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:3.6.1.3 (ATPase)
65,361 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Calponin from chicken gizzard consists of two principal components, possibly isoforms, separable by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and isoelectric focusing. Cleavage with 2-nitro-5-thiocyanobenzoic acid indicated that calponin contains 2 cysteine residues. Purified fragments of 30 and 21 kDa retained the following properties of the intact protein: actin-, tropomyosin- and calmodulin-binding, and ability to inhibit the actin-activated MgATPase activity of smooth muscle myosin. Both fragments, like intact calponin, were phosphorylated by protein kinase C which inhibited their binding to actin and relieved their inhibition of the ATPase. Tryptic digestion of calponin phosphorylated by protein kinase C generated 3 phosphopeptides with the following N-terminal sequences: FASQQGMTAYGTR, GASQQGMTVYGLP, and NHSGHVQ, each possessing a single phosphoserine.
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PMID:Structural and functional characterization of calponin fragments. 215 Oct 18

The effects and modes of action of certain lipid second messengers and protein kinase C regulators, such as sphingosine, lysophosphatidylcholine (lyso-PC), and oleic acid, on Na,K-ATPase and sodium pump were examined. Inhibition of purified rat brain synaptosome Na,K-ATPase by these lipid metabolites, unlike that by ouabain, was subject to membrane dilution (i.e. inhibition being counteracted by increasing amounts of membrane lipids). Kinetic analysis, using the purified enzyme, indicated that sphingosine and lyso-PC were likely to interact, directly or indirectly, with Na+-binding sites of Na,K-ATPase located at the intracellular face of plasma membranes, a conclusion also supported by studies on Na,K-ATPase and 22Na uptake using the inside-out vesicles of human erythrocyte membranes. The studies also showed that ouabain (but not sphingosine and lyso-PC) increased the affinity constant (K0.5) for K+, whereas sphingosine and lyso-PC (but not ouabain) increased K0.5 for Na+. Sphingosine and lyso-PC inhibited 86Rb uptake by intact human leukemia HL-60 cells at potencies comparable to those for inhibitions of purified Na,K-ATPase and protein kinase C. It is suggested that Na,K-ATPase (sodium pump) might represent an additional target system, besides protein kinase C, for sphingosine and possibly other lipid second messengers.
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PMID:Inhibition of Na,K-ATPase and sodium pump by protein kinase C regulators sphingosine, lysophosphatidylcholine, and oleic acid. 215 29

The kinetics of phosphorylation of an integral membrane enzyme, Na+/K(+)-ATPase, by calcium- and phospholipid-dependent protein kinase C (PKC) were characterized in vitro. The phosphorylation by PKC occurred on the catalytic alpha-subunit of Na+/K(+)-ATPase in preparations of purified enzyme from dog kidney and duck salt-gland and in preparations of duck salt-gland microsomes. The phosphorylation required calcium (Ka approximately 1.0 microM) and was stimulated by tumor-promoting phorbol ester (12-O-tetradecanoylphorbol 13-acetate) in the presence of a low concentration of calcium (0.1 microM). PKC phosphorylation of Na+/K(+)-ATPase was rapid and plateaued within 30 min. The apparent Km of PKC for Na+/K(+)-ATPase as a substrate was 0.5 microM for dog kidney enzyme and 0.3 microM for duck salt-gland enzyme. Apparent substrate inhibition of PKC activity was observed at concentrations of purified salt-gland Na+/K(+)-ATPase greater than 1.0 microM. Phosphorylation of purified kidney and salt-gland Na+/K+ ATPases occurred at both serine and threonine residues. The 32P-phosphopeptide pattern on 15% sodium dodecyl sulfate-polyacrylamide gel electrophoresis after hydroxylamine cleavage of pure 32P-phosphorylated alpha subunit was the same for the two sources of enzyme, which suggests that the phosphorylation sites are similar. The results indicate that Na+/K(+)-ATPase may serve as a substrate for PKC phosphorylation in intact cells and that the Na+/K(+)-ATPase could be a useful in vitro model substrate for PKC interaction with integral membrane proteins.
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PMID:Kinetics of phosphorylation of Na+/K(+)-ATPase by protein kinase C. 215 96

