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1.
A Zn 2+-glycerophosphocholine cholinephosphodiesterase was purified with a specific activity of 4.6 μmole/min·mg protein from bovine
brain membranes by procedures involving PI-PLC solubilization, concanavalin A affinity chromatography, CM-sephadex chromatography
and Sephadex G-150 chromatography. Based on molecular weight determination gel chromatography and SDS polyacrylamide gel electrophoresis,
the phosphodiesterase activity appears to be a dimeric protein (110 kDa) composed of two subunits with a molecular weight
of approximately 54 kDa. The Km value for p-nitrophenylphosphocholine and the optimum pH were found to be 16 μM and pH 10.5,
respectively. The phosphodiesterase was inhibited by Cu 2+, but not the other divalent metal ions. The activity of the apoenzyme was remarkably activated by Co 2+ or Zn 2+, but not Mn 2+ or Mg 2+. In addition, the inactivation of the enzyme in glycine buffer was prevented by Mn 2+ or Zn 2+, but not Co 2+ or Mg 2. In a separate experiment, comparing properties of the purified and membrane-bound phosphodiesterases, the forms of two enzymes
were quite similar except in stability. Both enzymes were more stable at pH 7.4 than pH 5 or 10. However, the membrane-bound
enzyme was more stable than the soluble enzyme at all three pHs. These data suggest that the activity of the phosphodiesterase
may be stabilized in-vivo. 相似文献
2.
Purified glutamine synthetase from pea seedlings was most active with Mg 2+ as the metal activator, but Mn 2+ and Co 2+ were 45 to 60% and 30 to 45% as effective, respectively, when assayed at the optimal pH for each cation. The Mg 2+ saturation curve was quite sigmoid, and evidence indicates that MgATP is the active ATP substance. Co 2+ also gave a sigmoidal saturation curve, but when Mn 2+ was varied only slightly sigmoidal kinetics were seen. Addition of Mn 2+, Ca 2+, or Zn 2+ at low concentrations sharply inhibited the Mg 2+ -dependent activity, partially by shifting the pH optimum. Addition of Co 2+ did not inhibit Mg 2+-dependent activity. The nucleotide triphosphate specificity changed markedly when Co 2+ or Mn 2+ replaced Mg 2+. Using the Mg 2+-dependent assay, the Michaelis constant (K m) for NH 4+ was about 1.9 × 10 −3 M. The K m for l-glutamate was directly proportional to ATP concentration and ranged from 3.5 to 12.4 m m with the ATP levels tested. The K m for MgATP also varied with the l-glutamate concentration, ranging from 0.14 m m to 0.65 m m. Ethylenediaminetetracetic acid activated the enzyme by up to 54%, while sulfhydryl reagents gave slight activation, occasionally up to 34%. 相似文献
3.
Ca 2+,Mg 2+- and Ca 2+,Mn 2+-dependent and acid DNases were isolated from spermatozoa of the sea urchin Strongylocentrotus intermedius. The enzymes have been purified by successive chromatography on DEAE-cellulose, phenyl-Sepharose, Source 15Q, and by gel filtration, and the principal physicochemical and enzymatic properties of the purified enzymes were determined. Ca 2+,Mg 2+-dependent DNase (Ca,Mg-DNase) is a nuclear protein with molecular mass of 63 kD as the native form and its activity optimum is at pH 7.5. The enzyme activity in the presence of bivalent metal ions decreases in the series (Ca 2+ + Mg 2+) > Mn 2+ = (Ca 2+ + Mn 2+) > (Mg 2+ + EGTA) > Ca 2+. Ca,Mg-DNase retains its maximal activity in sea water and is not inhibited by G-actin and N-ethylmaleimide, whereas Zn 2+ inhibits the enzyme. The endogenous Ca,Mg-DNase is responsible for the internucleosomal cleavage of chromosomal DNA of spermatozoa. Ca 2+,Mn 2+-dependent DNase (Ca,Mn-DNase) has molecular mass of 25 kD as the native form and the activity optimum at pH 8.5. The enzyme activity in the presence of bivalent metal ions decreases in the series (Ca 2+ + Mn 2+) > (Ca 2+ + Mg 2+) > Mn 2+ > (Mg 2+ + EGTA). In seawater the enzyme is inactive. Zinc ions inhibit Ca,Mn-DNase. Acid DNase of spermatozoa (A-DNase) is not a nuclear protein, it has molecular mass of 37 kD as a native form and the activity optimum at pH 5.5, it is not activated by bivalent metal ions, and it is inhibited by N-ethylmaleimide and iodoacetic acid. Mechanisms of the endonuclease cleavage of double-stranded DNA have been established for the three enzymes. The possible involvement of DNases from sea urchin spermatozoa in programmed cell death is discussed. 相似文献
4.
