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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)

The (Na+ + K+)-dependent ATPase exhibits substrate sites with both high affinity (Km near 1 microM) and low affinity (Km near 0.1 mM) for ATP. To permit the study of nucleotide binding to the high-affinity substrate sites of a canine kidney enzyme preparation in the presence as well as absence of MgCl2, the nonhydrolyzable beta-gamma imido analog of ATP, AMP-PNP, was used in experiments performed at 0-4 degrees C by a centrifugation technique. By this method the KD for AMP-PNP was 4.2 microM in the absence of MgCl2. Adding 50 microM MgCl2, however, decreased the KD to 2.2 microM; by contrast, higher concentrations of MgCl2 increased the KD until, with 2 mM MgCl2, the KD was 6 microM. The half-maximal effect of MgCl2 on increasing the KD occurred at approximately 1 mM. This biphasic effect of MgCl2 is interpreted as Mg2+ in low concentrations favoring AMP-PNP binding through formation at the high-affinity substrate sites of a ternary enzyme-AMP-PNP-Mg complex; inhibition of nucleotide binding at higher MgCl2 concentrations would represent Mg2+ acting through the low-affinity substrate sites. NaCl in the absence of MgCl2 increased AMP-PNP binding, with a half-maximal effect near 0.3 mM; in the presence of MgCl2, however, NaCl increased the KD for AMP-PNP. KCl decreased AMP-PNP binding in the presence or absence of MgCl2, but the simultaneous presence of a molar excess of NaCl abolished (or masked) the effect of KCl. ADP and ATP acted as competitors to the binding of AMP-PNP, although a substrate for the K+-dependent phosphatase reaction also catalyzed by this enzyme, p-nitrophenyl phosphate, did not. This lack of competition is consistent with formulations in which the phosphatase reaction is catalyzed at the low-affinity substrate sites.
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PMID:Binding to the high-affinity substrate site of the (Na+ + K+)-dependent ATPase. 626 Jul 67

The interaction of the cardiac glycoside [3H]ouabain with the Na+, K+ pump of resealed human erythrocyte ghosts was investigated. Binding of [3H]ouabain to high intracellular Na+ ghosts was studied in high extracellular Na+ media, a condition determined to produce maximal ouabain binding rates. Simultaneous examination of both the number of ouabain molecules bound per ghost and the corresponding inhibition of the Na+, K+-ATPase revealed that one molecule of [3H]ouabain inhibited one Na+, K+-ATPase complex. Intracellular magnesium or magnesium plus inorganic phosphate produced the lowest ouabain binding rate. Support of ouabain binding by adenosine diphosphate (ADP) was negligible, provided synthesis of adenosine triphosphate (ATP) through the residual adenylate kinase activity was prevented by the adenylate kinase inhibitor Ap5A. Uridine 5'-triphosphate (UTP) alone did not support ouabain binding after inhibition of the endogenous nucleoside diphosphokinase by trypan blue and depletion of residual ATP by the incorporation of hexokinase and glucose. ATP acting solely at the high-affinity binding site of the Na+, K+ pump (Km approximately 1 microM) promoted maximal [3H]ouabain binding rates. Failure of 5'-adenylyl-beta-gamma-imidophosphate (AMP-PNP) to stimulate significantly the rate of ouabain binding suggests that phosphorylation of the pump was required to expose the ouabain receptor.
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PMID:[3H]Ouabain binding and Na+, K+-ATPase in resealed human red cell ghosts. 630 99

1. Based on a detailed reaction scheme of the phosphorylation process of the sarcoplasmic transport ATPase the inhibition mechanisms of benzoctamine, DIO 9, AMP-PNP and of Ca2+-ions at relatively high concentrations (1 approximately 100 microM) were determined. 2. The inhibition mechanisms were analyzed by measuring the gamma-phosphate exchange between ATP and ADP and evaluated by applying conventional and an extended Dixon plot procedures. 3. The kinetic patterns of the inhibition were shown to be compatible with the assumed reaction scheme. 4. Each inhibitor combines with definite intermediates: Benzoctamine with the intermediate species Ca2MgE and Ca2Mg2E-ATP; AMP-PNP with Ca2Mg2E approximately P; DIO 9 with E and MgE and Ca2+ at relatively high concentrations with E. 5. The central intermediate blocked by benzoctamine can partially exist as Ca2Mg2E ADPP-benzoctamine which is detected as phosphoprotein after acid denaturation.
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PMID:Characterisation of the enzyme intermediates of the sarcoplasmic transport ATPase by the use of inhibitors. 630 40

Sarcoplasmic reticulum ATPase has been found to cleave the ATP analog adenyl-5'-yl imidodiphosphate in a calcium-dependent reaction. The reaction products were determined by 31P NMR to be inorganic phosphate and adenyl-5'-yl phosphoramidate (AMP-PN). AMP-PNP hydrolysis, like ATP hydrolysis, drives active Ca2+ accumulation by sarcoplasmic reticulum vesicles.
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PMID:Sarcoplasmic reticulum ATPase catalyzes hydrolysis of adenyl-5'-yl imidodiphosphate. 645 67

