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1.
The mechanism of anion inhibition of the reaction of the pork heart extramitochondrial aspartate aminotransferase (EC 2.6.1.1) with erythro-β-hydroxy-l-aspartate was investigated. This reaction produces a mixture of complexes, one of which is characterized by an absorption maximum at 492 nm. Spectrophotometric analysis of equilibrium mixtures of aspartate aminotransferase and erythro-β-hydroxy-l-aspartate, in different buffers, indicated that acetate, chloride, and cacodylate were competitive inhibitors of hydroxyaspartate binding. Pyrophosphate, however, was not a competitive inhibitor. Between pH 4.5 and 9.0 the affinity of the enzyme for the monovalent anions decreased as the pH increased. The data indicated that the anion binding group had a pKa in the range from pH 6 to 7, depending upon the anion studied. From pH 4.5 to 9.0, the substrate dissociation constant and the distribution of enzyme-substrate complexes were both unaffected by pH. By stopped-flow spectrophotometry, an initial rapid relaxation () was associated with an increase in absorbance at 492 nm, and this rate depended upon both substrate and buffer concentrations. A slower relaxation () was associated with a decrease in the absorbance at 492 nm to approximately 70% of the value attained in the first rapid reaction. The rate of this slower reaction was largely independent of substrate and buffer concentrations. Kinetic analysis of the rates of the first relaxation in several different concentrations of Tris-acetate buffer of pH 8 showed that the rate of association decreased with increasing acetate concentration whereas the reaction rate for dissociation was unaffected. Thus, acetate appears to exert its inhibitory effect by preventing the formation of the enzyme-substrate complex rather than by displacing the substrate from the enzyme. 相似文献
2.
The adenosine deaminase of the digestive diverticulum of the bay scallop was purified and electrophoresis of the purified enzyme yielded a single enzymatically active band at several different pH values. A molecular weight of 130,000 was estimated using gel filtration and sucrose density gradient centrifugation. The enzyme had spectral properties typical of simple proteins and its isoelectric point proved to be 4.8. The scallop enzyme was stable at room temperature from pH 5.0 to 7.0, and in this range it was exceptionally resistant to heat inactivation.The effect of the substrate, adenosine, on the reaction velocity was followed over a 10,000-fold concentration range, and no deviation from Michaelis-Menten kinetics was observed. The following rate equation applies to the enzyme: .The effect of pH on the reaction, using adenosine as the substrate, was studied; and it was found that pH had a much greater effect on the α parameter of the rate equation than on the β parameter and that pH had little effect on the apparent activation energy of either parameter. The apparent activation energy of the β parameter was 12.2 kcal with adenosine as the substrate, while the apparent activation energy of the α parameter was zero. The α parameter of the rate equation, using other substrates, was also insensitive to temperature. 相似文献
3.
C. Danzin P. Casara N. Claverie B.W. Metcalf M.J. Jung 《Biochemical and biophysical research communications》1983,116(1):237-243
It was previously shown that 5-hexyne-1,4-diamine is a potent enzyme-activated irreversible inhibitor of mammalian ornithine decarboxylase. However this compound has secondary pharmacological effects owing to its oxidation to 4-aminohex-5-ynoic acid, an irreversible inhibitor of 4-aminobutyrate aminotransferase. The first step of this oxidation is catalysed by mitochondrial monoamine oxidase. The monomethyl and dimethyl analogues of 5-hexyne-1,4-diamine, i.e. 6-heptyne-2,5-diamine and 2-methyl-6-heptyne-2,5-diamine, which cannot be substrate of monoamine oxidase, were tested as selective irreversible inhibitors of ornithine decarboxylase. Our results demonstrate that (2R,5R)-6-heptyne-2,5-diamine is greater than 10 times more potent, both and , than α-difluoromethylornithine, the most widely used irreversible inhibitor of this enzyme. 相似文献
4.
《Insect Biochemistry》1985,15(1):35-44
Isolation of glutathione from the New Zealand grass grub, is complicated by the marked loss of activity from crude homogenates. This loss may be due to proteolysis or to modification by endogenous chemicals. The effect may be minimized by immediate fractionation with ammonium sulphate and by inclusion of 5mM glutathione in homogenates.Two enzymes species, isoelectric at pH 8.7 and 5.9 respectively, could be isolated by ammonium sulphate fractionation, affinity chromatography, anion exchange chromatography and chromatography on hydroxyl apatite. They had different substrate specificities and had differing subunit structure. The pI 8.7 enzyme appeared to be a homodimer of subunits of 23,700 and the pI 5.9 enzyme one of subunit 22,500.A third major enzyme species, isoelectric at pH 4.3 differed from the other two enzymes in having low affinity for the affinity matrix. This preparation was heterogeneous. The enzymically active species in this preparation had the same molecular weight as that of the pI 8.7 enzyme, had a very similar substrate specificity to the basic enzyme species and was characterized by kinetic parameters almost identical to those of the pI 8.7 enzyme. 相似文献
5.
