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)

A membrane fraction enriched in plasma membrane marker enzymes K+-dependent p-nitrophenyl phosphatase, 5'-nucleotidase and alkaline phosphatase was prepared from rat parotid glands using Percoll self-forming gradient. This fraction contained an ATP-dependent CA2+ transport system which was distinct from those located on the endoplasmic reticulum and mitochondria of parotid glands. The Km for ATP was 0.57 +/- 0.07 mM (n = 3). Nucleotides other than ATP such as ADP, AMP, GTP, CTP, UTP or ITP were unable to support significant Ca2+ uptake. ATP-dependent Ca2+ uptake displayed sigmoidal kinetics with respect to free Ca2+ concentration with a Hill coefficient of 2.02. The K0.5 for Ca2+ was 44 +/- 3.1 nM (n = 3) and the average Vmax was 13.5 +/- 1.1 nmol/min per mg of protein. The pH optimum was 7.2. Trifluorperazine inhibited Ca2+ transport with half maximal inhibition observed at 30.8 microM. Complete inhibition was observed at 70 microM trifluorperazine. Exogenous calmodulin however had no effect on the rate of transport. Na+ and K+ ions activated Ca2+ transport at 20 to 30 mM ion concentrations. Higher concentrations of Na+ or K+ were inhibitory.
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PMID:Characterisation of an ATP-dependent Ca2+ transport system in a plasma membrane enriched fraction from rat parotid. 345 46

Although acute alterations in Ca2+ fluxes may mediate the skeletal responses to certain humoral agents, the processes subserving those fluxes are not well understood. We have sought evidence for Ca2+-dependent ATPase activity in isolated osteoblast-like cells maintained in primary culture. Two Ca2+-dependent ATPase components were found in a plasma membrane fraction: a high affinity component (half-saturation constant for Ca2+ of 280 nM, Vmax of 13.5 nmol/mg per min) and a low affinity component, which was in reality a divalent cation ATPase, since Mg2+ could replace Ca2+ without loss of activity. The high affinity component exhibited a pH optimum of 7.2 and required Mg2+ for full activity. It was unaffected by potassium or sodium chloride, ouabain or sodium azide, but was inhibited by lanthanum and by the calmodulin antagonist trifluoperazine. This component was prevalent in a subcellular fraction which was also enriched in 5'-nucleotidase and adenylate cyclase activities, suggesting the plasma membrane as its principal location. Osteosarcoma cells, known to resemble osteoblasts in their biological characteristics and responses to bone-seeking hormones, contained similar ATPase activities. Inclusion of purified calmodulin in the assay system caused small non-reproducible increases in the Ca2+-dependent ATPase activity of EGTA-washed membranes. Marked, consistent calmodulin stimulation was demonstrated in membranes exposed previously to trifluoperazine and then washed in trifluoperazine-free buffer. These results indicate the presence of a high affinity, calmodulin-sensitive Ca2+-dependent ATPase in osteoblast-like bone cells. As one determinant of Ca2+ fluxes in bone cells, this enzyme may participate in the hormonal regulation of bone cell function.
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PMID:A high affinity, calmodulin-responsive (Ca2+ + Mg2+)-ATPase in isolated bone cells. 613 20

