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)

A potent (Na,K)-ATPase inhibitor purified from "Sigma Grade* ATP has been identified as vanadium using electron probe microanalysis and confirmed by microwave-induced emission spectroscopy and electron paramagnetic resonance spectroscopy. Sodium orthovanadate (Na3 VO4) is identical with the purified inhibitor with respect to ultraviolet absorbance, migration on thin layer chromatography, and inhibition of (Na,K)-ATPase. The (Na,K)-ATPase is in-inhibited 50% by 40 nM Na3 VO4 under optimal conditions (28 mM Mg2+) and the inhibition is 100% reversible by millimolar concentrations of norepinephrine. The physiological significance of this inhibition is discussed in relation to vanadium concentrations in vivo.
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PMID:Vanadate is a potent (Na,K)-ATPase inhibitor found in ATP derived from muscle. 14 27

1. Modification of Lys residues of the Ca(2+)-ATPase from human red blood cells with methyl acetimidate (MA) inhibited up to 70% of the Ca(2+)-ATPase activity. Furthermore, calmodulin-activated p-nitrophenyl phosphatase activity was fully inhibited at non-limiting concentrations of MA. 2. Treatment with MA inhibited phosphorylation of the Ca(2+)-ATPase. 3. When the enzyme was treated with 7.2 mM-MA in the presence of 100 microM-Ca2+, Ca(2+)-ATPase activity was decreased by 33%, whereas when the membranes were treated with MA in the presence of 50 microM-VO4(3-), this activity was decreased by only 8%. 4. When membranes were either proteolysed or preincubated with 1 mM-Ca2+, MA quickly inactivated the Ca(2+)-ATPase (k = 1.2 min-1). On the other hand, inactivation of membranes preincubated in the absence of Ca2+ and Mg2+ was slow (k = 0.08 min-1). 5. When the activity was measured in the absence of calmodulin, MA decreased to the same extent the values of KCa (the apparent dissociation constant for Ca2+) and Vmax, but in the presence of calmodulin the treatment decreased Vmax. only. 6. The results are consistent with the idea that MA reacts readily with the Ca(2+)-ATPase when the enzyme is in an E1 conformation, but not an E2 conformation, and that, reciprocally, treatment of the enzyme with MA shifts the enzyme to E1. 7. Provided that Ca2+ is present, ATP, with low apparent affinity (K0.5 = 195 microM), protected against inactivation by MA. However, MA treatment did not change the Km values of either the high-affinity or the low-affinity site for ATP, suggesting that protection results from a shift to a conformation in which the Lys residues are inaccessible to MA.
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PMID:Differential reactivity of lysine residues of the red blood cell Ca2+ pump involved in the E1-E2 conformational equilibrium. 165 36

A well-characterized chicken osteoclast plasma membrane vesicle preparation manifested Mg2(+)-dependent ATP hydrolyzing activity of 0.213 mumol inorganic phosphate released per mg protein per minute (n = 7). The Mg2+ dependence showed a high-affinity component with a KMg of 1.293 microM and Vmax of 0.063 mumol Pi per mg protein per minute, and a low-affinity component with a KMg of 297.6 microM and a Vmax of 0.232 mumol Pi per mg protein per minute. The Mg2(+)-ATPase activity was inhibited by N,N'-dicyclohexylcarbodiimide (DCCD, 0.2 mM, 50.7%), N-ethylmaleimide (0.5 mM, 34.6%), nolinium bromide (1 mM, 29.9%), 4,4'-diisothiocyano-2,2'-stilbene sulfonic acid (DIDS, 1 mM, 45.1%), and p-chloromercuribenzoic acid (PCMB, 0.1 mM, 33.8%). Sodium orthovanadate (Na3 VO4) at 1 microM had no effect but caused 29.5% inhibition at 1 mM. Na+ could substitute for K+ without loss of activity, NO3- caused 19.5% inhibition when substituted for Cl-, and acetate replacement of Cl- resulted in 36.4% stimulation of Mg2(+)-ATPase. ATP, GTP, ITP, CTP, and ADP were all hydrolyzed effectively. DCCD (0.2 mM), NEM (0.5 mM), nolinium bromide (1 mM), and DIDS (50 microM) almost completely abolished proton transport as measured spectrofluorometrically by acridine orange quenching. Na3 VO4 (1 mM) had no effect, and duramycin (80 micrograms/ml) inhibited transport 52.7%. K+ replacement of Na+ caused a 79.2% increase in initial proton transport rate. NO3- and acetate substitution of Cl- resulted in a 46.1 and 55.7% decrease in transport, respectively. ATP supports transport far more effectively than the other nucleotides tested. ADP was ineffective. Experiments using the potassium ionophore, valinomycin, indicated that the proton pump functions electrogenically, with Cl- most likely cotransported by an anion transporter. The proton pump also seems to have at least one anion-sensitive site, elucidated by experiments in the presence of NO3- and Cl-.
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PMID:Biochemical characterization of an electrogenic vacuolar proton pump in purified chicken osteoclast plasma membrane vesicles. 216 21

