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1.
—The hydrolysis of ThTP by rat brain membrane-bound ThTPase is inhibited by nucleoside diphosphates and triphosphates. ATP and ADP are most effective, reducing hydrolysis by 50% at concentrations of 2 × 10?5m and 7·5 × 10?5m respectively. Nucleoside monophosphates and free nuclcosides as well as Pi have no effect on enzyme activity. ThMP and ThDP also fail to inhibit hydrolysis in concentrations up to 5 × 10?3m . Non-hydrolysable methylene phosphate analogs of ATP and ADP were used in further kinetic studies with the ThTPase. The mechanism of inhibition by these analogs is shown to be of mixed non-competitive nature for both compounds. An observed Ki, of 4 × 10?5m for the ATP analog adenosine-PPCP and 9 × 10?5m for the ADP analog adenosine-PCP is calculated at pH 6·5. Formation of the true enzyme substrate, the [Mg2+. ThTP] complex, is not significantly affected by concentrations of analogs producing maximal (>95%) inhibition of enzyme activity. Likewise the relationships between pH and observed Km and pH and Vmax are not shifted by the presence of similar concentrations of inhibitor.  相似文献   

2.
Phospholipase D activity (phosphatidylcholine phosphatidohydrolase, EC 3.1.4.4) was demonstrated in vitro in a solubilized preparation from rat brain particulate fraction which also possessed the transphosphatidylation activity. The preparation attacked a phosphatidylcholine microdispersion and cleaved the terminal phosphate diester bond of this phospholipid resulting in the formation of phosphatidic acid. The pH optimum for the phosphatidohydrolase activity was broad with an apparent peak aroung 6.0 whereas the transphosphatidylation showed a sharp pH optimum at 7.2 Ca2+ was not essential for the hydrolysis, but merely stimulated slightly with an optimum about 5 mm, however, it could be replaced by Mg2+. The hydrolysis of phosphatidylcholine by the enzyme was almost completely inhibited in the presence of either diethyl ether (20% by volume) or p-chloromercuriophenyl sulfonate (6 × 10 ?5m). The latter inhibition was reduced by the addition of dithiothreitol (6 × 10?4m). The result suggests an essential role of sulfhydryl group in the formation of the enzyme-substrate complex.The apparent Km for phsophatidylcholine for the phosphatidohydrolase activity was about 8.3 × 10 ?4m.  相似文献   

3.
Base-exchange activity was contrasted to the usual phosphatidohydrolase activity of commercial phospholipase D preparation from cabbage. The former activity was assayed by measuring the incorporation of labeled ethanolamine and choline into phospholipids. The latter activity was assayed by measuring the formation of phosphatidic acid with radioactive phosphatidylcholine microdispersion as substrate. The pH optimum for the base-exchange activity was about 9.0, whereas the phosphatidohydrolase activity had a pH optimum around 5.6. The incorporation of ethanolamine and choline into phospholipid was dependent upon the amount of acceptor asolectin microdispersion present. The optimum concentration of Ca2+ in the base-exchange reaction was about 4 mm, whereas the optimum concentration for the phosphatidohydrolase activity was greater than 28 mm. The incorporation of ethanolamine into phospholipid was decreased 50% by heating the enzyme preparation at 50°C for about 10 min, whereas the choline incorporation decreased approximately 20% and the phosphatidohydrolase activity decreased by about 10% under these conditions.Hemicholinium-3 was found to be a noncompetitive inhibitor for the incorporation of both ethanolamine and choline into phospholipid with respective Ki, values of 1.25 × 10?3 and 2.50 × 10?3m. The Km values for ethanolamine and choline in the base-exchange reaction were 1.25 × 10?3 and 2.50 × 10?3m, respectively. The apparent Km for phosphatidylcholine for the phosphatidohydrolase activity was about 1.5 × 10?3m, and there was no inhibition by hemicholinium-3.  相似文献   