The in vitro stimulation of human and rabbit erythrocyte membrane Ca2+-ATPase activity by physiological concentrations of thyroid hormone has recently been described. To extend these observations to a nucleated cell model, Ca2+-ATPase activity in a membrane preparation obtained from rabbit myocardium has been studied. Activity of 5'-nucleotidase in the preparation was increased 26-fold over that of myocardial homogenate, consistent with enrichment by sarcolemma. Mean basal enzyme activity in membranes from nine animals was 20.8 +/- 3.3 mumol Pi mg membrane protein-1 90 min-1, approximately 20-fold the activity described in rabbit red cell membranes. Exposure of heart membranes in vitro to L-thyroxine (T4) (10(-10)M) increased Ca2+-ATPase activity to 29.2 +/- 3.8 mumol Pi (P less than 0.001). Dose-response studies conducted with T4 showed that maximal stimulatory response was obtained at 10(-10) M). Hormonal stimulation was comparable for L-T4 and triiodo-L-thyronine (T3) (10(-10) M). Tetraiodothyroacetic acid was without biological activity, whereas triiodothyroacetic acid and D-T4, each at 10(-10) M, significantly decreased enzyme activity compared to control (basal) levels. The action of L-T4 on myocardial membrane Ca2+-ATPase activity was inhibited by trifluoperazine (100 microM) and the naphthalenesulfonamide W-7 (50-100 microM), compounds that block actions of calmodulin, the protein activator of membrane-associated Ca2+-ATPase. Radioimmunoassay revealed the presence of calmodulin (1.4 micrograms/mg membrane protein-1) in the myocardial membrane fraction and 0.35 micrograms/mg-1 in cytosol. Myocardial Ca2+-ATPase activity, apparently of sarcolemmal origin, is thus thyroid hormone stimulable. The hormonal responsiveness of this calcium pump-associated enzyme requires calmodulin.
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PMID:Rabbit myocardial membrane Ca2+-adenosine triphosphatase activity: stimulation in vitro by thyroid hormone. 623 Sep 96

1. Incubation of Schistosoma mansoni for 5 min in a phosphate-buffered medium, pH 7.4, released tegumental material containing the following phosphohydrolase activities: alkaline phosphatase, 5'-nucleotidase, glycerol-2-phosphatase, glucose 6-phosphatase, phosphodiesterase and ATPase. 2. Maximum activity of these enzymes was measured at pH 9.5; however, the phosphodiesterase and ATPase activities were also appreciable at pH 7.0. 3. Solubilization of the released tegumental material in 1% Triton X-100 followed by gel filtration distinguished three peaks of enzyme activity: an ATPase (mol.wt. greater than 1000 000), a phosphodiesterase (mol.wt. 1 000 000) and an alkaline phosphomonoesterase with broad specificity (mol.wt. 232 000). 4. The ATPase activity was highly activated by 10 mM-Mg2+ or 1 mM-Ca2+ and was inhibited by chelating agents. Ouabain, Na+ and K+ had little effect on enzyme activity, whereas activity was increased by 50% in the presence of calmodulin. The phosphodiesterase activity was highest in the presence of 100 mM-Na+ or -K+, and 10 mM-Mg2+ or -Ca2+. Alkaline phosphatase activity was also stimulated by 100 mM-Na+ or -K+, and 10 mM-Mg2+; however Ca2+ inhibited at greater than 1 mM. 5. Surface iodination of parasites followed by detergent solubilization and gel filtration of the released tegumental membranes indicated that these enzymes were not accessible. A major surface component, apparent mol.wt. 80 000, was iodinated. 6. Rabbit anti-(mouse liver 5'-nucleotidase) antibodies did not inhibit the phosphohydrolase activities. However, an immunoglobulin G fraction from sera of mice chronically infected with S. mansoni partially inhibited alkaline phosphatase activity, but was without effect on the phosphodiesterase and ATPase activities. 7. The location of the enzymes in the double membrane of the tegument and their significance in host-parasite interactions is discussed.
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PMID:Properties of a series of tegumental membrane-bound phosphohydrolase activities of Schistosoma mansoni. 627 49

Two substrate proteins for cAMP-dependent protein kinase detected in a rat heart sarcolemma preparation displayed molecular weights of 24,000 and 9000 in sodium dodecyl sulfate gels and were shown to be interconvertible. The 9000-dalton protein could readily be separated from other low molecular weight phosphoproteins (mol. wt. 14,000 and 7000) by the use of 15% polyacrylamide gels. In addition to an endogenous cAMP-dependent protein kinase the membrane preparation also contained a protein-phosphorylation system that required Ca2+ and calmodulin. It appeared that both 24,000- and 55,000-dalton proteins were substrates for the endogenous Ca2+- and calmodulin-dependent protein kinase. Contaminating sarcoplasmic reticulum vesicles, first loaded with calcium oxalate, could be separated from the enriched sarcolemma preparation by sucrose gradient centrifugation. The separation was confirmed by comparative analysis of 5'-nucleotidase, Na+ -Ca2+ antiporter, and (Ca2+ + Mg2+)-dependent ATPase activities and by determination of gel electrophoretic (phospho)protein composition, sialic acid, cholesterol, and phospholipid contents. The 24,000-dalton phosphoprotein complex was equally distributed between sarcolemmal and sarcoplasmic reticulum fractions, whereas the 55,000- and 7000-dalton proteins were predominantly found in the sarcolemmal fraction. The 24,000-dalton protein was most likely phospholamban, because no other phosphoprotein was found in the 20,000 molecular weight range.
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PMID:Phosphorylation of low-molecular-weight proteins in preparations of rat heart sarcolemma and sarcoplasmic reticulum. 630 73