Different conformational states of the purified plasma membrane Ca2+-ATPase from pig erythrocytes have been detected by circular dichroism (CD) and fluorescence spectroscopy. The helical content of the enzyme decreased by about 10% in the transition from the Ca2+ high-affinity form (10 microM free Ca2+ = E1 state) to the VO4(3-)-inhibited state (20 microM VO4(3-) = E2 state). The changes in the CD spectra did not show full reversibility upon reversing the E1-E2 transition, whereas those in the fluorescence spectra did. A temperature-dependent loss of alpha-helical content in the presence of Ca2+ was also observed. Intrinsic fluorescence measurements revealed an increase in fluorescence intensity upon addition of Ca2+. The change was fully reversed by ethylene glycol bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid. The increase in fluorescence intensity was partly reversed by adding ATP, an effect which is suggested to correspond to the "Ca2+-occluded" form of the ATPase. The steady-state level of the fluorescence intensity was stable for several minutes in the presence of 100 microM ATP. By contrast, the decrease of fluorescence intensity induced by limiting concentrations of ATP (= 1 microM) was only transient, indicating the decomposition of the phosphorylated intermediate of the ATPase and the reestablishment of the Ca2+ high-affinity form of the enzyme.
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PMID:Conformational differences between the E1 and E2 states of the calcium adenosinetriphosphatase of the erythrocyte plasma membrane as revealed by circular dichroism and fluorescence spectroscopy. 295 86

The effect of vanadate (orthovanadate, VO4-) on water and ion transport was studied in rat jejunum. Water transport was tested by single-pass perfusion in vivo and ion fluxes by the Ussing-chamber technique in vitro. The results suggest that vanadate has two actions on ion and water transport: At low concentrations (10(-4) M) it causes Cl-, Na+ and water secretion by stimulation of adenylate cyclase; At higher concentrations (10(-3) and 10(-2) M) it decreases net absorption of Na+ and Cl- by inhibition of (Na+ + K+)-ATPase.
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PMID:Effects of vanadate on water and electrolyte transport in rat jejunum. 302 23