4.
The observed equilibrium constants (Kobs) for the reactions of d-2-phosphoglycerate phosphatase, d-2-Phosphoglycerate3? + H2O → d-glycerate? + HPO42?; d-glycerate dehydrogenase (EC 1.1.1.29), d-Glycerate? + NAD+ → NADH + hydroxypyruvate? + H+; and l-serine:pyruvate aminotransferase (EC 2.6.1.51), Hydroxypyruvate? + l-H · alanine± → pyruvate? + l-H · serine±; have been determined, directly and indirectly, at 38 °C and under conditions of physiological ionic strength (0.25 m) and physiological ranges of pH and magnesium concentrations. From these observed constants and the acid dissociation and metal-binding constants of the substrates, an ionic equilibrium constant (K) also has been calculated for each reaction. The value of K for the d-2-phosphoglycerate phosphatase reaction is 4.00 × 103m [ΔG0 = ?21.4 kJ/mol (?5.12 kcal/mol)]([H20] = 1). Values of Kobs for this reaction at 38 °C, [K+] = 0.2 m, I = 0.25 M, and pH 7.0 include 3.39 × 103m (free [Mg2+] = 0), 3.23 × 103m (free [Mg2+] = 10?3m), and 2.32 × 103m (free [Mg2+] = 10?2m). The value of K for the d-glycerate dehydrogenase reaction has been determined to be 4.36 ± 0.13 × 10?13m (38 °C, I = 0.25 M) [ΔG0 = 73.6 kJ/mol (17.6 kcal/mol)]. This constant is relatively insensitive to free magnesium concentrations but is affected by changes in temperature [ΔH0 = 46.9 kJ/mol (11.2 kcal/mol)]. The value of K for the serine:pyruvate aminotransferase reaction is 5.41 ± 0.11 [ΔG0 = ?4.37 kJ/mol (?1.04 kcal/mol)] at 38 °C (I = 0.25 M) and shows a small temperature effect [ΔH0 = 16.3 kJ/ mol (3.9 kcal/mol)]. The constant showed no significant effect of ionic strength (0.06–1.0 m) and a response to the hydrogen ion concentration only above pH 8.5. The value of Kobs is 5.50 ± 0.11 at pH 7.0 (38 °C, [K+] = 0.2 m, [Mg2+] = 0, I = 0.25 M). The results have also allowed the value of K for the d-glycerate kinase reaction (EC 2.7.1.31), d-Glycerate? + ATP4? → d-2-phosphoglycerate3? + ADP3? + H+, to be calculated to be 32.5 m (38 °C, I = 0.25 M). Values for Kobs for this reaction under these conditions and at pH 7.0 include 236 (free [Mg2+] = 0) and 50.8 (free [Mg2+] = 10?3m).  相似文献   

5.
Fructokinase (Fraction III) of Pea Seeds   总被引:5,自引:4,他引:1       下载免费PDF全文
A second fructokinase (EC 2.7.1.4) was obtained from pea seed (Pisum sativum L. var. Progress No. 9) extracts. The enzyme, termed fructokinase (fraction III), was specific for fructose and had little activity with glucose. With fructose concentrations above 0.25 millimolar, there was strong substrate inhibition at the optimum pH (8.0) and also at pH 6.6. The apparent Km values at pH 8.0 for fructose and glucose were 0.06 millimolar and 0.14 millimolar, respectively. The apparent Km for Mg adenosine 5′-triphosphate (MgATP) was 0.06 millimolar and excess MgATP was inhibitory. Mg2+ was essential for activity but the enzyme was inhibited by excess Mg2+ or ATP. Mg adenosine 5′-pyrophosphate was also inhibitory. Activity was stimulated by the addition of monovalent cations: of those tested K+, Rb+, and NH4+ were the most effective. The possible role of fructokinase (fraction III) is discussed.  相似文献   

6.
An extract from Jerusalem artichoke shoots exhibited important adeninephosphoribosyltransferase activity. Only partial purification was possible because of the great instability of the enzyme. Phosphate ions and thiol reducing substances were necessary to stabilize it. The optimal temperature and pH were 40–45° and 5.5 to 6.5. The enzyme showed an absolute requirement for divalent cation: Mn2+ > Mg2+ = CO2+ = Zn2+ ? Ca2+. Kinetic studies gave Km values of 6.4 × 10?1 M for phosphoribosylpyrophosphate (PRPP) and 5.5 × 10?6 M for adenine. AMP exercised a strong product inhibition, competitive towards PRPP (Ki = 10?4 M). Inhibition by phosphate and pyrophosphate ions was also observed. The results suggested that the adeninephosphoribosyltransferase of Helianthus tuberosus has a key role in the purine salvage pathway.  相似文献   