Cyclic AMP-dependent phosphorylation of rat liver 6-phosphofructo 2-kinase, fructose 2,6-bisphosphatase 总被引:8,自引:0,他引:8
M R El-Maghrabi E Fox J Pilkis S J Pilkis 《Biochemical and biophysical research communications》1982,106(3):794-802
Incorporation of 32P from [γ-32P]ATP into a homogeneous preparation of rat hepatic 6-phosphofructo 2-kinase/fructose 2,6-bisphosphatase was catalyzed by a homogeneous preparation of the catalytic subunit of the cyclic AMP dependent protein kinase from rat liver. Approximately 2 mol of phosphate were incorporated per mol of the dimeric enzyme and this was associated with inhibition of the phosphotransferase activity and activation of the phosphohydrolase activity. Acid hydrolysis of the enzyme that was phosphorylated , revealed that only seryl residues were labeled. Fructose 2,6-bisphosphate inhibited the initial rate of phosphorylation of the enzyme. It is concluded that both activities of this bifunctional enzyme are regulated in a reciprocal manner by cyclic AMP-dependent phosphorylation and that this phosphorylation can be modulated by fructose 2,6-bisphosphate. 相似文献
6.
Masataka Mori Kazumasa Aoyagi Masamiti Tatibana Tsutomu Ishikawa Hisashi Ishii 《Biochemical and biophysical research communications》1977,76(3):900-904
δ-(Phosphonacetyl)-L-ornithine, a transition state analogue for the reaction catalyzed by ornithine carbamoyltransferase (EC 2.1.3.3), was synthesized. It strongly inhibited bovine liver ornithine carbamoyltransferase. The inhibition was competitive with respect to carbamoyl-phosphate; the apparent m values for carbamoyl-phosphate were 15 μM in 0.05 M -2-hydroxyethylpiperazine--2-ethanesulfonate (pH 7.2) and 33 μM in 0.1 M Tris-HCl (pH 8.5), and the inhibition constants at pH 7.2 and 8.5 were 7.1 and 4.7 nM, respectively. The inhibition was non-competitive with L-ornithine, the other substrate of the enzyme. This analogue may provide an effective reagent for the elucidation of carbamoyl-phosphate metabolism and its regulation in the liver of ureotelic animals. 相似文献
7.
Toluene dioxygenase: purification of an iron-sulfur protein by affinity chromatography 总被引:26,自引:0,他引:26
V Subramanian T N Liu W K Yeh D T Gibson 《Biochemical and biophysical research communications》1979,91(3):1131-1139
Toluene dioxygenase, from , oxidizes toluene to (+)--1(S),2(R)-dihydroxy-3-methylcyclohexa-3,5-diene. The oxygenase-component of this multienzyme system was purified to homogeneity by a two-step procedure that utilized affinity and ion exchange chromatography. The purified enzyme would oxidize toluene only in the presence of NADH, ferrous iron and partially purified preparations of NADH cytochrome c reductase and an iron-sulfur protein (ferredoxinTOL). Spinach NADPH cytochrome c reductase and NADPH could substitute for the reductase and NADH. The molecular weight of the oxygenase-component was determined to be 151,000 and polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate indicated that the enzyme is composed of two subunits with molecular weights of 52,500 and 20,800. The absorption spectrum showed maxima at 550 (Shoulder), 450, 326 and 278 nm and preliminary experiments have indicated the presence of 2 gram atoms of iron and 2 gram atoms of acid-labile sulfur per mole of protein. The results indicate that the oxygenase-component of the toluene dioxygenase enzyme system is an iron-sulfur protein that has been designated ISPTOL. 相似文献
8.