1. The mechanisms involved in the responses induced by sodium vanadate (Va3 VO4) on cat cerebral and femoral arteries were studied. The possibility that these responses were due to Na+, K+-ATPase inhibition was investigated by measuring the effect of vanadate on [3H]-ouabain binding to arterial membrane fractions, K+-induced vasodilatation and ouabain-sensitive 86Rb+ uptake. 2. The vanadium compounds (Na3VO4, VOSO4, VCl3 and O5V3) induced similar, concentration-dependent contractions in each kind of artery, the cerebral vessels being the most sensitive to these compounds. 3. Exposure of the arteries to a low-Na+ (25 mM) solution suppressed the contraction caused by vanadate in femoral but not in cerebral arteries. 4. Vanadate-induced contractions were reduced in Ca2+-free medium but remained unaffected by 3 x 10(-6) M phentolamine, reserpine pretreatment or 3 x 10(-6) M verapamil in both kinds of artery. 5. The addition of 7.5 mM K+ to the arteries immersed in a K+-free solution induced vasodilatation, which was not modified by 10(-3) M vanadate. 6. The consecutive administration of ouabain (10(-4) M) and vanadate (10(-3) M) (or vice versa), or the simultaneous administration of both agents (10(-8) to 10(-3) M) appeared to produce an additive contraction in both types of artery. 7. Vanadate (10(-7) to 10(-3) M) did not displace the [3H]-ouabain binding to arterial membrane fractions of these arteries, whereas 10(-4) M ouabain did. 8. In both kinds of artery, total 86Rb+ uptake was reduced by ouabain (10(-8) to 10(-3) M), in a concentration-dependent manner, whereas it was not modified by vanadate (10(-8)-10(-3) M). 9. These results suggest that vanadate induces contraction in both types of artery by a mechanism unrelated to Na+, K+-ATPase inhibition. Such a mechanism is likely to be related to inhibition of the Ca2-ATPase of the cell membrane and/or the sarcoplasmic reticulum.
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PMID:Actions of vanadate on vascular tension and sodium pump activity in cat isolated cerebral and femoral arteries. 334 33

Plasma membrane vesicles isolated from rat liver exhibited an azide-insensitive Mg2+-ATP-dependent Ca2+ pump which accumulated Ca2+ at a rate of 5.1 +/- 0.5 nmol of calcium/mg of protein/min and reached a total accumulation of 33.2 +/- 2.6 nmol of calcium/mg of protein in 20 microM Ca2+ at 37 degrees C. Equiosmotic addition of 50 mM Na+ resulted in a loss of accumulated calcium. Measurement of Mg2+-ATP-dependent Ca2+ uptake in the presence of 50 mM Na+ revealed no effect of Na+ on the initial rate of Ca2+ uptake, but a decrease in the total accumulation. The half-maximal effect of Na+ on Ca2+ accumulation was achieved at 14 mM. The Ca2+ efflux rate constant in the absence of Na+ was 0.16 +/- 0.01 min-1, whereas the efflux rate constant in the presence of 50 mM Na+ was 0.25 +/- 0.02 min-1. Liver homogenate sedimentation fractions from 1,500 to 105,000 X g were assayed for azide-insensitive Mg2+-ATP-dependent Ca2+ accumulation. Na+-sensitive Ca2+ uptake activity was found to specifically co-sediment with the plasma membrane-associated enzymes, 5'-nucleotidase and Na+/K+-ATPase, whereas Na+-insensitive Ca2+ uptake was found to co-sediment with the endoplasmic reticulum-associated enzyme, glucose-6-phosphatase. The plasma membrane Ca2+ pump was also distinguished from the endoplasmic reticulum Ca2+ pump by its sensitivity to inhibition by vanadate. Half-maximal inhibition of plasma membrane Ca2+ uptake occurred at 0.8 microM VO4(3-), whereas half-maximal inhibition of microsomal Ca2+ uptake occurred at 40 microM.
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PMID:Liver plasma membrane calcium transport. Evidence for a Na+-dependent Ca2+ flux. 348 13