7.
Two l-lactate dehydrogenase isoenzymes and one dl-lactate dehydrogenase could be separated from potato tubers by polyacrylamide-gel electrophoresis. The enzymes are specific for lactate, while β-hydroxybutyric acid, glycolic acid, and glyoxylic acid are not oxidized. Their pH optima are pH 6.9 for the oxidation and 8.0 for the reduction reaction.The Km values for l-lactate for the two isoenzymes are 2.00 × 10?2 and 1.82 × 10?2, m. In the reverse reaction the affinities for pyruvate are 3.24 × 10?4 and 3.34 × 10?4, m. Both enzymes have similar affinities for NAD and NADH (3.00 × 10?4; 4.00 × 10?4, and 8.35 × 10?4; 5.25 × 10?4, m).The dl-lactate oxidoreductase may transfer electrons either to NAD or N-methyl-phenazinemethosulfate. The Km values of this enzyme for l-lactate are 4.5 × 10?2, m and for d-lactate 3.34 × 10?2, m. Its affinity for pyruvate is 4.75 × 10?4, m. The enzyme is inhibited by excess NAD (Km = 1.54 × 10?4, M) and has an affinity toward NADH (Km = 5.00 × 10?3, M) which is about one tenth of that of the two isoenzymes of l-lactate dehydrogenase.  相似文献   

8.
R.K. Sindhu  H.V. Desai 《Phytochemistry》1979,18(12):1937-1938
Agmatine iminohydrolase was purified ca 375-fold from groundnut cotyledons. The enzyme exhibited an optimum pH between 5.5 and 8.5 and the energy of activation was 22 kcal/mol. The Km for agmatine was (7.57 ± 0.77) × 10?4 M. The enzyme was inhibited by tryptamine, putrescine, cadaverine, spermidine and spermine. Inhibition by cadaverine and spermidine was competitive. The Ki values for cadaverine and spermidine were 4.1 × 10?3 and 7.5 × 10?4 M, respectively.  相似文献   