The effects and modes of action of certain antineoplastic phospholipid analogues (racemic 1-O-octadecyl-2-O-methyl glycero-3-phosphocholine, BM 41.440, JH-1, CV-3988, and HePC) on (sodium plus potassium)-activated adenosine triphosphatase (Na,K-ATPase) and sodium pump activities were investigated. Inhibition of Na,K-ATPase in purified rat brain synaptosomal membranes by these lipids, in contrast to ouabain, was subject to membrane surface dilution and unaffected by whether the reaction was started with KCl, NaCl, or ATP. Kinetic analysis indicated that the analogues, again dissimilar to ouabain, were likely to interact directly or indirectly with sodium-binding sites of Na,K-ATPase located at the intracellular surface of the plasma membrane, a conclusion also supported by studies using the inside-out vesicles of human erythrocyte membranes. The studies also showed that ouabain (but not the lipids) increased the affinity constant of Na,K-ATPase for K+, whereas the lipids (but not ouabain) increased that for Na+. The lipids also inhibited 86Rb uptake by intact human leukemia HL60 cells at potencies quite comparable to those seen for inhibition of purified protein kinase C or Na,K-ATPase. It is suggested that Na,K-ATPase (sodium pump) might represent a hitherto unrecognized site of action for the lipid analogues, and that the antineoplastic effects of the agents might be due to, in part, inhibition of both protein kinase C and Na,K-ATPase and perhaps other membrane-associated enzymes.
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PMID:Inhibition of protein kinase C, (sodium plus potassium)-activated adenosine triphosphatase, and sodium pump by synthetic phospholipid analogues. 215 69

Calponin isolated from chicken gizzard smooth muscle inhibits the actin-activated MgATPase activity of smooth muscle myosin in a reconstituted system composed of contractile and regulatory proteins. ATPase inhibition is not due to inhibition of myosin phosphorylation since, at calponin concentrations sufficient to cause maximal ATPase inhibition, myosin phosphorylation was unaffected. Furthermore, calponin inhibited the actin-activated MgATPase of fully phosphorylated or thiophosphorylated myosin. Although calponin is a Ca2(+)-binding protein, inhibition did not require Ca2+. Furthermore, although calponin also binds to tropomyosin, ATPase inhibition was not dependent on the presence of tropomyosin. Calponin was phosphorylated in vitro by protein kinase C and Ca2+/calmodulin-dependent protein kinase II, but not by cAMP- or cGMP-dependent protein kinases, or myosin light chain kinase. Phosphorylation of calponin by either kinase resulted in loss of its ability to inhibit the actomyosin ATPase. The phosphorylated protein retained calmodulin and tropomyosin binding capabilities, but actin binding was greatly reduced. The calponin-actin interaction, therefore, appears to be responsible for inhibition of the actomyosin ATPase. These observations suggest that calponin may be involved in regulating actin-myosin interaction and, therefore, the contractile state of smooth muscle. Calponin function in turn is regulated by Ca2(+)-dependent phosphorylation.
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PMID:Smooth muscle calponin. Inhibition of actomyosin MgATPase and regulation by phosphorylation. 216 34

The short term regulation of the activity of the Na,K-pump (Na+,K(+)-ATPase) is just beginning to be understood. By using single microdissected proximal tubule segments (PCT) (permeabilized in order to clamp Na entry), it was possible to study regulation of Na+,K(+)-ATPase activity in its own environment and in a well defined cell population. The Na+,K(+)-ATPase activity can be regulated over a short term via guanidine triphosphate (GTP) dependent regulatory proteins. However the guanidine proteins are not directly coupled to the Na,K-pump and the mechanism involves the activation of complex intracellular signalling system. Locally produced dopamine induces a dose dependent inhibition of Na+,K+ ATPase activity. This inhibition is mediated by a complex mechanism that requires the activation of both membrane dopamine receptors, DA-1 and DA-2. It involves the activation of a pertussis toxin sensitive GTP-binding protein and activation of protein kinase C. A DA-2 agonist only inhibits Na+,K(+)-ATPase activity when it is incubated together with dibutyryl cAMP or Forskolin. We have therefore concluded that an increase in cellular cAMP levels plays a permissive role for DA-2 inhibition of Na+,K(+)-ATPase activity. A fully differentiated cell is required for dopamine inhibition of Na+,K(+)-ATPase activity. An abnormal regulation of proximal tubule Na+,K(+)-ATPase activity might be of importance in the pathogenesis of certain types of hypertension.
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PMID:Short-term regulation of Na+,K(+)-ATPase activity by dopamine. 216 34