Experiments were carried out to test the hypothesis that membrane injury associated with Ca2+ depletion of the heart is involved in the development of Ca2+ overload during the Ca2+ paradox phenomenon. Biochemical properties of sarcolemma (SL) and sarcoplasmic reticulum (SR) isolated from the rabbit heart after 10 min of Ca2+-free perfusion were examined. This treatment predisposes hearts to the Ca2+ paradox as assessed by perfusate creatine kinase (CK) activity and heart contractility during reperfusion with Ca2+. Homogenates prepared from Ca2+ depleted heart were examined for their capacity to accumulate Ca2+ in the presence of ATP, which is mainly a property of the SR Ca2+ pump. The initial rate of Ca2+ pumping was 55% less in the Ca2+ depleted heart. The activities of 5'-nucleotidase, Na+/K+-ATPase and Na+/Ca2+ antiporter, and the Ca2+ permeability were studied in isolated SL vesicles. Na+/K+-ATPase activity was 75% less in SL isolated from Ca2+ depleted hearts, no significant changes were observed with the other parameters studied. Calmodulin (CaM) content in SL, assayed by radioimmunoassay, was unchanged. However, a 98% increase was observed in homogenates prepared from Ca2+ depleted hearts. The possible involvement of CaM in the Ca2+ paradox phenomenon is discussed. The data provide evidence that the net Ca2+ gain of myocardial cells in Ca2+ repletion may in part be associated with a loss of the ability of the SL and SR to remove Ca2+ from the cytosol.
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PMID:Diminished Na+/K+ and Ca2+ pump activities in the Ca2+ depleted heart: possible role in the development of Ca2+ overload during the Ca2+ paradox. 631 44

Sarcolemmal fractions of vascular smooth muscles were prepared from porcine thoracic aortae by differential and sucrose density gradient centrifugation. In these fractions, there was a high activity of 5'-nucleotidase, a putative marker enzyme of plasma membrane, and a low activity of rotenone insensitive NADH-cytochrome c reductase a marker of sarcoplasmic reticulum. In these fractions, the Ca2+ uptake was ATP-dependent. A low concentration of saponin which inhibited Ca2+ uptake by the plasma membrane but not by the sarcoplasmic reticulum, inhibited 65% of the Ca2+ uptake of this fraction. The Ca2+ uptake of this fraction was enhanced by cAMP- and cGMP-dependent protein kinases, and by calmodulin. The cAMP-dependent protein kinase enhanced the phosphorylation of 28 and 22 kDa proteins, while the cGMP-dependent protein kinase phosphorylated the 35 kDa protein. The phosphorylation of 100, 75, 65, 41 and 22 kDa proteins was enhanced by Ca2+ and calmodulin. These results indicate that cAMP- and cGMP-dependent protein kinases as well as calmodulin play important roles in Ca2+ transport in the sarcolemma, and that the phosphorylated proteins may be associated with an enhancement of Ca2+ transport in the sarcolemma.
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PMID:Effects of cAMP- and cGMP-dependent protein kinases, and calmodulin on Ca2+ uptake by highly purified sarcolemmal vesicles of vascular smooth muscle. 632 80

