Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: HUMANGGP:019083 (5'-nucleotidase)
 3,019 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

A procedure for the isolation of primate skeletal microsomal membranes was initiated. Membranes exhibited specific enzymatic markers such as 5'-nucleotidase, Ca++,Mg(++)-adenosine triphosphatase and an ATP-dependent calcium uptake. Baboon skeletal microsomes bound specifically with high-affinity potent Ca++ channel blockers such as dihydropyridine, phenylalkylamine and benzothiazepine derivatives. Scatchard analysis of equilibrium binding assays with [3H](+)-PN 200-110, [3H](-)-desmethoxyverapamil [( 3H](-)-D888) and [3H]-d-cis-dilitiazem were consistent with a single class of binding sites for the three radioligands. The pharmacological profile of SR 33557, an original compound with calcium antagonist properties, was investigated using radioligand binding studies. SR 33557 totally inhibited the specific binding of the three main classes of Ca++ channel effectors and interacted allosterically with them. In addition, SR 33557 bound with high affinity to a homogeneous population of binding sites in baboon skeletal muscle.
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PMID:Interaction of SR 33557 with skeletal muscle calcium channel blocker receptors in the baboon: characterization of its binding sites. 185 30

Plasma membrane vesicles were purified from rat aortic myocytes by centrifugation in a discontinuous sucrose gradient. Vesicles were prepared in the presence or absence of five proteinase inhibitors (aprotinin, benzamidine, leupeptin, pepstatin A and phenylmethylsulfonyl fluoride). The proteinase inhibitors decreased the Vmax by 3.4-fold and had no effect on the Km for Ca2+ of Na+ gradient-dependent 45Ca2+ influx. The proteinase inhibitors had no direct effect on exchange activity, and they had no effect on membrane purity as indicated by 5'-nucleotidase activity. Removing the proteinase inhibitors or adding trypsin or chymotrypsin increased exchange activity approx. 2-fold. The Vmax of exchange activity in intact aortic myocytes is approx. 10-fold higher than the Vmax in plasma membrane vesicles prepared in the presence of proteinase inhibitors. Exchange activity in plasma membrane vesicles is only a sixtieth of the expected value, because the vesicles have approx. 7-fold higher 5'-nucleotidase activity and approx. 6-fold higher specific exchange activity than the crude homogenate. The large loss of exchange activity may be caused by a change in a regulatory domain of the exchanger because endogenous proteolysis restores some of the activity lost during vesicle preparation.
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PMID:Sodium-calcium exchange in membrane vesicles from aortic myocytes: stimulation by endogenous proteolysis masks inactivation during vesicle preparation. 189 60

Soluble low Km 5'-nucleotidase from human seminal plasma has been purified to homogeneity by one affinity and two gel-filtration chromatographic steps. The pure enzyme had a specific activity of 2000 nmol min-1 mg-1. Sodium dodecyl sulphate polyacrylamide gel electrophoresis of purified low Km 5'-nucleotidase revealed a single polypeptide band of 40 +/- 7 kDa and a tetrameric structure of 160 +/- 10 kDa has been proposed for the native enzyme. The kinetic properties of low Km 5'-nucleotidase have been determined and rather unique characteristics have been found for this soluble low Km 5'-nucleotidase: the substrate efficiency was slightly higher for IMP with an optimum pH at 7.5; the enzyme showed an absolute dependence on Mg2+ ions. Ca2+ could replace Mg2+ ions for activity while other divalent cations could not substitute for Mg2+; the enzymes were equally activated by ATP and ADP up to 0.1 mM concentrations. At higher concentrations up to 1 mM, ADP was still an activator while ATP caused a gradual decrease of activation to the native activity. This effect could not be related to the Mg-ATP = complexes since the enzymic preparation Mg(2+)-free still showed the same biphasic pattern of activation.
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PMID:Purification and partial characterization of the soluble low Km 5'-nucleotidase from human seminal plasma. 195 33

5'-Nucleotidase I (N-I) from rabbit heart was purified to homogeneity. After ammonium sulfate precipitation, the purification involved chromatography on phosphocellulose, DEAE-Sepharose, AMP-agarose, and ADP-agarose. The pure enzyme has a specific activity of 318 mumol (mg of protein)-1 min-1. Polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate yields a subunit molecular weight of 40,000. N-I is activated by ADP but not by ATP, in contrast to the 5'-nucleotidase (N-II) purified by Itoh et al. (1986), which is activated by ATP and, less well, by ADP. N-I displays sigmoidal saturation kinetics in the absence of ADP and hyperbolic kinetics in the presence of ADP. Partially purified N-I was previously shown to prefer AMP over IMP as substrate (Truong et al., 1988); this has been confirmed for pure N-I. Comparison of AMP and ADP concentrations reported to occur in heart with the kinetic behavior of N-I implicates N-I as the enzyme responsible for producing adenosine under conditions leading to a rise in ADP and AMP, such as hypoxia or increased workload. N-I is not activated by the ADP analogue adenosine 5'-methylenediphosphonate (AOPCP) and is only weakly inhibited by relatively high concentrations of AOPCP, in contrast to 5'-nucleotidase from plasma membrane, which is powerfully inhibited by this analogue. N-I shows an absolute dependence on Mg2+ ions. Mn2+ and Co2+ ions can replace Mg2+ ions as activator; Ni2+ and Fe2+ are much less effective, while Ca2+, Ba2+, Zn2+, and Cu2+ fail to activate the enzyme.
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PMID:5'-Nucleotidase I from rabbit heart. 199 69

Thirty-three different flavonoids were screened for their ability to influence ATP-dependent Ca2+ uptake by rat liver plasma membrane vesicles. Nine of the flavonoids, at a concentration of 100 microM inhibited Ca2+ uptake by more than 20%. The remaining 24 flavonoids exhibited little or no effect. The relative order of potency of the more biologically active flavonoids was myricetin greater than butein greater than phloretin = luteolin greater than eriodictyol = silybin. Myricitrin and phloridzin, the glycosides of myricetin and phloretin, respectively, had no effect. The degree of inhibition caused by myricetin was concentration dependent and was also affected by the preincubation time. After 10 min of preincubation, 52 microM myricetin lowered the initial rate of 45Ca uptake by 50%. The inhibition by myricetin was non-competitive with respect to Mg-ATP and of a mixed type with respect to Ca2+. At a concentration of 100 microM, myricetin had no effect on several plasma membrane enzymes such as 5'-nucleotidase, alkaline phosphatase and a Ca2(+)-activated ATPase but inhibited K(+)-dependent p-nitrophenyl phosphatase by 83%. The ATP-dependent Ca2+ transport systems located on the plasma membrane or endoplasmic reticulum derived from other tissues were also inhibited by myricetin. Analysis of the structure-activity relationship revealed that lipid solubility and polyhydroxylation particularly at positions 5,7,3' and 4' of the flavonoid ring structure enhanced the ability of the flavonoid to inhibit Ca2+ uptake. The results suggest that inhibition of Ca2+ transport activity probably involves the interaction of the phenolic groups of the flavonoid with the Ca2+ transporting protein.
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PMID:Effect of myricetin and other flavonoids on the liver plasma membrane Ca2+ pump. Kinetics and structure-function relationships. 199 24

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


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