Evidence in this and other reports from this laboratory suggest that adrenergic nerves in rat heart ventricle slices incubated in a Na+-deprived (choline+) medium containing Ca++ (Ch+--Ca++), transport (by a cocaine-sensitive mechanism) 3H-norepinephrine outwardly from synaptic vesicles attached or fused to the plasma membrane. The 3H-amine secretion was not inhibited by probenecid, an anion transport inhibitor which may prevent exocytosis. The 3H-amine release was rapidly inhibited by exogenous nucleotides ATP, UTP, and GTP greater than ADP greater than AMP greater than the nucleoside adenosine. Magnesium++ tended to increase and reserpine to decrease the effect of ATP. Neither increasing the [Ca++] nor [Mg++] (to compete with Ca++ for ATP) decreased the effect of 3 mM ATP. After secretion began, lowering the Ca++ concentration by ommission, or by the inclusion of either a low concentration of EDTA or the Ca++-binding, but non-energy-conserving synthetic analogs of ATP: AMP--PCP and AMP--PNP, gradually lowered the rates of secretion. By comparison, the rapid effects of the energy-conserving nucleotides suggested that their effects were at least partially independent of chelation, and were energy dependent. ATP, unlike cocaine, did not inhibit the uptake of NE in a Krebs HCO3 medium. Inhibition of (Na+ + K+)-ATPase by ouabain neither inhibited the release by Ch+--Ca++, nor antagonizes the release inhibiting effect of ATP. Hence, ATP did not increase apparent retention of NE by stimulating the uptake of released NE. The ATP-inhibited secretion was not increased by theophylline.
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PMID:The effect of exogenous adenosinetriphosphate on the choline-calcium stimulated release of 3H-norepinephrine in rat heart ventricle slices. 668 77

To show adenylate cyclase (AC) activity in rat calvaria, it is necessary first to decalcify the specimen. In hard tissues, several enzymes (adenosine triphosphatase (ATPase), alkaline phosphatase (APase), adenylate cyclase (AC) and perhaps pyrophosphatase (PPiase) are able to degrade adenosine triphosphate (ATP). The presence of sodium fluoride (NaF) in the incubation medium reduces the quantity of precipitate formed, compared to that observed using a NaF-free incubation medium. Levamisole, used under the same conditions, gives similar results. Possibly NaF inhibits pyrophosphohydrolase and/or phosphatases which mask the AC activity. Adenylylimidophosphate (AMP-PNP), which is a specific AC substrate, confirms the results obtained with ATP. AC activity is demonstrated cytochemically in the osteoblast and preosteoblast membranes, at the junction between two osteoblasts and along the cytoplasmic processes of the osteoblast which penetrate into the osteoid matrix. The osteocytes never show a precipitate, except those which present some osteoblastic features and then only on the membrane facing the osteogenic layer. An intracellular reaction is also evident and is discussed. Parathyroid hormone (PTH) does not reveal new sites of AC activity but increases the quantity of precipitate observed.
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PMID:An attempt at localizing adenylate cyclase in rat calvaria. Influence of sodium fluoride and parathyroid hormone. 700 93

ATP and ADP stimulated the release of specific prostaglandin products from the perfused rabbit kidney heart. The two nucleotides produced the same qualitative profile of prostaglandin products. In kidney, prostaglandin E2 was the major product, whereas in heart 6-keto prostaglandin F1 alpha and prostaglandin E2 predominated. ATP was a slightly more potent than ADP. ATP administered into the perfused heart to kidney was rapidly hydrolyzed to ADP and AMP. The prostaglandin E2 generating activity of ATP was increased 6-10 fold when ATP was given together with AMP-PCP or AMP-PNP which competitively inhibit the activity of vascular ATPase. Thus, the rapid hydrolysis of ATP reduces its agonistic activity for prostaglandin release. ATP and ADP administered together at maximal stimulating doses produced an additive response for prostaglandin E2 release. These results and the results of tachyphylaxis experiments indicate that ATP and ADP interact independently with different types of purinergic receptors.
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PMID:Evidence for different purinergic receptors for ATP and ADP in rabbit kidney and heart. 732 99