9.
The observed equilibrium constants (Kobs) for the l-phosphoserine phosphatase reaction [EC 3.1.3.3] have been determined under physiological conditions of temperature (38 °C) and ionic strength (0.25 m) and physiological ranges of pH and free [Mg2+]. Using Σ and square brackets to indicate total concentrations Kobs = Σ L-serine][Σ Pi]Σ L-phosphoserine]H2O], K = L-H · serine±]HPO42?][L-H · phosphoserine2?]H2O]. The value of Kobs has been found to be relatively sensitive to pH. At 38 °C, K+] = 0.2 m and free [Mg2+] = 0; Kobs = 80.6 m at pH 6.5, 52.7 m at pH 7.0 [ΔGobs0 = ?10.2 kJ/mol (?2.45 kcal/mol)], and 44.0 m at pH 8.0 ([H2O] = 1). The effect of the free [Mg2+] on Kobs was relatively slight; at pH 7.0 ([K+] = 0.2 m) Kobs = 52.0 m at free [Mg2+] = 10?3, m and 47.8 m at free [Mg2+] = 10?2, m. Kobs was insignificantly affected by variations in ionic strength (0.12–1.0 m) or temperature (4–43 °C) at pH 7.0. The value of K at 38 °C and I = 0.25 m has been calculated to be 34.2 ± 0.5 m [ΔGobs0 = ?9.12 kJ/mol (?2.18 kcal/ mol)]([H2O] = 1). The K for the phosphoserine phosphatase reaction has been combined with the K for the reaction of inorganic pyrophosphatase [EC 3.6.1.1] previously estimated under the same physiological conditions to calculate a value of 2.04 × 104, m [ΔGobs0 = ?28.0 kJ/mol (?6.69 kcal/mol)] for the K of the pyrophosphate:l-serine phosphotransferase [EC 2.7.1.80] reaction. Kobs = [Σ L-serine][Σ Pi][Σ L-phosphoserine][H2O], K = [L-H · serine±]HPO42?][L-H · phosphoserine2?]H2O. Values of Kobs for this reaction at 38 °C, pH 7.0, and I = 0.25 m are very sensitive to the free [Mg2+], being calculated to be 668 [ΔGobs0 = ?16.8 kJ/mol (?4.02 kcal/mol)] at free [Mg2+] = 0; 111 [ΔGobs0 = ?12.2 kJ/mol (?2.91 kcal/mol)] at free [Mg2+] = 10?3, m; and 9.1 [ΔGobs0 = ?5.7 kJ/mol (?1.4 kcal/mol) at free [Mg2+] = 10?2, m). Kobs for this reaction is also sensitive to pH. At pH 8.0 the corresponding values of Kobs are 4000 [ΔGobs0 = ?21.4 kJ/mol (?5.12 kcal/mol)] at free [Mg2+] = 0; and 97.4 [ΔGobs0 = ?11.8 kJ/ mol (?2.83 kcal/mol)] at free [Mg2+] = 10?3, m. Combining Kobs for the l-phosphoserine phosphatase reaction with Kobs for the reactions of d-3-phosphoglycerate dehydrogenase [EC 1.1.1.95] and l-phosphoserine aminotransferase [EC 2.6.1.52] previously determined under the same physiological conditions has allowed the calculation of Kobs for the overall biosynthesis of l-serine from d-3-phosphoglycerate. Kobs = [Σ L-serine][Σ NADH][Σ Pi][Σ α-ketoglutarate][Σ d-3-phosphoglycerate][Σ NAD+][Σ L-glutamat0] The value of Kobs for these combined reactions at 38 °C, pH 7.0, and I = 0.25 m (K+ as the monovalent cation) is 1.34 × 10?2, m at free [Mg2+] = 0 and 1.27 × 10?2, m at free [Mg2+] = 10?3, m.  相似文献   

10.
The effect of mersalyl and of antibodies, directed against smooth-muscle myosin and skeletal muscle myosin, on the (Ca2+ + Mg2+)-activated adenosine triphosphatase (Ca,Mg)ATPase) system of mouse liver plasma membranes has been studied. Antismooth-muscle myosin inhibited by 38.6% at optimum substrate concentration the (Ca,Mg)ATPase with a Km of 0.88 × 10?3m. Mersalyl (0.5 mm) also inhibited this enzyme, the percentage inhibition being 44.6% at optimal substrate concentration. These results suggest the presence of a smooth-muscle myosin-like protein in the plasma membrane of mouse liver cells which has an associated (Ca,Mg)ATPase activity.  相似文献   

11.
The possible role of fructosyl transferase in the biosynthesis of fructosans in Agave americana was investigated. This enzyme was extracted from A. americana stem and purified 17.5-fold by salt fractionation and DEAE-cellulose chromatography. The optimum conditions for the enzyme were pH 6. 1, temperature 37°, substrate concentration 20% and Km 3.6 × 10?1 M; Ag+, Pb 2+, Hg2+, Al3+, Sn2+, CN? acted as inhibitors and Ca2+, Mg2+, Co2+ and Li+ actemd as activators. Only sugars of the type F ~ R (R-aidose), e.g. sucrose and raffinose acted as substrates for the enzyme. The donor acceptor specificity of the enzyme was studied extensively. Sugars sucrose. None of the intermediates of fructosan biosynthesis from sucrpse acted as fructose donors. The possible acceptors from sucrose and raffinose. The enzyme was capable of building up oligosaccharides up to FIOG from sucrose. None of the intermediates of fructosan biosynthesis from sucrose acted as fructose donors. The possible mechanism of fructosan biosynthesis from sucrose is discussed.  相似文献   