The effect of various metals and regucalcin, a calcium-binding protein isolated from rat liver cytosol, on (Ca 2+–Mg 2+)-ATPase activity in the plasma membranes of rat liver was investigated. Of various metals (Zn 2+, Cu 2+, Ni 2+, Mn 2+, Co 2+ and Al 3+; 100 M as a final concentration), Mn 2+ and Co 2+ increased markedly (Ca 2+–Mg 2+)-ATPase activity, while other metals had no effect. When Ca 2+ was not added into enzyme reaction mixture, Mn 2+ and Co 2+ (25–100 M) did not significantly increase the enzyme activity, indicating that heavy metals act on Ca 2+-stimulated phosphorylation of the enzyme. Meanwhile, regucalcin (0.25–1.0 M) caused a remarkable elevation of (Ca 2+–Mg 2+)-ATPase activity. This increase was not inhibited by the presence of 100 M vanadate, although the effects of Mn 2+ and Co 2+ (100 M) were inhibited by vanadate. Also, the inhibition of the Mn 2+ and Co 2+ effects by vanadate was not seen in the presence of regucalcin. Moreover, regucalcin (0.5 M) increased significantly the enzyme activity in the absence of Ca 2+. This effect of regulcalcin was not altered by increasing concentrations of Ca 2+ added, indicating that the regucalcin effect does not depend on Ca 2+. The present results suggest that regucalcin activates directly (Ca 2+–Mg 2+)-ATPase in liver plasma membranes, and that the activation is not involved in the Ca 2+-dependent phosphorylation of the enzyme. 相似文献
5.
Summary The toxicity of chromium and tin on growth, photosynthetic carbon-fixation, oxygen evolution, heterocyst differentiation and nitrogenase activity of Anabaena doliolum and its interaction with bivalent cations has been studied. Some interacting cations, viz. Ca 2+, Mg 2+ and Mn 2+, substantially antagonised the toxic effects of chromium and tin with reference to growth, heterocyst differentiation and nitrogenase activity in the following hierarchal sequence: Ca 2+ > Mg 2+ > Mn 2+. However, the sequence of hierarchy was Mg 2+ > Ca 2+ > Mn 2+ for carbon fixation and Mn 2+ > Mg 2+ > Ca 2+ for photosynthetic oxygen evolution. Synergistically inhibitory patterns were noticed for all the parameters, viz. growth, 14CO 2 uptake, oxygen evolution, heterocyst differentiation and nitrogenase activity of A. doliolum when Ni 2+, Co 2+ and Zn 2+ were combined with the test metals in the growth medium. These cations followed the following sequence of synergistic inhibition: Ni 2+ > Co 2+ > Zn 2+. Among all the interacting cations, Ca 2+, Mg 2+ and Mn 2+ exhibited antagonistic effects which relieved the test cyanobacterium from metal toxicity. In contrast to this, Ni 2+, CO 2+ and Zn 2+ showed synergistic inhibition which potentiating the toxicity of test metals in the N 2-fixing cyanobacterium A. doliolum. It is evident from the present study that bivalent cations, viz. Ca 2+, Mg 2+, Mn 2+, Ni 2+, Co 2+ and Zn 2+, may appreciably regulate the toxicity of heavy metals in N 2-fixing cyanobacteria if present in aquatic media. 相似文献
6.
The relation that exist between the Pi-PPi exchange reaction and pyrophosphate hydrolysis by the membrane-bound pyrophosphatase of chromatophores of Rhodospirillum rubrum was studied. The two reactions have a markedly different requirement for pH. The optimal pH for hydrolysis was 6.5 while the Pi-PPi exchange reaction was at 7.5; the pH affects mainly the K
m
of Mg 2+ or Pi for the enzyme; Mn 2+ and Co 2+ support the Pi-PPi exchange reaction partially (50%), but the reaction is slower than with Mg 2+; other divalent cations like Zn 2+ or Ca 2+ do not support the exchange reaction. In the hydrolytic reaction, Zn 2+, at low concentration, substitutes for Mg 2+ as substrate, and Co 2+ also substitutes in limited amount (50%). Other cations (Ca 2+, Cu 2+, Fe 2+, etc.) do not act as substrates in complex with PPi. The Zn 2+ at high concentrations inhibited the hydrolytic reaction, probably due to uncomplexed free Zn 2+. In the presence of high concentration of substrate for the hydrolysis (Mg-PPi) the divalent cations are inhibitory in the following order: Zn 2+>Mn 2+>Ca 2+Co 2+>Fe 2+>Cu 2+>Mg 2+. The data in this work suggest that H + and divalent cations in their free form induced changes in the kinetic properties of the enzyme. 相似文献
7.
