Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:3.1.3.5 (5'-nucleotidase)
3,167 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The plasma membrane fraction of chicken osteoclasts was purified utilizing 20% continuous Percoll gradients. Biochemical marker enzyme analysis (ouabain-sensitive Na+,K(+)-ATPase and 5'-nucleotidase) indicated that plasma membrane enrichment was 11.87-fold and 7.25-fold, respectively, and contamination with mitochondria, endoplasmic reticulum, and lysosomes was low as determined by succinic dehydrogenase, NADH dehydrogenase, and N-acetylglucosaminidase activities, respectively. SDS latency of Na+,K(+)-ATPase and 5'-nucleotidase activities of the isolated plasma membranes revealed that 43-50% of vesicles were sealed, with 10-16% in the inside-out orientation, depending on the membrane fraction used. Electron microscopy confirmed the vesicular nature of the plasma membrane fraction. The plasma membrane Ca2(+)-ATPase had a high-affinity (KCa = 0.22 microM; Vmax = 0.16 mumol/mg per min) and a low-affinity (KCa = 148 microM; Vmax = 0.37 mumol/mg per min) component. Calmodulin (0.12 microM) had no effect on Ca2(+)-ATPase activity. However, trifluoperazine (0.1 mM), a calmodulin antagonist, strongly inhibited especially the high-affinity component of the enzyme. Vanadate and lanthanum also caused inhibition. In the presence of CDTA, a potent Ca2+ and Mg2+ chelating agent, high-affinity Ca2(+)-ATPase activity was abolished, indicating that trace Mg2+ was essential for activity. The Ca2(+)-ATPase substrate curve using ATP showed a high-affinity (Km = 12.3 microM; Vmax = 0.022 mumol/mg per min) and a low-affinity (Km = 43.8 microM; Vmax = 0.278 mumol/mg per min) component. These results demonstrate that osteoclasts have a plasma membrane Ca2(+)-ATPase with characteristics similar to the enzyme responsible for active calcium extrusion in other cells.
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PMID:Characterization of a Ca2(+)-ATPase in osteoclast plasma membrane. 214 47

A (H+ + K+)-ATPase-enriched membrane fraction derived from the fundic portion of hog gastric mucosa was obtained by a combination of differential and repeated 7% Ficoll gradient centrifugation. The microsomal membrane fraction isolated by repeated 7% Ficoll gradient centrifugation was free of ouabain-sensitive (Na+ + K+)-ATPase, 5'-nucleotidase and succinate dehydrogenase; and it was highly enriched in (H+ + K+)-ATPase and K(+)-stimulated p-nitrophenylphosphatase (p-NPPase). The (H+ + K+)-ATPase had a pH optimum of 7.4 and was stimulated by Tl+, K+, Rb+ and NH4+ with Ka values of 0.0667, 0.526, 0.667 and 3.03 mM, respectively, at this pH. On the other hand, monovalent cations such as Na+, Li+ and (CH3)4N+ as well as divalent cations such as Cu2+, Ca2+, Ba2+, Sr2+ and Cd2+ inhibited this enzyme activity concentration-dependently. Ouabain and oligomycin had no effect, whereas omeprazole, a specific (H+ + K+)-ATPase inhibitor, inhibited this enzyme activity in a pH-dependent manner. Sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis showed a major band (greater than or equal to 90% of protein) at 97,400 daltons, which was phosphorylated in the presence of Mg2+ and [gamma-32P]-ATP and dephosphorylated in the presence of K+. The present method was very simple, and the (H+ + K+)-ATPase activity of the microsomal fraction obtained by this method was much higher compared with those obtained by other methods such as free-flow electrophoresis.
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PMID:Purification and characterization of (H+ + K+)-ATPase from hog gastric mucosa. 215 97