Kinesin is a 'motor' molecule, consisting of two head domains, an alpha-helical coiled coil rod, and a tail part that binds to its cargo. When expressed in a bacterial system, the head domain is functional, and can bind to microtubules with the stoichiometry of one head per tubulin dimer. Kinesin moves along microtubules by means of a cyclic process of nucleotide binding, hydrolysis and product release. We have used negative-stain electron microscopy and image analysis to study the structures of microtubules and tubulin sheets decorated with the motor domain (head) of kinesin in three states: in the presence of an unhydrolysable ATP analogue, 5'-adenylylimidodiphosphate (AMP-PNP); without nucleotides; and with adenosine 5'-diphosphate (ADP). A single kinesin head bound to a microtubule has a pear-shaped structure, with the broader end towards the 'plus' end of the microtubule under all conditions; the reverse motor, ncd, is similarly oriented. Three-dimensional maps reveal that kinesin heads have a spike that is assumed to form the attachment to the tail of a complete kinesin molecule. This spike is perpendicular to the microtubule axis in the presence of ADP, but points towards the plus end (approximately 45 degrees) in the presence of AMP-PNP or absence of nucleotides. Our results provide direct evidence for a conformational change of the kinesin motor domain during the ATPase cycle.
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PMID:Nucleotide-dependent angular change in kinesin motor domain bound to tubulin. 761 26

We have demonstrated that caldesmon does not alter the affinity of weak binding actomyosin complexes when it inhibits actin-tropomyosin activation at physiological ratios (1 per 14 actins), and we proposed that it acts upon the strong binding complexes in the same way that troponin-tropomyosin does. We therefore compared the effect of caldesmon, caldesmon fragments, and troponin upon the interaction of the strongly bound complexes S-1.ADP, S-1.adenylyl imidodiphosphate (AMP.PNP), and N-ethylmaleimide-treated myosin subfragment-1 (NEM-S-1) with actin-tropomyosin. In 0.17 M ionic strength buffer [14C]iodoacetamide-labeled S1.ADP bound to actin-smooth muscle tropomyosin with no evidence of cooperativity; Kd = 0.8 +/- 0.3 microM (n = 5). Inhibitory concentrations of sheep aorta caldesmon or rabbit skeletal muscle troponin made the binding highly cooperative. At low levels of saturation the apparent Kd was 10-40 microM with 10 microM caldesmon and 8-20 microM with 6 microM troponin; at > 50% saturation the binding was indistinguishable from actin-tropomyosin alone. A similar result was obtained for the binding of [14C]iodoacetamide-labeled S-1.AMP.PNP to actin-smooth muscle tropomyosin at 0.03 M ionic strength (Kd = 0.47 +/- 0.05 microM). Binding was slightly cooperative and became highly cooperative in the presence of inhibitory concentrations of troponin, caldesmon, and the human caldesmon fragments H7 (amino acids 622-767) and H9 (amino acids 726-793). We conclude that caldesmon and troponin both act as allosteric effectors of the "on"/"off" equilibrium of actin-tropomyosin. 0.1 NEM-S-1/actin potentiated actin-smooth muscle tropomyosin activation of myosin MgATPase 7-fold at 0.03 M ionic strength. Caldesmon inhibited the ATPase in the presence and absence of 0.5 microM NEM-S-1. NEM-S-1 reactivated actin-tropomyosin, which had been inhibited by troponin, caldesmon, H7, or H9. This is compatible with opposing effects of NEM-S-1 and caldesmon or troponin upon the actin-tropomyosin on/off equilibrium.
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PMID:Smooth muscle caldesmon controls the strong binding interaction between actin-tropomyosin and myosin. 779 5

Solubilized Rhodospirillum rubrum RrF1-ATPase, depleted of loosely bound nucleotides, retains 2.6 mol of tightly bound ATP and ADP/mol of enzyme. Incubation of the depleted RrF1 with Mg(2+)-ATP or Mg(2+)-AMP-PNP, followed by passage through two successive Sephadex centrifuge columns, results in retention of a maximal number of 4 mol of tightly bound nucleotides/mol of RrF1. They include 1.5 mol of nonexchangeable ATP, whereas all tightly bound ADP is fully exchangeable. A similar retention of only four out of the six nucleotide binding sites present on CF1 has been observed after its passage through one or two centrifuge columns. These results indicate that the photosynthetic, unlike the respiratory, F1-ATPases have faster koff constants for two of the Mg-dependent nucleotide binding sites. This could be the reason for the tenfold lower Mg2+ than Ca(2+)-ATPase activity observed with native RrF1, as with epsilon-depleted, activated CF1. An almost complete conversion of both RrF1 and CF1 from Ca(2+)- to Mg(2+)-dependent ATPases is obtained upon addition of octylglucoside, at concentrations below its CMC, to the ATPase assay medium. Thus, octylglucoside seems to affect directly the RrF1 and CF1 divalent cation binding site(s), in addition to its proposed role in relieving their inhibition by free Mg2+ ions. The RrF1-ATPase activity is 30-fold more sensitive than CF1 to efrapeptin, and completely resistant to either inhibition or stimulation by the CF1 effector, tentoxin. Octylglucoside decreases the inhibition by efrapeptin and tentoxin, but exposes on CF1 a low-affinity, stimulatory site for tentoxin.
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PMID:Tight nucleotide binding sites and ATPase activities of the Rhodospirillum rubrum RrF1-ATPase as compared to spinach chloroplast CF1-ATPase. 789 72


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