The Ca 2+/Mg 2+ ATPase of the rat heart sarcolemmal particles was solublized with Triton X-100 after treating the membranes with trypsin and purified by high speed centrifugation, ammonium sulfate fractionation, hydrophobic chromatography and gel filtration. The purified enzyme was seen as a single protein band in nondenaturing polyacrylamide gel electrophoresis and its molecular weight by gel filtration was found to be about 240000. The enzyme utilized Ca-ATP or Mg-ATP as a substrate with high affinity sites (Km = 0.12 – 0.16 mM) and low affinity sites (Km = 1 mM). The enzyme also utilized CTP, GTP, ITP, UTP and ADP as substrates but at a lower rate in comparison to ATP. The enzyme was activated by Ca 2+ (Ka = 0.4 mM) and Mg 2+ (Ka = 0.2 mM) as well as by other cations in the order Ca 2– > Mg 2+ > Mn 2+ > Sr 2+ > Ba 2+ > Ni 2+ > Cu 2+. The ATPase activity in the presence of Ca 2+ was markedly inhibited by Mg 2+, Mn 2+, Ni 2+ and Cu 2+ whereas the monovalent cations such as Na + and K + were without effect. The enzyme did not exhibit Ca 2+ stimulated Mg 2+ dependent ATPase activity and was insensitive to calmodulin, ouabain, verapamil, D-600, oligomycin, azide and vanadate. Optimum pH for Ca 2+ or Mg 2+ ATPase activity was 8.5 – 9.0. In view of the possible ectoenzyme nature of the ATPase, its role in adenine nucleotide and Ca 2+ metabolism in the myocardium is discussed. 相似文献
8.
Sealed microsomal vesicles were prepared from corn ( Zea mays, Crow Single Cross Hybrid WF9-Mo17) roots by centrifugation of a 10,000 to 80,000 g microsomal fraction onto a 10% dextran T-70 cushion. The Mg 2+-ATPase activity of the sealed vesicles was stimulated by Cl − and NH 4+ and by ionophores and protonophores such as 2 micromolar gramicidin or 10 micromolar carbonyl cyanide p-trifluoromethoxyphenyl hydrazone (FCCP). The ionophore-stimulated ATPase activity had a broad pH optimum with a maximum at pH 6.5. The ATPase was inhibited by NO 3−, was insensitive to K +, and was not inhibited by 100 micromolar vanadate or by 1 millimolar azide. Quenching of quinacrine fluorescence was used to measure ATP-dependent acidification of the intravesicular volume. Quenching required Mg2+, was stimulated by Cl−, inhibited by NO3−, was insensitive to monovalent cations, was unaffected by 200 micromolar vanadate, and was abolished by 2 micromolar gramicidin or 10 micromolar FCCP. Activity was highly specific for ATP. The ionophore-stimulated ATPase and ATP-dependent fluorescence quench both required a divalent cation (Mg2+ ≥ Mn2+ > Co2+) and were inhibited by high concentrations of Ca2+. The similarity of the ionophore-stimulated ATPase and quinacrine quench and the responses of the two to ions suggest that both represent the activity of the same ATP-dependent proton pump. The characteristics of the proton-translocating ATPase differed from those of the mitochondrial F1F0-ATPase and from those of the K+-stimulated ATPase of corn root plasma membranes, and resembled those of the tonoplast ATPase. 相似文献
9.
Phosphodiesterase production with bis- p-nitrophenyl phosphate as a substrate by alkalophilic Bacillus No. A-40-2 increased with increasing Mn 2+ concentration, showing maximum productivity at 10 m m. The enzyme production was negligible in the medium without Mn 2+. The simultaneous addition of 10 m m Mn 2+ and one of the several cations Mg 2+, Co 2+, Mo 6+, and Pb 2+ at suitable concentrations stimulated the enzyme production 1.8-fold at most over that with only 10 m m Mn 2+. Inorganic phosphate hardly repressed the enzyme production. The enzyme was purified homogeneously. The purified enzyme had the optimum pH of 7.5 and was fairly stable from pH 7–11. The enzyme hydrolyzed 2′,3′-cyclic-nucleotides and 3′-nucleotides, but did not hydrolyze 3′,5′-cyclic-nucleotides or 5′-nucleotides, indicating it to be a 2′,3′-cyclic-nucleotide 2′-phosphodiesterase (EC 3.1.4.16). The enzyme had activity without metals, but Mg 2+, Ca 2+, Ba 2+, and Mo 6+ activated the enzyme reaction. 相似文献
10.