Purified transverse tubule membranes from normal and dystrophic chicken skeletal muscle were isolated by a calcium-loading procedure. Normal and dystrophic T-tubules were similar in cholesterol content and (Na+,K+)-ATPase and 5'-nucleotidase activities but a significant decrease of Mg2(+)-ATPase activity was observed in dystrophic membranes. A comparative analysis of the enzyme properties revealed that the kinetic parameters were altered in dystrophic T-tubules and the ATP-hydrolyzing activity was differently affected by the ionic strength. However, the influence of temperature and the regulatory effect of concanavalin A were the same as in normal T-tubules. Membrane fluidity was similar in both preparations as estimated by fluorescence polarization of 1,6-diphenyl-1,3,5-hexatriene and trimethylammonium diphenylhexatriene. These results point to an impairment in the function of Mg2(+)-ATPase due to structural alterations of the enzyme.
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PMID:Abnormal properties of Mg2(+)-ATPase in transverse tubule membranes from dystrophic chicken. 215 57

There is need for a reliable index of zinc status in humans. Considering the importance of zinc in membrane function, activities of erythrocyte membrane enzymes have been measured in animals of low and normal zinc status as possible indices. Immature rats and neonatal pigs were fed low and adequate zinc diets; the latter was fed both ad libitum and restricted so as to control for food intake effects. Low rates of gain and plasma zinc concentrations demonstrated that animals fed the low zinc diets were of low zinc status. Erythrocyte membranes were prepared and assayed for Na,K-ATPase, 5'-nucleotidase, and calcium-ATPase activities. Na,K-ATPase activity was not affected by zinc status, but 5'-nucleotidase was significantly lower in deficient animals of both species than in controls, whose food intake was restricted to maintain comparable weight (2.76 vs 3.94 nmol/hr/mg of protein in rats and 60.5 vs 119 in pigs). The basal calcium-ATPase activities were also decreased by low zinc status in both species. Addition of calmodulin in vitro stimulated activity two-fold to four-fold and resulted in the same maximal activities for all treatments. The results show that erythrocyte membrane 5'-nucleotidase activity is an index of zinc status in these species. It is suggested that the decreased membrane calcium-ATPase activity in zinc deficiency is caused by a defect in calmodulin metabolism.
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PMID:Effect of zinc deficiency on enzyme activities in rat and pig erythrocyte membranes. 217 96

Basolateral and brush-border membranes were prepared from the intestines and kidneys of spontaneously hypertensive (SHR) and normotensive (WKY) rats fed on a calcium-adequate diet and assayed for their enzyme activities. In intestinal basolateral membranes the activities of Na+ K(+)-ATPase (EC 3.6.1.37) Ca2(+)-ATPase (EC 3.6.1.38) and alkaline phosphatase (EC 3.1.3.1) were lower in SHR rats when compared with WKY rats, whilst 5'-nucleotidase (EC 3.1.3.5) (a marker for basolateral membranes) was unaffected. In kidney basolateral membranes all enzymes were similar in activity in SHR and WKY rats. In intestinal brush-border membranes the activities of Ca2(+)-ATPase and alkaline phosphatase were lower in SHR rats when compared with WKY rats, whilst microvillus aminopeptidase (EC 3.4.11.2) (a marker for brush-border membranes) was unaffected. In kidney brush-border membranes all enzymes were similar in activity in SHR and WKY rats. The blood pressures of the SHR rats were considerably higher than those of the WKY rats. When SHR rats were fed on a Ca-deficient diet the activities of Na+K(+)-ATPase, Ca2(+)-ATPase and alkaline phosphatase in basolateral membranes and Ca2(+)-ATPase and alkaline phosphatase in brush-border membranes were all increased in the intestine when compared with SHR rats fed on a Ca-adequate diet. The equivalent enzymes in the kidneys of SHR rats, and the intestines and kidneys of WKY rats, were not affected by altering the Ca in the diet. The blood pressures of SHR rats fed on a Ca-deficient diet were higher than in those fed on a Ca-adequate diet. Blood pressures of WKY rats were not affected by altering the diet in this way. The results indicate that the absorption of Ca by active mechanisms may be reduced in SHR rats compared with WKY rats. Changing the level of Ca in the diet modified both blood pressure and the activities of enzymes which catalyse active Ca transport. The implications of these results to the aetiology, and possible nutritional treatment, of essential hypertension are discussed.
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PMID:The effect of diets adequate and deficient in calcium on blood pressures and the activities of intestinal and kidney plasma membrane enzymes in normotensive and spontaneously hypertensive rats. 231 78

