine 3-monooxygenase have been investigated. Using a partially purified tyrosine 3-monooxygenase from bovine adrenal medulla, 3′,4′-deoxynorlaudanosolinecarboxylic acid was found to be a mixed inhibitor against the cofactor (Ki = 122 μM), equipotent with norepinephrine. Norlaudanosolinecarboxylic acid inhibited tyrosine 3-monooxygenase competitively with respect to the cofactor (Ki = 126 μM). When tyrosine 3-monooxygenase activity in catecholamine-free striatal homogenates was studied, again 3′,4′-deoxynorlaudanosolinecarboxylic acid (Ki = 40 μM) behaved as a mixed inhibitor whereas norlaudanosolinecarboxylic acid (Ki = 136 μM) was competitive. When the rat striatal tyrosine 3-monooxygenase was subjected to phosphorylating conditions in vitro, decreases in the Ki of norlaudanosolinecarboxylic acid and in that of 3′,4′-deoxynorlaudanosolinecarboxylic acid were observed, whereas the Ki of dopamine was increased. Tyrosine 3-monooxygenase activity in rat striatal synaptosomes was also inhibited by 3′,4′-deoxynorlaudanosolinecarboxylic acid (IC50 = 100 μm) and phosphorylating conditions affected only that inhibition produced by dopamine, but not that by the tetrahydroisoquinolinecarboxylic acids. The results are discussed in relation to the structure of the tetrahydroisoquinolinecarboxylic acids and their possible role in vivo. 相似文献
14.
Hypoxanthine transport by Chinese hamster lung fibroblasts: Kinetics and inhibition of nucleosides 总被引:1,自引:0,他引:1
The transport of []hypoxanthine was studied in monolayer cultures of mutant Chinese hamster lung fibroblasts lacking hypoxanthine-guanine phosphoribosyltransferase. Initial rates of transport were determined by rapid uptake experiments (8 to 20 s); a Michaelis constant of 0.68 ± 0.09 mm for hypoxanthine was derived from linear, monophasic plots of against v. Nucleosides are competitive inhibitors of this process; adenosine and thymidine give respective Ki values of 86 and 300 μm. The corresponding bases give much higher inhibition constants with adenine and thymine yielding values of 3100 and 1700 μm, respectively. A similar pattern was observed for competitive inhibition of hypoxanthine transport by inosine, adenine arabinoside, uridine, cytidine, and two ribofuranosylimidazo derivatives of pyrimidin-4-one; in every case the nucleoside exhibited a lower Ki value than the corresponding homologous base. The inhibition constants observed for nucleosides are remarkably similar to their Km values for nucleoside transport by cultured cells recently reported by others. Hypoxanthine transport was also blocked by the 6-(2-hydroxy-5-nitrobenzylthio) derivatives of inosine and guanosine and by dipyridamole; these agents are also inhibitors of nucleoside transport. These results indicate a closer relationship between base and nucleoside transport than previously recognized and suggest that these two transport processes may involve identical or very similar transport proteins. 相似文献
15.
A 3-O-methyltransferase which catalyzes the methylation of caffeic acid to ferulic acid using S-adenosyl-l-methionine as methyl donor has been isolated and purified about 60-fold from cell suspension cultures of soybean (Glycine max L., var. Mandarin). The enzyme utilized, in addition to caffeic acid (Km = 133 μM), 5-hydroxyferulic acid (Km = 55 μM), 3,4,5-trihydroxy-cinnamic acid (Km = 100 μM), and protocatechualdehyde (Km = 50 μM) as substrates. Methylation proceeded only in the meta position. The enzyme was unable to catalyze the methylation of ferulic acid, of ortho-, meta-, and para-coumaric acids, and of the flavonoid compounds quercetin and luteolin. The methylation of caffeic acid and 5-hydroxyferulic acid showed a pH optimum at 6.5–7.0. No stimulation of the reaction velocity was observed when Mg2+ ions were added. EDTA did not inhibit the reaction. The Km for S-adencsyl-l-methionine was 15 μm. S-Adenosyl-l-homocysteine was a potent competitive inhibitor of S-adenosyl-l-methionine (Ki = 6.9 μM). 相似文献
16.
The Involvement of Aspartate and Glutamate in the Decarboxylation of Malate by Isolated Bundle Sheath Chloroplasts from Zea mays 总被引:1,自引:1,他引:0
Aspartate or glutamate stimulated the rate of light-dependent malate decarboxylation by isolated Zea mays bundle sheath chloroplasts. Stimulation involved a decrease in the apparent Km (malate) and an increased maximum velocity of decarboxylation. In the presence of glutamate other dicarboxylates (succinate, fumarate) competitively inhibited malate decarboxylation by intact chloroplasts with respect to malate with an apparent Ki of about 6 millimolar. For comparison the Ki for inhibition of nicotinamide adenine dinucleotide phosphate-malic enzyme from freshly lysed chloroplasts by these dicarboxylates was 15 millimolar. A range of compounds structurally related to aspartate stimulated malate decarboxylation by intact chloroplasts. Ka values for stimulation at 5 millimolar malate were 1.7, 5, and 10 millimolar for l-glutamate, l-aspartate, and β-methyl-dl-aspartate, respectively. Certain compounds, notably cysteic acid, which stimulated malate decarboxylation by intact chloroplasts inhibited malate decarboxylation by nicotinamide adenine dinucleotide phosphate-malic enzyme obtained from lysed chloroplasts and assayed under comparable conditions. It was concluded that aspartate, glutamate, and related compounds affect the transport of malate into the intact chloroplasts and that malate translocation does not take place on the general dicarboxylate translocator previously reported for higher plant chloroplasts. 相似文献
17.