The catalytic activity of guanylate cyclase (GCase) coupled to atrial natriuretic peptide (ANP) receptor depends on the metal co-factor, Mn 2+ or Mg 2+. ATP synergistically stimulates the ANP-stimulated GCase in the presence of Mg 2+. We have now shown the ATP regulation of the ANP-stimulated GCase in the presence of Mn 2+ in rat lung membranes. ANP stimulated the GCase 2.1-fold compared to the control. ATP enhanced both the basal (basal-GCase) and the ANP-stimulated GCase maximally 1.7- and 2.3- fold compared to the control, respectively, at a concentration of 0.1 mM. The stimulation by ATP was smaller in the presence of Mn 2+ than in the presence of Mg 2+. The addition of inorganic phosphate to the reaction mixture altered the GCase activities in the presence of Mn 2+ with or without ANP and/or ATP. In the presence of 10 mM phosphate, ATP dose-dependently stimulated the basal GCase 5-fold compared to the control at a concentration of 1 mM and augmented the ANP-stimulated GCase, which was 4.2-fold compared to the basal-GCase, 5.5-fold compared to the control at a concentration of 0.5 mM. Protein phosphatase inhibitors, okadaic acid (100 nM), H8 (1 M) and staurosporin (1 M), did not alter the activity. Orthovanadate (1 mM), an inorganic phosphate analogue, significantly stimulated both the basal-GCase and the ANP-stimulated GCase, which were inhibited by ATP. It was assumed that phosphate and orthovanadate might interact with the GCase to regulate the activity in the opposite manner. This was the first report that inorganic phosphate and orthovanadate affected the ATP-regulation of the ANP-stimulated GCase in the presence of Mn 2+. 相似文献
11.
Two barley chloroplast nuclease fractions were separated by the affinity chromatography and gel electrophoresis. Both were
about 2 times more active to RNA than to native DNA and about half as active to denaturated DNA as to native DNA. Both fractions
were as active to UV-irradiated (270 J m -2) native DNA as to intact DNA but their action was inhibited by apurinic sites. The enzyme activities were inhibited by high
concentrations of EDTA, NaCl, Mn 2+, Ca 2+, Zn 2+ ions and by N-ethylmaleimide. They do not require Mg 2+ ions but are stimulated or at higher concentration inhibited by their presence. Both RNase and DNase were active over a wide
pH range (5.5–9), the optimum for DNase action in the presence of Mg 2+ being 6.5, for RNA decomposing activity at pH 8.0. As no mononucleotides were detected in acid soluble form, it seems likely
that DNase acts in the endonucleolytic way. 相似文献
12.
Buffalo milk galactosyltransferase is a single poly-peptide of molecular weight 55,000 to 56,000. The enzyme is specific for
glucose as an acceptor substrate in the presence of 8-lactalbumin, L-Arabinose. L-xylose, D-ribose and D-fructose did not
serve as acceptor substrates even at concentration as high as 0.13 M, while N-acetylglucosamine and ovalbumin served as good
acceptors of galactosyl moiety in the absence of ∞ -lactalbumin. UDP-galacturonic acid did not serve as a donor substrate;
on the contrary, it inhibited the reaction. Lactose synthetase reaction was inhibited by D-ribose, L-arabinose and L-xylose,
whereas D-fructose did not show any inhibition. Buffalo milk ∞ -lactalbumin enhanced the synthesis of lactose but inhibited
the synthesis of N-acetyllactosamine. Cations like Ca 2+, Mg 2+, Cu 2+, Ba 2+ and Co 2+ could not replace Mn 2+ in the N-acetyllactosamine synthetase reaction. Except Co 2+, these cations had no effect on this reaction. Co 2+ was found to be a competitive inhibitor of Mn 2+. The observed inhibition of the reaction by-EDTA also confirmed the absolute requirement of Mn 2+ for the reaction. Lactose synthetase reaction had an optimum pH of 8.5, whereas N-acetyllactosamine synthetase reaction was
maximal at pH 8.0. 相似文献
13.