Vanadate (VO4(-3] produces a positive inotropic effect in rats and also promotes diuresis as well as natriuresis. Although the mechanism(s) of these effects is uncertain, in the kidney, VO4(-3) may act through inhibition of (Na+ + K+)-ATPase activity, whereas in the heart, other or additional mechanisms are likely. Under the assay conditions used in the present study, microsomal (Na+ + K+)-ATPase activities from rat kidney cortex and medulla were inhibited to a greater extent than was left ventricular (Na+ + K+)-ATPase activity over a range of VO4(-3) concentrations. The apparent dissociation constant for left ventricular (Na+ + K+)-ATPase (10.95 +/- 1.26 X 10(-7)M VO4(-3] was significantly greater than that of (Na+ + K+)-ATPase from the cortex (3.46 +/- 0.96 X 10(-7)M VO4(-3] or the medulla (3.32 +/- 0.7 X 10(-7)M VO4(-3), N = 6, P less than .05) whereas there were no significant differences between the effects of VO4(-3) on (Na+ + K+)-ATPase from the cortex and medulla. The greater inhibition by VO4, of (Na+ + K+)-ATPase from the cortex relative to that of the left ventricle, occurred over a range of Na+ and K+ concentrations, and K+ enhanced the inhibition by VO4(-3) to a greater extent for (Na+ + K+)-ATPase from the cortex than the left ventricle. These results suggest that renal (Na+ + K+)-ATPase is more sensitive than left ventricular (Na+ + K+)-ATPase to inhibition by VO4(-3) and would, therefore, be more likely to be modulated in vivo.
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PMID:Greater sensitivity to vanadate of rat renal relative to cardiac (Na+ + K+)-ATPase. 609 7

Orthovanadate (VO4) has been shown to cause a marked natriuresis in rats. This has been ascribed to its inhibitory action on renal Na-K-ATPase activity. Because virtually all nephron segments possess Na-K-ATPase activity the administration of VO4 should alter renal tubular transport along the entire nephron. To examine this possibility, adult rats were anesthetized and infused with VO4 (10 mumol.kg body wt-1.h-1 i.v.). This dose had no effect on glomerular filtration rate, effective renal plasma flow, and blood pressure, whereas urine flow and sodium and water excretion rose markedly. Potassium excretion remained unaltered. VO4 depressed only maximal bicarbonate and glucose reabsorption without causing a glucose or bicarbonate "leak" at normal levels of blood glucose or bicarbonate. In acutely thyroparathyroidectomized rats VO4 produced a striking phosphaturia, not accompanied by an increase in nephrogenous cAMP excretion. Both free water clearance in Brattleboro rats and free water reabsorption in normal rats was significantly depressed by VO4. These data demonstrate that VO4 depresses tubular reabsorption in proximal and distal nephron segments. We conclude that VO4 exerts its effect on tubular function by inhibition of Na-K-ATPase activity.
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PMID:Effect of vanadate on renal tubular function in rats. 626 95

The effect of vanadium oxides on living systems may involve the in vivo conversion of vanadate and vanadyl ions. The addition of 5 mM orthovanadate (VO4(3-), V(V)), a known inhibitor of the (Na,K)-ATPase, to yeast cells stopped growth. In contrast, the addition of 5 mM vanadyl (VO2+, V(IV) stimulated growth. Orthovanadate addition to whole cells is known to stimulate various cellular processes. In yeast, both ions inhibited the plasma membrane Mg2+ ATPase and were transported into the cell as demonstrated with [48V]VO4(3-) and VO2+. ESR spectroscopy has been used to measure the cell-associated paramagnetic vandyl ion, while 51V NMR has detected cell-associated diamagnetic vanadium (e.g. V(V)). Cells were exposed to both toxic (5 mM) and nontoxic (1 mM) concentrations of vanadate in the culture medium. ESR showed that under both conditions, vanadate became cell associated and was converted to vanadyl which then accumulated in the cell culture medium. 51V NMR studies showed the accumulation of new cell-associated vanadium resonances identified as dimeric vanadate and decavanadate in cells exposed to toxic amounts of medium vanadate (5 mM). These vanadate compounds did not accumulate in cells exposed to 1 mM vanadate. These studies confirm that the inhibitory form of vanadium usually observed in in vitro experiments is vanadate, in one or more of its hydrated forms. These data also support the hypothesis that the stimulatory form of vanadium usually observed in whole cell experiments is the vanadyl ion or one or more of its liganded derivatives.
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PMID:Metabolism of added orthovanadate to vanadyl and high-molecular-weight vanadates by Saccharomyces cerevisiae. 638 12


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