To elucidate the regulation mechanisms for sarcolemmal Ca2(+)-pumping ATPase of vascular smooth muscle, the preparation of the membrane fraction of porcine aorta with which the enzyme activity could be analyzed was attempted. A Ca2(+)-activated, Mg2(+)-dependent ATPase [Ca2(+)+Mg2+)-ATPase) activity with high affinity for Ca2+ (Km = 79 +/- 18 nM) was found in a sarcolemma-enriched fraction obtained from digitonin-treated microsomes that possessed the essential properties of plasma membrane (PM) Ca2(+)-pumping ATPases, as determined for the erythrocyte and cardiac muscle enzymes. The activity was stimulated by calmodulin and inhibited by low concentrations of vanadate. Saponin had a stimulatory effect on it. The existence of the PM enzyme in the membrane fraction was substantiated by the Ca2(+)-dependent, hydroxylamine sensitive phosphorylation of a 130K protein, which could be selectively enhanced by LaCl3. The enzyme activity was potentiated by either cGMP or a purified G-kinase. Purified protein kinase C potentiated the enzyme activity. However, none of these agents stimulated the activity of the enzyme purified from microsomes by calmodulin affinity chromatography. The results suggest that the sarcolemmal Ca2(+)-pumping ATPase of vascular smooth muscle is regulated by these protein kinases not through phosphorylation of the enzyme itself but through phosphorylation of membrane components(s) other than the enzyme. Phosphatidylinositol phosphate was found to stimulate the enzyme, suggesting its role in mediation of the stimulatory effects of the protein kinases.
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PMID:Sarcolemmal (Ca2(+)+Mg2+)-ATPase of vascular smooth muscle and the effects of protein kinases thereupon. 216 73

The efflux of GSH has been shown previously to be a saturable process in both isolated rat hepatocytes and perfused liver, suggesting a carrier-mediated transport mechanism. The possibility in hormonal regulation of this process has been raised by recent reports. Our present work examined the role of hormones known to affect intracellular signal transduction mechanisms on GSH efflux in cultured rat hepatocytes and perfused rat livers. We found that cAMP-dependent factors, such as cholera toxin (CT), dibutyryl cAMP, forskolin, and glucagon all stimulated GSH efflux in cultured rat hepatocytes. The efflux kinetics were compared in cultured cells incubated with or without CT; the stimulation of GSH efflux was related to a near doubling of the Vmax while exhibiting no significant alteration of the Km. The increase in intracellular cAMP level associated with the threshold for this stimulatory effect was 25% above control. The stimulatory effect of CT could not be blocked by cyclohexamide pretreatment or reversed by colchicine treatment. The stimulatory effect of glucagon was abolished in the presence of ouabain but not in the presence of barium. On the other hand, hormones which act through Ca2+ and protein kinase C, such as phenylephrine and vasopressin, had no effect on GSH efflux in the cultured cells. In the perfused liver model, glucagon (10 nM) and dibutyryl cAMP (8 microM) stimulated sinusoidal GSH efflux to 130 and 144% of control values, respectively, and increased bile flow while not affecting biliary GSH efflux. Finally, the physiological significance of glucagon-mediated stimulation of sinusoidal GSH efflux was assessed by both plasma GSH and glucose levels in response to in vivo glucagon infusion. The threshold dose of glucagon for significant increase in plasma GSH (5.21 pmol/min) was lower than for glucose (15.61 pmol/min). At the highest glucagon infusion rate (261 pmol/min), plasma GSH level doubled while glucose level increased 80%. In conclusion, increased cAMP stimulates GSH efflux in cultured rat hepatocytes and perfused livers. The stimulatory effect of cAMP is exerted at the sinusoidal pole and appears to be mediated by hyperpolarization of hepatocytes by stimulation of Na(+)-K(+)-ATPase. In vivo studies confirmed the importance of cAMP-mediated stimulation of sinusoidal GSH efflux as it resulted in significant elevation of the plasma GSH level.
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PMID:Hormonal regulation of glutathione efflux. 216 79