Margareta Warholm Claes Guthenberg Bengt Mannervik Christer von Bahr 《Biochemical and biophysical research communications》1981,98(2):512-519
A new glutathione -transferase from human liver has been purified to homogeneity in good yield by use of ion-exchange chromatography on DEAE-cellulose, affinity chromatography on -hexylglutathione coupled to epoxy-activated Sepharose 6B, and chromatography on hydroxyapatite. This new enzyme, transferase μ, is present in high concentration, but only in some individuals. It has an isoelectric point at about pH 6 to 6.5 and a different substrate specificity than the previously described alkaline transferases α-ε from human liver. Especially noteworthy is the finding of high activity against benzo(α)pyrene-4,5-oxide. Glutathione -transferase μ has about 20-fold higher activity with this substrate than have the alkaline transferases. The most pronounced difference was found with -4-phenyl-3-buten-2-one which was >100-fold better as substrate for transferase μ than for the previously described transferases. 相似文献
9.
Tore Skotland Torbjørn Ljones Torgeir Flatmark 《Biochemical and biophysical research communications》1978,84(1):83-88
The enzyme-bound copper of dopamine was reduced by ascorbate and the enzyme separated from excess reductant by gel filtration. The reduced enzyme did not hydroxylate substrate at a rate consistent with the turnover time. This observation supports a sequential mechanism. 相似文献
10.
Jan A. Miernyk David T. Dennis 《Biochemical and biophysical research communications》1982,105(3):793-798
The plastid isozyme of phosphofructokinase from developing castor oil seeds is stimulated by low concentrations of fructose 2,6-bisphosphate when assayed at pH 7.0. The stimulation involves a shift in fructose 6-phosphate kinetics from sigmoidal to near hyperbolic. The plastid isozyme is unaffected by fructose 2,6-bisphosphate when assayed at pH 8.0, and the cytosolic isozyme is unaffected at either pH 7.0 or 8.0. There is no interaction between fructose 2,6-bisphosphate and the other regulators of the phosphofructokinases; phosphoenolpyruvate, 2-phosphoglycerate, 3-phosphoglycerate and inorganic phosphate. 相似文献
11.
T Yagi H Kagamiyama M Nozaki 《Biochemical and biophysical research communications》1979,90(2):447-452
Aspartate aminotransferases from pig heart cytosol and mitochondria, B and accepted L-cysteine sulfinate as a good substrate. The mitochondrial isoenzyme and the enzyme showed higher activity toward L-cysteine sulfinate than toward the natural substrates, L-glutamate and L-aspartate. The cytosolic isoenzyme catalyzed the L-cysteine sulfinate transamination at 50% the rate of L-glutamate transamination. The enzyme had the same reactivity toward the three substrates. Antisera against the two isoenzymes and the enzyme inactivated almost completely cysteine sulfinate transamination activity in the crude extracts of pig heart muscle and B, respectively. These results indicate that cysteine sulfinate transamination is catalyzed by aspartate aminotransferase in these cells. 相似文献
12.
The NADP+ specific glutamate dehydrogenase from wild-type forms a stable binary complex with NADPH. This can combine with L-glutamate, α-ketoglutarate or the substrate analogue D-glutamate to form ternary complexes which can be distinguished by their different fluorescence properties. The affinity of the enzyme for NADPH diminishes with increases in pH or ionic strength of the solution. Experimental data obtained using modified glutamate dehydrogenases from mutant strains of suggest that the reduced-coenzyme binding sites observed fluorimetrically are the same as those observed by enzyme kinetics. 相似文献
13.
The inhibition of prostaglandin (PG) synthetase by nonsteroidal anti-inflammatory drugs (NSAID) is not well understood. Co-factors (glutathione and hydroquinone) are needed for maximum enzymatic activity , and we suggest that NSAID might inhibit PG synthetase partly by interfering with co-factor induced stimulation of the enzyme. This hypothesis was tested by:A) Examining the effect of glutathione, noradrenaline and hydroquinone on bull seminal vesicle (BSV) PG synthetase . The stimulatory effects were concentration-dependent.B) Three structurally distinct NSAID, indomethacin, aspirin and paracetamol, inhibited the stimulation by each co-factor in a concentration-related manner. Drug effectiveness also depended on the concentration of co-factor. 相似文献
14.
Anthony L. Tarentino Frank Maley 《Biochemical and biophysical research communications》1975,67(1):455-462
The substrate specificities of the endo-β-N-acetylglucosaminidases from and were compared and found to differ considerably. The enzyme from released Asn-GlcNAc-Fuc-containing glycopeptides from exoglycosidase-treated acidic IgM glycopeptides but was limited in its capacity to hydrolyze ovalbumin glycopeptides larger than Asn(GlcNAc)2(Man)5. In contrast, the enzyme from hydrolyzed this and larger neutral oligosaccharides but could not hydrolyze the above fucose-containing IgM glycopeptides. Removal of the fucose residue, however, converted the latter to an active substrate for the enzyme, thus broadening its substrate range to encompass most of those substrates hydrolyzed by the endoglycosidase. 相似文献
15.