Z B Rose 《Archives of biochemistry and biophysics》1973,158(2):903-910
Erythrocyte diphosphoglycerate mutase is inhibited by several inorganic salts, the extent of the effect being characteristic of the anionic component, i.e., at ionic strength of about 0.1, SO42? > Cl? > CH3COO?. Using a partially purified enzyme preparation from human red blood cells, kinetic constants were determined in the presence of 0.1 m KCl to simulate the ionic environment of the cell. At pH 7.5, the addition of salt caused a 10-fold increase in the Km of 1,3-diphosphoglycerate and a 46-fold increase in the Ki of 2,3-diphosphoglycerate. There was no effect of salt on the Km of 3-phosphoglycerate or on the maximal velocity of the reaction. In the presence of 0.1 m KCl, the i of inorganic phosphate increased from 0.3 mm to 0.6 mm. The Km of 1,3-diphosphoglycerate was pH dependent, the values obtained being 3.6 μm at pH 6.75, 3.1 μm at pH 7.24, and 6.7 μm at pH 7.75. The Ki values for 2,3-diphosphoglycerate under the same conditions were: 12 μm at pH 6.75, 20μm at pH 7.24, and 53 μm at pH 7.75. The relative maximal velocity of the reaction has been evaluated over the same pH range. The maximal activity of the enzyme measured at 25 °C and pH 7.5 was 2 units/min/ml of packed red cells. From these studies, it is concluded that the effective enzymatic rate increases fourfold when the pH increases from 6.75 to 7.75. 相似文献
18.
A relatively rapid five-step procedure was used in purifying to apparent homogeneity the glutamine synthetase from roots and one form of the enzyme (GSI) from leaves of rice. The steps were: preparation of crude extracts, ammonium sulfate precipitation, filtration on Sepharose 4B, fractionation on DEAE-Sephadex A25, and affinity chromatography on ADP-Sepharose 4B. The purified protein appeared as a single band on polyacrylamide gel electrophoresis. Leaf GSI and the second type of leaf glutamine synthetase (GSII) formed distinct peaks when eluted from DEAE-Sephadex (step 4). The root enzyme and leaf GSI were similar in all the properties which were examined. Both enzymes bound to ADP-Sepharose, had similar biosynthetic (18 μmol P/img protein/min) and transferase (1324 and 1156 μmol γ-glutamyl hydroxamate/mg protein/min) activities, and the same or nearly the same Km values for glutamate (2.17 mm), Mg2+ (4.5 and 5.0 mm), ATP (286 μm), NH4+ (210 and 135 μm), and ADP (3.8 and 5.3 μm). In contrast, leaf GSII did not bind to ADP-Sepharose and had much higher Km values for glutamate (8.3 mm), Mg2+ (15 mm), NH4+ (684 μm), and ADP (33 μm). 相似文献
19.
A S Levi 《Archives of biochemistry and biophysics》1975,168(1):115-121
Lactate dehydrogenase enzyme was immobilized by binding to a cyanogen bromideactivated Sepharose 4B-200 in 0.1 m phosphate buffer, pH 8.5. The immobilized enzyme was found to have lower Km values for its substrates. Km values for pyruvate and lactate were 8 × 10 ?5m and 4 × 10?3m, respectively, an order of magnitude less than the value for the native (free) enzyme. Chicken heart (H4) lactate dehydrogenase was found to lose nearly all its substrate inhibition characteristics as a result of immobilization. The covalently bound muscle-type subunits of lactate dehydrogenase showed more favorable interaction with the muscle type than with the heart type subunits. An increase in thermal and acid stability of the dogfish muscle (M4) lactate dehydrogenase as well as a decrease in the percentage of inhibition of enzyme activity by rabbit antisera and in the complement fixation was observed as a result of immobilization. The changes in the properties of the enzyme as a result of immobilization may be attributable to hindrance produced by the insoluble matrix as well as conformational changes in the enzyme molecules. 相似文献
20.
A S Liacouras T Q Garvey F K Millar E P Anderson 《Archives of biochemistry and biophysics》1975,168(1):74-80
The initial velocity pattern has been determined for uridine-cytidine kinase purified from the murine mast cell neoplasm P815. With either uridine or cytidine as phosphate acceptor, and ATP as phosphate donor, the pattern observed was one of intersecting lines, ruling out a ping-pong reaction mechanism, and suggesting that the reaction probably proceeds by the sequential addition of both substrates to the enzyme to form a ternary complex, followed by the sequential release of the two products. This pattern was obtained whether the reaction was run in 0.01 m potassium phosphate buffer, pH 7.5, or in 0.1 m Tris-HCl, pH 7.2. When analyzed by the Sequen computer program, the data indicated an apparent Km of the enzyme for uridine of 1.5 × 10?4m, an apparent Km for cytidine of 4.5 × 10?5m, and a Km for ATP, with uridine or cytidine as phosphate acceptor, of 3.6 × 10?3m or 2.1 × 10?3m, respectively. The V was 1.83 μmol phosphorylated/min/mg enzyme protein for the uridine kinase reaction and 0.91 μmol for the cytidine kinase reaction. 相似文献