ATP promotes 45Ca uptake by the microsomal fraction from the longitudinal smooth muscle of guinea-pig ileum and this uptake is stimulated by oxalate. As the microsomal fraction is made up of various subcellular entities, we examined the localization of the Ca2+-transport activity by density gradient centrifugation, taking advantage of the selective effect of digitonin (at low concentration) on the density of plasmalemmal elements. When the 45Ca-uptake activity was measured in the absence of oxalate, its behavior in subfractionation experiments closely paralleled that of the plasmalemmal marker 5'-nucleotidase. In contrast, the additional Ca2+-transport activity elicited by oxalate behaved like NADH-cytochrome c reductase, a putative endoplasmic reticulum marker. The endoplasmic reticulum vesicles constituted only a small part of the membranes in the microsomal fraction, which explains that their Ca2+-storage capacity was not detectable in the absence of Ca2+-trapping agent. Low digitonin concentrations selectively increased the Ca2+ permeability of the plasmalemmal vesicles. The two Ca2+-transport activities were further differentiated by their distinct sensitivity of K+, vanadate and calmodulin. In this respect, the oxalate-insensitive and oxalate-stimulated Ca2+-transport systems resembled, respectively, the sarcolemmal and sarcoplasmic reticulum Ca2+ pumps in cardiac and skeletal muscle, in accordance with the subcellular locations established by density gradient centrifugation.
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PMID:Differentiation of Ca2+ pumps linked to plasma membrane and endoplasmic reticulum in the microsomal fraction from intestinal smooth muscle. 645 27

A Ca2+- and calmodulin-dependent casein kinase specific for dephosphorylated bovine kappa-casein was identified in a microsomal fraction of mammary acini prepared from rats in late lactation. This phosphorylation has an absolute requirement for Mg2+ for either the basal or the Ca2+- and calmodulin-dependent activity. One-half of the maximal stimulation is achieved at a calmodulin concentration of 204nM in the presence of Ca2+. The Ca2+- and calmodulin-dependent kinase activity (but not the basal activity) is inhibited by trifluoperazine. The casein kinase is associated with a microsomal fraction enriched in markers for plasma membrane and Golgi (5'-nucleotidase and galactosyltransferase respectively). The activity of this casein kinase remains relatively constant throughout lactation, but declines dramatically in 24h when rats are removed from their pups. This activity may represent the physiological activity responsible in part or whole for kappa-casein phosphorylation occurring before micelle formation and milk secretion.
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PMID:Calcium-ion and calmodulin-dependent kappa-casein kinase in rat mammary acini. 659 96

Cell swelling and elevated intracellular Ca2+ increase K+ permeability in lymphocytes. Experiments were performed to test whether these effects can also be elicited in isolated plasma membrane vesicles. Rabbit thymocytes, used as a source of membrane vesicles, were found to regain their volume after swelling in hypotonic, low-K+ media. This regulatory volume decrease (RVD) was inhibited by quinine and trifluoperazine, but not affected by ouabain. Both efflux and uptake of K+ (86Rb) were stimulated by hypotonicity. Addition of A23187 plus Ca2+ also increased 86Rb fluxes. Ca2+- and volume-induced 86Rb fluxes were also studied in isolated membranes. A plasma membrane-rich vesicle fraction, enriched over 11-fold in 5'-nucleotidase, was isolated from thymocytes. The vesicles were about 35% inside-out and trapped 86Rb in an osmotically active compartment of approximately 1.3 microliter/mg protein. Equilibrium exchange fluxes of 86Rb in the vesicles were unaffected by Ca2+ with or without A23187. Calmodulin had no effect on 86Rb permeability but stimulated ATP-dependent Ca2+ accumulation. Hypotonic swelling increased both uptake and efflux of 86Rb from vesicles. However, this increase was not blocked by either quinine or trifluoperazine, was not specific for K+ (86Rb), and is probably unrelated to RVD. It is concluded that components essential for the volume- and Ca2+-induced changes in K+ permeability are lost or inactivated during membrane isolation. An intact cytoarchitecture may be required for RVD.
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PMID:Induction of 86Rb fluxes by Ca2+ and volume changes in thymocytes and their isolated membranes. 660 61


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