Synopsis Acid phosphatase activities against p-nitrophenyl phosphate, -naphthyl phosphate -naphthyl phosphate, naphthol AS-BI phosphate, -glycerophosphate and -glycerophosphate were studied in whole homogenates and subcellular fractions of rat testes. The mitochondrial-lysosomal fraction (the 9000 g sediment) was the most active against all these substrates, but a high specific activity was also present in the soluble fraction (105,000 g supernatant). The effect of various inhibitors and activators on p-nitrophenyl phosphate hydrolysis was different in these fractions. The soluble enzyme was markedly stimulated by Co 2+, Mg 2+, Mn 2+, Ni 2+ and Zn 2+. The particulate enzyme was inhibited by sodium fluoride, sodium tartrate and sodium molybdate, whereas the soluble activity was sensitive to Cd 2+, Cu 2+ and Sn 2+ as well as to formaldehyde and glutaraldehyde. The soluble acid phosphatase is mainly localized within the seminiferous tubules. The high sensitivity of the soluble activity to the commonly used fixatives may interfere in the histochemical demonstration of the enzyme. 相似文献
14.
Two non mitochondrial systems involved in ATP-dependent Ca 2+ accumulation have been described and characterized in two membrane fractions from pea internodes purified on a metrizamide-sucrose discontinuous gradient. In the lighter membrane fraction an ATP-dependent Ca 2+ accumulation system, which shows the characteristics of an ATP-dependent H +/Ca 2+ antiport, predominates. This system is inhibited by FCCP and nigericin and stimulated by 50 mM KCl. It is saturated by 0.8–1.0 mM MgSO 4-ATP, strictly requires ATP and is severely inhibited by an excess of free Mg 2+ or Mn 2+. A second system of ATP-dependent Ca 2+ accumulation, recovered mainly in the heavier membrane fraction, is insensitive to FCCP, is saturated by 8–10 mM MgSO 4-ATP, can utilize also ITP or other nucleoside triphosphates although at lower rate than ATP and is only scarcely affected by an excess of free Mg 2+ or Mn 2+. This system is interpreted as corresponding to the (Ca 2+ + Mg 2+)-ATPase described by Dieter, P. and Marmé, D. ((1980) Planta 150, 1–8). 相似文献
15.
A solubilized preparation with activity for catalyzing the incorporation of free myo-inositol into phosphatidyl inositol was obtained from a rat liver microsomal fraction. The incorporation took place both in the presence and in the absence of cytidine diphosphodiglyceride (CDP-DG). The pH optimum of the incorporation in the absence of CDP-DG was 7.4–7.5, while that of the incorporation in its presence was 8.5–8.6. The incorporation in the absence of CDP-DG was activated by Mn 2+ but not by Mg 2+, while that in the presence of CDP-DG was activated by either Mn 2+ or Mg 2+. These results indicated that the incorporation in the absence of CDP-DG and the incorporation in its presence were catalyzed by different enzymes. Before Solubilization, the CDP-DG-independent enzyme was bound to endoplasmic reticulum. The CDP-DG-dependent enzyme also was bound mainly to endoplasmic reticulum and, to a minor extent, to plasma membrane. The CDP-DG-independent enzyme was more easily solubilized by sodium cholate than the CDP-DG-dependent enzyme. There were also differences between these two enzyme activities of the solubilized preparation with respect to their sensitivity to various detergents and their dependence on exogenous lipids. The CDP-DG-independent incorporation was inhibited by CDP-DG, by some nucleotides, and by phosphatidyl serine, while the CDP-DG-dependent incorporation was not inhibited by these substances. Both activities were both inhibited by thiol-reactive compounds. 相似文献
16.
An effective method of preparation involving sonication was developed for cell-free mycobacillin synthetase from Bacillus subtilis. The enzyme showed optimum activity at a buffer concentration of 50 mM (Tris-HCl) and pH 7.5. ATP and Mg 2+ which were essential for synthesis showed an optimum requirement at a ratio of 1∶1. The synthetase was markedly inhibited
by ADP whereas AMP was without any effect. ATP or ATP-generating system could not be replaced by GTP, UTP or CTP. Co 2+ and Mn 2+ could to some extent substitute Mg 2+. Mercapto reagents inhibited the antibiotic synthesis. Exogenous addition of pantothenic acid had no effect. 相似文献
17.