12.
Oligomeric structure and kinetic properties of NADP-malic enzyme, purified from sugarcane (Saccharam officinarum L.) leaves, were determined at either pH 7.0 and 8.0. Size exclusion chromatography showed the existence of an equilibrium between the dimeric and the tetrameric forms. At pH 7.0 the enzyme was found preferentially as a 125 kilodalton homodimer, whereas the tetramer was the major form found at pH 8.0. Although free forms of l-malate, NADP+, and Mg2+ were determined as the true substrates and cofactors for the enzyme at the two conditions, the kinetic properties of the malic enzyme were quite different depending on pH. Higher affinity for l-malate (Km = 58 micromolar), but also inhibition by high substrate (Ki = 4.95 millimolar) were observed at pH 7.0. l-Malate saturation isotherms at pH 8.0 followed hyperbolic kinetics (Km = 120 micromolar). At both pH conditions, activity response to NADP+ exhibited Michaelis-Menten behavior with Km values of 7.1 and 4.6 micromolar at pH 7.0 and 8.0, respectively. Negative cooperativity detected in the binding of Mg2+ suggested the presence of at least two Mg2+ - binding sites with different affinity. The Ka values for Mg2+ obtained at pH 7.0 (9 and 750 micromolar) were significantly higher than those calculated at pH 8.0 (1 and 84 micromolar). The results suggest that changes in pH and Mg2+ levels could be important for the physiological regulation of NADP-malic enzyme.  相似文献   

13.
High-affinity cyclic AMP phosphodiesterase purified to homogeneity from dog kidney was studied with respect to its stability, its catalytic and kinetic properties, and its sensitivity to pharmacological agents. The enzyme was shown to rapidly lose activity upon dilution to low protein concentrations in aqueous media, but this activity loss was largely prevented by the presence of bovine serum albumin or ethylene glycol. Similarly, maximum activity required bovine serum albumin to be present during incubation for activity analysis. Enzyme activity required a divalent cation; Mg2+, Mn2+, and Co2+ each supported activity, but highest activity was obtained with Mg2. The temperature optimum ranged from 30 to 45 °C and depended on substrate concentration; the Ea = 10,600 cal/mol. The pH optimum of the enzyme was broad, with a maximum from pH 8.0 to 9.5. The enzyme exhibits linear Michaelis-Menton kinetics for hydrolysis of cyclic AMP at all substrate concentrations tested and for hydrolysis of cyclic GMP at > 20 μm. The Km for cyclic AMP hydrolysis was 2 μm, and that for cyclic GMP hydrolysis was 312 μm. The Ki values for the competitive inhibition of hydrolysis of each substrate by the other were similar to their Km values suggesting a single active site. Cyclic AMP hydrolysis was weakly inhibited by cyclic GMP, cyclic IMP, adenine, and adenosine, but was not inhibited by the mono-, di, or trinucleotides of adenosine, guanosine, or inosine. Activity was competitively inhibited with Ki values in the micromolar range by drugs representative of methylxanthines, isoquinolines, pyrazolopyridines, imidazolidinones, triazolopyrimidines, pyridylethylenediamines, phenothiazines, and calcium antagonists. The results are discussed with reference to the similarities and differences between high- and low-affinity phosphodiesterase forms.  相似文献   

14.
《Insect Biochemistry》1991,21(3):239-242
Alkaline phosphatase from the excretory system of the grasshopper, Poekilocerus bufonius was purified with ammonium sulphate fractionation and chromatography on Bio-Gel A-0.5 m. The specific activity of the enzyme is 152 units/mg of protein. The enzyme is a tetramer and the Mr value of the subunit is 72,000 ± 2500 as shown by gel filtration and SDS-polyacrylamide gel electrophoresis. The enzyme has a pH optimum of 9.6 and an apparent Km value of 0.28 × 10−3 M. The activity of the enzyme reached a maximum at 75°C and the enzyme showed stability at 65°C. The enzyme was inhibited by Ca2+, Na+ and Fe3+ and was stimulated by Zn2+, Mn2+ and Mg2+.  相似文献   