KT5926, (8R*,9S*,11S*)-(-)-9-hydroxy-9-methoxycarbonyl-8-methyl-14-n-propoxy-2,3 ,9, 10-tetrahydro-8,11-epoxy, 1H,8H, 11H-2,7b,11a-triazadibenzo[a,g]cycloocta[cde] trinden-1-one, was found to be a potent and selective inhibitor of myosin light chain kinase. The compound inhibited both Ca2+/calmodulin-dependent and -independent smooth muscle myosin light chain kinases to a similar extent. The inhibition was not affected by the concentration of calmodulin. Kinetic analyses showed that the mode of inhibition was of the competitive type with respect to ATP (Ki, 18 nM) and of the noncompetitive type with respect to myosin light chain (Ki, 12 nM). These results indicated that KT5926 directly interacted with the enzyme at the catalytic site. KT5926 also inhibited other protein kinases, but with relatively high Ki values; the values for protein kinase C, cAMP-dependent protein kinase, and cGMP-dependent protein kinase were 723, 1200, and 158 nM, respectively. Ca2(+)-ATPase, Na+/K(+)-ATPase, hexokinase, and 5'-nucleotidase were not inhibited by KT5926 at less than 10 microM. The effect of KT5926 on serotonin secretion and protein phosphorylation induced by platelet-activating factor or phorbol ester was examined in rabbit platelets. KT5926 inhibited the phosphorylation of a 20-kDa protein but had no effect on the phosphorylation of a 40-kDa protein, thereby indicating that the compound exerts its selective inhibition of myosin light chain kinase in intact cells. The compound inhibited serotonin secretion induced by platelet-activating factor, but its potency was significantly less than that of K-252a, (8R*,9S*,11S*)-(-)-9-hydroxy-9-methoxycarbonyl-8-methyl-2,3,9, 10-tetrahydro-8,11-epoxy-1H,8H,11H-2,7b, 11a-triazadibenzo[a,g]cycloocta [cde]trinden-1-one, which inhibited the phosphorylation of both the 20-kDa protein and the 40-kDa protein. Phorbol ester-induced secretion was not suppressed by KT5926. These results provide the evidence that both the 20-kDa protein phosphorylation by myosin light chain kinase and the 40-kDa protein phosphorylation by protein kinase C substantially contribute to the secretion response in platelets.
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PMID:KT5926, a potent and selective inhibitor of myosin light chain kinase. 232 35

Inositol 1,4,5-trisphosphate [Ins(1,4,5)P3], arising from hydrolysis of phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P2], is proposed as the link between membrane-receptor activation and mobilization of Ca2+ from intracellular sites in hormone-secreting cells. The location of Ins(1,4,5)P3-sensitive membranes was investigated in cultured neonatal beta-cells. Membranes were obtained after lysis of cells attached to positively charged Sephadex. After lysis the presence of the enzyme markers 5'-nucleotidase, glucose-6-phosphatase, NADH-cytochrome c reductase, UDP-galactosyltransferase and succinate dehydrogenase indicated the mixed nature of the preparation. After sonication, however, UDP-galactosyltransferase and succinate dehydrogenase activities were undetectable, but 4.8% of total cellular glucose-6-phosphatase and 3.4% of total cellular NADH-cytochrome c reductase remained with 5'-nucleotidase in the preparation, indicating endoplasmic-reticulum association. ATP-dependent 45Ca2+ accumulation was shown in this preparation (410 +/- 24 pmol/mg of protein at 150 nM free Ca2+) and was inhibited by vanadate (100 microM). Ca2+ release was effected by Ins(1,4,5)P3, with half-maximal release at 0.5 +/- 0.14 microM-Ins(1,4,5)P3, t1/2 11.2 +/- 1.1 s. GTP- and guanosine 5'-[beta gamma-imido]triphosphate (p[NH]ppG)-promoted release of 45Ca2+ was demonstrated in this preparation, but the kinetics of release (half-maximal Ca2+ release at 5.4 +/- 0.7 microM, with t1/2 77.3 +/- 6.9 s, and at 51.1 +/- 4.2 microM, with t1/2 19.0 +/- 2.2 s, for GTP and p[NH]ppG respectively), and the ability of neomycin sulphate to block p[NH]ppG-induced release only, are indicative of separate release mechanisms after treatment with these agents. A close association between plasma membrane and elements of the endoplasmic reticulum is indicated in this model, providing a possible mechanism for local alterations in free Ca2+ in the sub-plasma-membrane region.
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PMID:GTP- and inositol 1,4,5-trisphosphate-induced release of 45Ca2+ from a membrane store co-localized with pancreatic-islet-cell plasma membrane. 245 19