Intestinal guanylate cyclase C is activated by guanylin, an endogenous peptide. This activity seems to be modulated by adenine nucleotides, the ions Mg 2+ and Mn 2+, and pH. In this study, we report an ultracytochemical method for the localization of guanylate cyclase C activity at the electron microscope level. We studied the enzymatic activity in the presence or absence of guanylin and/or ATP, in the presence of the ions Mg 2+ or Mn 2+, and at different pH levels. The greatest distribution of enzymatic activity was detected in samples incubated at pH 8 and 7.4 in the presence of guanylin, Mg 2+ and ATP. Guanylate cyclase C activity was detected at the surface epithelium of stomach and intestine, and in liver, exocrine pancreas and parotid gland. In the intestine, enzymatic activity was more widely distributed in the duodenum than in the jejunum–ileum and colon. In the small intestine, activity was more evident in the upper portion than in the basal portion of the villus. In samples incubated at pH 8 and 7.4 in the absence of ATP, enzymatic activity was detected only in small intestine, liver and exocrine pancreas. Enzymatic activity was present in duodenum incubated at pH 8 and 7.4 in the presence of Mn 2+ and in the presence or absence of ATP. No samples incubated in all these experimental conditions but at pH 5 or samples incubated in the presence of guanylin only or in the absence of guanylin, displayed guanylate cyclase C activity. Our results suggest that a complete ultracytochemical detection of guanylate cyclase C activity requires guanylin as stimulator, and incubation in the presence of Mg 2+ and ATP atbreak pH 8 and 7.4. 相似文献
18.
A novel thermostable, halostable carboxymethylcellulase (CMCase) from a marine bacterium Bacillus licheniformisAU01 was purified 10.4-fold with 18% yield with a specific activity of 88.43 U/mg and the molecular weight was estimated as
37 kDa. The enzyme was optimally active at pH 9–10 and temperature 50–60°C and it was most stable up to pH 12 and 20–30% of
NaCl concentration. The enzyme activity was reduced when treated with Hg 2+, Fe 2+ and EDTA and stimulated by Co 2+, Mn 2+, Mg 2+ and Ca 2+. Various cationic, anionic detergents and commercial detergents were not much affected CMCase activity. 相似文献
19.
Monovalent ion stimulated ATPase activity from oat ( Avena sativa) roots has been found to be associated with various membrane fractions (cell wall, mitochondrial and microsomal) of oat roots. The ATPase requires Mg 2+ (or Mn +2) but is further stimulated by K + and other monovalent ions. The monovalent ions are ineffective in the absence of the divalent activating cation. The ATPase has been described with respect to monovalent ion specificity, temperature, pH, substrate specificity, and Mg 2+ and K + concentrations. It was further shown that oligomycin inhibits a part of the total ATPase activity and on the basis of the oligomycin sensitivity it appears that at least 2 membrane associated ATPases are being measured. The mitochondrial fraction is most sensitive to oligomycin and the microsomal fraction is least sensitive to oligomycin. The oligomycin insensitive ATPase appears to be stimulated more by K + than the oligomycin sensitive ATPase. 相似文献
20.
Adenosine kinase (ATP:adenosine 5′-phosphotransferase, EC 2.7.1.20) from Lupinus luteus seeds has been obtained with good yield in almost homogeneous state by ammonium sulfate fractionation, chromatography on aminohexyl-Sepharose, and gel filtration. Active enzyme is a single polypeptide chain with a molecular weight of about 38,000 as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and gel nitration. Estimated molecular activity is 156. The enzyme exhibits a strict requirement for divalent metal ions. Among several ions tested the following appeared to be active as cofactors: Co 2+ ? Mn 2+ > Mg 2+ = Ca 2+ ? Ni 2+ > Ba 2+. The optimal metal ion concentrations were as follows: Mn 2+, 0.5 mm, Mg 2+ and Ca 2+, 1 mm, Co 2+, 1.5 mm. The adenosine kinase shows optimum activity at pH 7.0–7.5. Km values for adenosine and ATP are 1.5 × 10 ?6 and 3 × 10 ?4m, respectively. Lupin adenosine kinase is completely inhibited by antisulfhydryl reagents. ATP is the main phosphate donor and among other nucleoside triphosphates ITP, dATP, GTP, and XTP can substitute it but less effectively. Among the ribo- and deoxyribonucleosides occurring in nucleic acids adenosine is phosphorylated effectively and 2′-deoxyadenosine at a lower rate. Of other adenosine analogs tested all adenine d-nucleosides and purine derivative ribosides, besides those with a hydroxyl group at C-6, were found to be substrates for lupin adenosine kinase. Pyrimidine ribo- and deoxyribonucleosides were not phosphorylated. 相似文献
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