15.
Magnesium-dependent adenosine triphosphatase, purified from sheep kidney medulla using digitonin, has been characterized in a series of kinetic and magnetic resonance studies. Kinetic studies of divalent metal activation using either Mg2+ or Mn2+ indicate a biphasic response to divalent cations. Apparent Km values of 23 μm for free Mg2+ and 3.3 μm for free Mn2+ are obtained at low levels of added metal, while Km values of 0.50 mm for free Mg2+ and 0.43 mm for free Mn2+ are obtained at much higher levels of divalent cations. In all cases the kinetic data indicate that the binding of divalent metals is independent of the substrate, ATP. Kinetic studies of the substrate requirements of the Mg2+-ATPase also yield biphasic Lineweaver-Burk plots. At low ATP concentrations, kinetic studies yield apparent Km values for free ATP of 6.0 and 1.4 μm with Mg2+ and Mn2+, respectively, as the activating divalent metals. At much higher levels of ATP the response of the enzyme to ATP changes so that Km values for free ATP of 8.0 and 2.0 mm are obtained for Mg2+ and Mn2+, respectively. In both cases, however, the binding of ATP is independent of added metal. ADP inhibits the Mg2+-ATPase and the kinetic data indicate that ADP competes with ATP at both the high and low affinity sites. Dixon plots of the data are consistent with competitive inhibition at both ATP sites, with Ki values of 10.5 μm and 4.5 mm. Electron paramagnetic resonance and water proton relaxation rate studies show that the enzyme binds 1 g ion of Mn2+ per 469,000 g of protein. The Mn2+ binding studies yield a KD for Mn2+ at the single high affinity site of 2 μm, in good agreement with the kinetically determined activator constant for Mn2+ at low Mn2+ levels. Moreover, the EPR binding studies also indicate the existence of 34 weak sites for Mn2+ per single high affinity Mn2+ site. The KD for Mn2+ at these sites is 0.55 mm, in good agreement with the kinetic activator constant for Mn2+ of 0.43 mm, consistent with additional activation of the enzyme by the large number of weaker metal binding sites. The enhancement of water proton relaxation by Mn2+ in the presence of the enzyme is also consistent with the tight binding of a single Mn2+ ion per 469,000 Mr protein and the weaker binding of a large number of divalent metal ions. Analysis of the data yields a value for the enhancement for bound Mn2+ at the single tight site, ?b, of 5 and an enhancement at the 34 weak sites of 11. The frequency dependence of water proton relaxation by Mn2+ at the single tight site yields a dipolar correlation time (constant from 8–60 MHz) of 3.18 × 10?9 s. The kinetics and metal binding studies, together with the effect of temperature on ATPase activity at high and low levels of ATP, are consistent with the existence in this preparation of a single Mg2+-ATPase, with high and low affinity sites for divalent metals and for ATP. Observations of both high and low affinities for ATP have been made with two other purified ATPases. The similarities of these systems to the Mg2+-ATPase described here are discussed.  相似文献   

16.
Two esteroproteolytic enzymes (A and D) have been isolated from the mouse submaxillary gland and shown to be pure by ultracentrifugation, immunoelectrophoresis, acrylamide-gel electrophoresis, and amino acid analyses. The enzymes have molecular weights of approximately 30,000 and are structurally and antigenically related. Narrow pH optima between 7.5 and 8.0 are exhibited by both enzymes. The “pK1's” are between 6.0 and 6.5 and the “pK2's” are near 9.0. A marked preference for arginine-containing esters is shown by both enzymes. The maximum specific activity of enzyme A on p-tosylarginine methyl ester (TAME) at pH 8 was 2500–3000 μm min?1 mg?1 and for enzyme D, 400–600 μm min?1 mg?1. With TAME as substrate, the Km for enzyme A was 8 × 10?4m at 25 °C and 6 × 10?4m at 37 °C. For D, Km was 3 × 10?4 at 25 °C and 2 × 10?4m at 37 °C.An apparent activation of enzyme D by tosylarginine (TA), a product of TAME hydrolysis, and all α-amino acids examined was due to removal of an inhibitor by chelation. This effect could be duplicated by 8-hydroxyquinoline and diethyldithiocarbamate but not by EDTA. Enzyme A was not affected by these substances to any remarkable extent. Several divalent ions proved to be potent inhibitors of enzyme D. Both enzymes are inactivated by the active site reagents diisopropyl phosphofluoridate and tosyllysine chloromethylketone but much less rapidly than is trypsin. Nitrophenyl-4-guanidionobenzoate reacts with a burst of nitrophenol liberation but with a rapid continuing hydrolysis. One active site per molecule is indicated. Enzyme D is inactivated by urea, reversibly at 10 m and with maximal permanent losses at 6 m. Autolysis of the unfolded form by the native enzyme when they coexist at intermediate urea concentrations appears to occur.Identity of enzyme D and the epithelial growth factor binding protein is demonstrated.  相似文献   