The dnaK protein of Escherichia coli has been shown to possess both autophosphorylating and 5'-nucleotidase activities. The dnaK protein has been shown to bind avidly to ATP, but hydrolyzing it slowly. In vitro autophosphorylation occurs at a threonine residue when either ATP or GTP are used as phosphate donors. The extent of autophosphorylation is low; only a few percent of the molecules are phosphorylated. This activity is stimulated at least tenfold in the presence of Ca2+ ions with either ATP or GTP as the donor. The autophosphorylating activity of the mutant dnaK756 protein in the presence or absence of Ca2+ is reduced compared to that of the wild type.
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PMID:Biochemical properties of the Escherichia coli dnaK heat shock protein and its mutant derivatives. 251 98

Membrane-bound 5'-nucleotidase from Vibrio parahaemolyticus was solubilized and purified using a nonionic detergent, heptyl-beta-D-thioglucoside, and was characterized. This enzyme required Mg2+ for activity, maximum activity being observed at 5 and 20 mM Mg2+ with AMP and ATP, respectively, as substrates. Of the divalent cations tested, Mn2+ and Co2+ were able to replace Mg2+ partially, whereas Ca2+ was ineffective. Zinc strongly inhibited the enzyme activity and Ni2+ caused partial inhibition. This enzyme required Cl- for activity, the optimal concentration being 20 mM or more. The order of effectiveness of anions was Cl- greater than Br- greater than I- approximately NO3-. Sulfate and acetate were ineffective. The optimal pH was 8.0. The activity of the purified enzyme was stimulated by the addition of lipid to the assay mixture. This enzyme hydrolyzed all 5'-nucleotides tested, but did not hydrolyze 3'-nucleotides or ribose 5-phosphate. On sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the enzyme appeared to be a single polypeptide, with a molecular weight of 72 kDa.
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PMID:Purification and characterization of membrane-bound 5'-nucleotidase of Vibrio parahaemolyticus. 254 26

Inositol 1,4,5-trisphosphate (IP3) binding to, and Ca2+ uptake and release by plasma membrane- and endoplasmic reticulum-enriched fractions of rat liver were measured after continuous Escherichia coli endotoxin (ET) administration in vivo. IP3 binding to both fractions was significantly reduced by ET treatment. This was associated with decreased Ca2+ uptake and impaired IP3-dependent Ca2+ release. A decrease of 5'-nucleotidase specific activity of plasma membrane-enriched fraction was also observed in ET treated rats. The results suggest that previously observed impairments in the ability of hepatocytes to mobilize Ca2+, to activate glycogen phosphorylase and to respond--when saponin permeabilized--by Ca2+ release upon IP3 addition during chronic endotoxemia are due to alterations in both IP3 binding to the subcellular fractions that are imputed to be targets of IP3, and a decrease in the size of IP3-sensitive pool of releasable Ca2+.
Cell Calcium
PMID:Alterations in binding of inositol 1,4,5-trisphosphate to subcellular structures of rat liver during chronic endotoxemia. 255 Jan 37


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