P P Hipps M R Eveland M H Laird W R Sherman 《Biochemical and biophysical research communications》1976,68(4):1133-1138
-Inositol:NAD(P)+ oxidoreductase (-inositol oxidoreductase) has been identified in bovine brain. This enzyme elutes from DEAE cellulose with 0.3 M KCl in 50 mM Tris buffer, pH 7.5. Using NADH as cofactor -inosose-2 is reduced selectively to -inositol. With NADPH the enzyme forms both -inositol and -inositol, however, at a lower rate. The enzyme was chromatographed on G-100 Sephadex and found to have an apparent molecular weight of 74,000. This enzyme differs in DEAE binding, molecular weight and cofactor specificity from the previously described -inositol oxidoreductase which utilizes NADPH exclusively to produce 3 fold more -inositol than -inositol. 相似文献
16.
C H Reynolds R Y Hsu B Matthews T A Pry K Dalziel 《Archives of biochemistry and biophysics》1978,189(2):309-316
The oxidative decarboxylation of l-malate catalyzed by malic enzyme has been studied by stopped-flow spectrophotometry and by initial rate measurements with large concentrations of NADP+, malate, and Mn2+. The results show that hybride transfer is fast, ms. The formation of enzyme-bound NADPH in an amount equivalent to about half of the enzyme active center concentration is followed by turnover at a rate which is initially faster than the steady-state rate, under conditions such that substrate inhibition by malate is observed in the steady state. The steady-state rate is reached after about 0.5 s. It is suggested that a conformational change in the abortive complex of enzyme, manganese, NADPH, and malate is responsible for the malate inhibition and for the slow approach to the true steady state. The relief of malate inhibition by increasing Mn2+ concentrations is described, and the results are described in relation to other evidence of nonidentical binding sites for, or negatively cooperative binding of, substrate and activator and possible half-of-the-sites reactivity. 相似文献
17.
Charles D. Boyer Jack Preiss 《Biochemical and biophysical research communications》1978,80(1):169-175
Purification of starch branching enzymes from kernels of two nonlinked mutants of maize, and , showed the basis of the two mutations to be associated with branching enzymes I and IIb, respectively. Branching enzyme I from kernels purified as nonmutant branching enzyme I, but had an altered pattern of activity when amylose was used as a substrate. In addition to the typical fall in absorbance at high wavelengths (550–700 nm) of the amylose-iodine complex, branching of amylose by branching enzyme I caused an increase in absorbance at low wavelengths (400–550 nm). Branching enzyme IIb was undetected in extracts of kernels, while branching enzymes I and IIa appeared unaltered. Low umprimed starch synthase activity was also observed in DEAE-cellulose fractions of maize, but this activity was regenerated by the addition of any branching enzyme. 相似文献
18.
Activity of a penicillin-insensitive -endopeptidase that splits the -alanyl-meso-2,6-diaminopimelyl linkage in peptidoglycan was demonstrated in a sonic extract of . The protein with this activity was partially purified. The activity was inhibited by 3 μg per ml of deoxyribonucleic acid, suggesting that this cell wall hydrolytic enzyme is regulated by deoxyribonucleic acid or its fragments. 相似文献
19.
20.
The dependence on pH of the kinetic parameters for the hydrolysis of phenyl acetate catalyzed by pig liver carboxylesterase was examined for purified high-isoelectric point and low-isoelectric point fractions of enzyme that were separated by isoelectric focusing. The values of kcat are half-maximal at pH 4.3 and 5.1 for the high- and low-isoelectric point forms, respectively, and show a shallow dependence on pH with a value of n = 0.5. The absence of a change in the pH dependence of kcat for the high-isoelectric point enzyme in the presence of high concentrations of methanol, which reacts with the acetyl-enzyme intermediate to give methyl acetate, provides evidence that the pH dependence is not caused by a change in rate-determining step. This means that if an imidazole group is involved in catalysis its pK must be perturbed downward by 2–3 units. The pH dependence of is biphasic with apparent pK values for dissociations of the free enzyme near 7 and 4 for both the high- and low-isoelectric point enzymes. Inhibition by a second molecule of substrate and by methanol are strongest for high-pH forms of the enzyme. 相似文献