17.
Hexokinase was partially purified from the leaves of Dendrophthoe falcata. The optimum pH for the enzyme was 8.5. The enzyme was sensitive to p-CMB and the inhibition could be reversed by 2-mercaptoethanol. The optimum temperature was 40° and energy of activation 6900 cal/mol. The enzyme had an absolute requirement for a divalent metal ion. Although Mg2+ was the preferred metal, it could be partially replaced by Mn2+ and Ca2+. ATP was the most effective phosphoryl donor. Glucose was the best substrate, the Km values of 0.14 and 0.26 mM were obtained at saturated and sub-saturated ATP concentration. Phosphorylation coefficients show the following order of reactivity of sugars: glucose mannose 2-deoxy D-glucose fructose glucosamine galactose ribose. The Km value for ATP was 0.16 mM, which increased to 0.35 mM in the presence of 0.5 mM ADP. ADP and 5′-AMP were competitive inhibitors with respect to ATP, and Ki values were 0.4 and 1.2 mM respectively.  相似文献   

18.
ATP sulfurylase from Penicillium chrysogenum was purified to homogeneity. The enzyme binds 8 mol of free ATP (Ks = 0.53 mM) or AMP (Ks = 0.50 mM) per 440,000 g. The results are consistent with our earlier report that the enzyme is composed of eight identical subunits of Mr 55,000 (J. W. Tweedie and I. H. Segel, 1971, Prep. Biochem. 1, 91–117; J. Biol. Chem. 246, 2438–2446). In the absence of cosubstrates, the purified enzyme catalyzes the hydrolysis of MgATP (to AMP and MgPPi) and adenosine 5′-phosphosulfate (APS) (to AMP and SO42?). MgATP hydrolysis is inhibited by nonreactive sulfate analogs such as nitrate, chlorate, and formate (uncompetitive with MgATP). In spite of the hydrolytic reactions it is possible to observe the binding of MgATP and APS to the enzyme in a qualitative (nonequilibrium) manner. Neither inorganic sulfate (the cosubstrate of the forward reaction) nor formate or inorganic phosphate (inhibitors competitive with sulfate) will bind to the free enzyme in detectable amounts in the absence or in the presence of Mg2+, Ca2+, free ATP, or a nonreactive analog of MgATP such as Mg-α,β-methylene-ATP. Similarly, inorganic pyrophosphate (the cosubstrate of the reverse reaction) will not bind in the absence or in the presence of Mg2+ or Ca2+. The induced binding of 32Pi (presumably to the sulfate site) can be observed in the presence of MgATP. The results are consistent with the obligately ordered binding sequence deduced from the steady-state kinetics (J. Farley et al., 1976, J. Biol. Chem. 251, 4389–4397) and suggest that the subsites for SO2?4 or MgPPi appear only after nucleotide cleavage to form E~AMP · MgPPi or E~AMP · SO4 complexes. The suggestion is supported by the relative values of Kia (ca. 1 mm for MgATP) and Kiq (ca. 1 αm for APS) and by the inconsistent value of k?1 calculated from VfKiaKmA (The value is considerably less than Vr) Purified ATP sulfurylase will also catalyze a Mg32PPi-MgATP exchange in the absence of SO42?. A 35SO42?-APS exchange could not be demonstrated in the absence or presence of MgPPi. This result was not unexpected: The rate of APS hydrolysis (or conversion to MgATP) is extremely rapid compared to the expected exchange rate. Also, the pool of APS at equilibrium is extremely small compared to the sulfate pool. The V values for molybdolysis, APS hydrolysis (in the absence of PPi), ATP synthesis (from APS + MgPPi), and Mg32PPi-MgATP exchange at saturating sulfate are all about equal (12–19 μmol × min?1 × mg of enzyme?1). The rates of Mg32PPi-MgATP exchange in the absence of sulfate, APS synthesis (from MgATP + sulfate), and MgATP hydrolysis (in the absence of sulfate) are considerably slower (0.10 – 0.35 μmol × min?1 × mg of enzyme?1). These results and the fact that k4 calculated from VrKiqKmQ is considerably larger than Vf suggest that the rate-limiting step in the overall forward reaction is the isomerization reaction E~AMP-SO2?4 → EAPS. In the reverse direction the rate-limiting step may be SO2?4 release or isomerization of the E~AMP · MgPPi · SO42? complex. (The reaction appears to be rapid equilibrium ordered.) Reactions involving the synthesis or cleavage of APS are specific for Mg2+. Reactions involving the synthesis or cleavage of ATP will proceed with Mg2+, with Mn2+, and, at a lower rate, with Co2+. The results suggest that the enzyme possesses a Mg2+-preferring divalent cation (activator) binding site that is involved in APS synthesis and cleavage and is distinct from the MeATP or MePPi site. The equilibrium binding of about one atom of 45Ca2+ per subunit (possibly to the activator site) could be demonstrated (Ks = 1.4 mM).  相似文献   