The present study was undertaken to examine the cellular interaction between a Na+/K(+)-ATPase inhibitor, ouabain, and arginine vasopressin (AVP) in rat vascular smooth muscle cells (VSMC) in culture. Preincubation with 10(-5) M ouabain for 60 min increased basal cytosolic free Ca2+ [( Ca2+]i) concentration and intracellular 45Ca2+ uptake. Ouabain, however, did not affect basal 45Ca2+ efflux or AVP-stimulated 45Ca2+ efflux. As assessed by cell shape change, preincubation with 10(-5) M ouabain for 60 min also enhanced the sustained cellular contractile effect of a submaximal (10(-8) M AVP, 21.5% vs. 30.5%, P less than 0.01) but not maximal dose of 10(-6) M AVP. Preincubation with 10(-5) M ouabain for 60 min did not change AVP-induced V1-specific surface receptor binding or AVP-induced inositol phosphate production but did however potentiate the mobilization of [Ca2+]i induced by a submaximal (10(-8) M AVP, 301 vs. 385 nM, P less than 0.01) but not a maximal dose of AVP. These effects of ouabain on the mobilization of [Ca2+]i were abolished by incubation in Ca2(+)-free buffer or 5 X 10(-5) M verapamil. Ouabain (10(-5) M) also enhanced the sustained cellular contractile effect of a direct protein kinase C activator, phorbol 12-myristate 13-acetate. The present results therefore indicate that the inhibition of Na+/K(+)-ATPase may enhance the vascular action of AVP, and perhaps other vasoconstrictors, by increasing the AVP-induced mobilization of [Ca2+]i and by potentiating the activity of protein kinase C stimulated by AVP through enhancing basal and AVP-stimulated cellular Ca2+ uptake.
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PMID:Effect of inhibition of Na+/K(+)-adenosine triphosphatase on vascular action of vasopressin. 217 Apr 49

Agonist occupancy of the cloned human serotonin (5-HT)1A receptor expressed in HeLa cells stimulates Na+/K+ ATPase activity as assessed by rubidium uptake. The purpose of the study was to determine which of the receptor-associated signaling mechanisms was responsible for this effect. 5-HT stimulated Na+/K+ ATPase 38% at 2 mM extracellular potassium, an effect characterized by a decrease in apparent K0.5 from 2.8 +/- 0.3 to 1.8 +/- 0.3 mM potassium without a significant change in apparent Vmax. The EC50 for the transport effect was approximately 3 microM 5-HT. The response was pertussis toxin-sensitive but did not involve inhibition of adenylate cyclase, as stimulation of Na+/K+ ATPase by 5-HT was observed in the presence of excess dibutyryl cAMP. Protein kinase C was not required for the response since short-term incubation with the phorbol esters phorbol 12 myristate, 13 acetate (PMA) and phorbol 12,13-dibutyrate (PDBu) did not mimic the 5-HT effect. Moreover, 5-HT increased Na+/K+ ATPase activity after inactivation of protein kinase C by overnight incubation with PMA. 5-HT and the sesquiterpene lactone thapsigargin increased cytosolic calcium in this cell model, and the EC50 for 5-HT corresponded with that for stimulation of Na+/K+ ATPase. Both thapsigargin and A23187, a calcium ionophore, also increased Na+/K+ ATPase activity in a dose-responsive fashion. The response to 5-HT, thapsigargin, and A23187 was blocked by conditions that removed the cytosolic calcium response. By two-dimensional gel electrophoresis, we established evidence for a calcium-sensitive but protein kinase C-independent signaling pathway. We conclude that the 5-HT1A receptor, which we have previously shown to stimulate phosphate uptake via protein kinase C, stimulates Na+/K+ ATPase via a calcium-dependent mechanism. This provides evidence for regulation of two separate transport processes by a single receptor subtype via different signaling mechanisms.
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PMID:Short-term regulation of Na+/K+ adenosine triphosphatase by recombinant human serotonin 5-HT1A receptor expressed in HeLa cells. 217 7


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