19.
Protein methylase II (S-adenosylmethionine:protein—carboxyl methyltrans-ferase), which modifies free carboxyl residues of protein, was purified from both rat and human blood, and properties of the enzymes were studied. The pH optima for the reaction were dependent on the substrate proteins used; pH 7.0 was found with endogenous substrate, 6.1 with plasma, 6.5 with γ-globulin, and 6.0 with fibrinogen. The molecular weight of the enzymes from both rat and human erythrocytes were identical (25,000 daltons) determined by Sephadex G-75 chromatography. Partially purified enzyme from rat erythrocytes showed three peaks on electrofocusing column at pH 4.9, 5.5 and 6.0. The Km values of the enzymes from rat and human erythrocytes showed 3.1 × 10?6m and 1.92 × 10?6m at pH 6.0, 1.96 × 10?6m and 1.78 × 10?6m at pH 7.2, respectively, for S-adenosyl-l-methionine. It is also found that S-adenosyl-l-homocysteine is a competitive inhibitor for protein methylase II with Ki value of 1.6 × 10?6m.  相似文献   

20.
Ouabain binding was studied in isolated adult dog heart myocytes. The binding was correlated with the inhibition of K+-activated para-nitrophenylphosphatase (K+-PNPPase) activity and the beating response. It was shown that: (i) the specific binding was dependent upon Mg2+ and was inhibited by K+; (ii) the maximal binding capacity (Bmax) was 7.4 × 105 ouabain molecules per cell, or 410 pmol ouabain/K+-PNPPase unit (μmol/min); (iii) in the presence of Mg2+ (5 mm), there were two components in the Scatchard plot, i.e., a high-affinity component with a Kd value of 5.6 × 10?8m and a low-affinity component with a Kd value of 6.7 × 10?7m; (iv) the Hill coefficient (n′) for ouabain binding was 0.72 with a S0.5 value of 7.1 × 10?7m; these values were compatible with the values obtained from studies of K+-PNPPase inhibition by ouabain (n′ = 0.55, S0.5 = 3.6 × 10?7 m) and remained unchanged in the presence of physiological concentrations of Na+ plus K+; (v) in the presence of Mg2+ and K+, the high-affinity component tended to conform to the low-affinity component with an apparent decrease in Bmax; (vi) in the presence of Mg2+ and para-nitrophenylphosphate, the low-affinity component was changed to the high-affinity component with no change in Bmax; (vii) the dissociation rate of the labeled ouabain in the highly dilute medium was not altered in the presence of excess amounts of unlabeled ligand; this eliminated the possibility that the apparent negative cooperativity was due to a site-to-site interaction between receptors; (viii) ouabain increased the number of beating cells and the frequency of beating. Based on these findings, it is concluded that: (i) isolated myocytes possess functional receptors for ouabain; (ii) the binding of ouabain is associated with its inhibition of K+-PNPPase activity; (iii) ouabain receptors in isolated myocytes are of one class with at least two interconvertible conformational states.  相似文献   

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