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Query: UNIPROT:P20020 (adenosine triphosphatase)
 3,299 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

A new method for the assay of Na+, K+-adenosine triphosphatase (Na+, K+-ATPase) inhibition has been devised involving the determination of enzymatically produced adenosine diphosphate (ADP) and unchanged adenosine triphosphate (ATP) by high-performance liquid chromatography (HPLC). The substrate, ATP, was incubated with the enzyme preparation in the presence of an inhibitor. The incubation mixture was filtered through a membrane filter, and ADP and ATP in the filtrate were separated by (HPLC). The inhibitory effect of a cardiac steroid on the enzymic reaction was estimated by measuring the peak area ratio of ADP to ADP plus ATP on the chromatogram. The proposed assay method has proved to be satisfactory with respects to simplicity, sensitivity, and reproducibility.
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PMID:A high-performance liquid chromatographic method for the assay of Na+, K+-adenosine triphosphatase inhibition. 298 79

Membrane-bound (H+ + K+)-ATPase purified from hog gastric mucosa was exposed to limited papain digestion. Such treatment resulted in a rapid inhibition of the K+-stimulated adenosine triphosphatase and p-nitrophenyl phosphatase activities, with about 90% of these activities lost after 3 min incubation at 37 degrees C with 0.1 units of papain per mg of enzyme protein. Parallel to the inhibition of the enzyme activities, there was a production of a 77 kDa membrane-bound fragment containing the aspartyl phosphate residue of the phospho-intermediate. This fragment accounted for about 45% of the total enzyme protein after the 3 min papain treatment. The digestion barely affected the steady-state level of phosphorylation, allowed the aspartyl phosphate of the 77 kDa fragment to undergo the transition to the E2P form, and did not significantly alter the fraction of ADP-sensitive phosphoenzyme. The presence of KCl, however, depressed the steady-state level of phosphoenzyme formed from [gamma-32P]ATP considerably less than that of the control enzyme. With further exposure to papain the 77 kDa peptide became fragmented into a 28 kDa soluble peptide that retained the phosphorylating site. Binding of fluorescein 5'-isothiocyanate (FITC) to the native enzyme did not affect the sites of papain hydrolysis because the same peptide fragments were obtained. The FITC reaction site was also in the 28 kDa soluble peptide fragment.
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PMID:Papain fragmentation of the gastric (H+ + K+)-ATPase. 303 Apr 30

Microsomes from guinea-pig cerebral cortex contain a system capable of exchanging ADP with ATP at rates of about 20mumoles/mg. of protein/hr. The ADP-ATP-exchange reaction requires Mg(2+) for activity. The reaction is not stimulated by Na(+) or K(+) and is not inhibited by ouabain, in contrast with the Na(+)-plus-K(+)-stimulated adenosine triphosphatase. The pH optimum also differs from that of the adenosine triphosphatase. The ADP-ATP-exchange reaction is stimulated two- to three-fold by non-ionic, anionic and cationic detergents, even when these agents are inhibiting the adenosine-triphosphatase reaction. This reaction may represent a component of the Na(+)-plus-K(+)-stimulated adenosine-triphosphatase reaction but is more likely to be due to other enzyme systems present in microsomal subfractions.
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PMID:The adenosine diphosphate--adenosine triposphate-excange reaction of cerebral microsomes and its relation to he sodium ion-stimulatd adenosine-triphosphatase reaction. 422 69

1. A microsomal fraction from ox cerebral cortex catalysed [(14)C]ADP-ATP exchange at a speed similar to that at which it liberated P(i) from ATP in the presence of Na(+), K(+) and Mg(2+). 2. Repeated washing the fraction with MgATP solutions solubilized most of the exchange activity and left the adenosine triphosphatase insoluble and little changed in activity. The exchange activity was accompanied by negligible adenosine-triphosphatase activity and was enriched by precipitation at chosen pH and by DEAE-Sephadex. At no stage was its activity affected by Na(+), K(+) or ouabain. 3. The washed microsomal fraction was exposed to a variety of reagents; a sodium iodide-cysteine treatment increased both adenosine-triphosphatase and exchange activities, as also did a synthetic zeolite. Preparations were obtained with exchange activities less than 3% of their Na(+)-plus-K(+)-stimulated adenosine-triphosphatase activity. Some contribution to the residual exchange activity was made by an adenylate kinase. 4. Thus over 95% of the microsomal ADP-ATP-exchange activity does not take part in the Na(+)-plus-K(+)-stimulated adenosine-triphosphatase reaction. Participation of some of the residual 3% of the ADP-ATP-exchange activity has not been excluded, but there appears no firm evidence for its participation in the adenosine triphosphatase; the bearing of this conclusion on mechanisms proposed for the Na(+)-plus-K(+)-stimulated adenosine triphosphatase is indicated.
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PMID:Separation of adenosine diphosphate--adenosine triphosphate-exchange activity from the cerebral microsomal sodium-plus-potassium ion-stimulated adenosine triphosphatase. 422 77

Rottem, Shlomo (Hebrew University, Jerusalem, Israel), and Shmuel Razin. Adenosine triphosphatase activity of mycoplasma membranes. J. Bacteriol. 92:714-722. 1966.-Adenosine triphosphatase activity of Mycoplasma laidlawii, M. gallisepticum, and Mycoplasma sp. strain 14 was confined to the cell membrane. The enzymatic activity was dependent on magnesium, but was not activated by sodium and potassium. Ouabain did not inhibit the adenosine triphosphatase activity of the mycoplasmas, and did not interfere with the active accumulation of potassium by M. laidlawii cells. Sulfhydryl-blocking reagents and fluoride inhibited the enzymatic activity, whereas 2,4-dinitrophenol was without any effect. Membranes of M. laidlawii hydrolyzed other nucleotide triphosphates and adenosine diphosphate (ADP), but at a lower rate than adenosine triphosphate (ATP). Nucleoside-2'-(3')-phosphates, ribose-5-phosphate, glucose-6-phosphate, and pyrophosphate were not hydrolyzed by the membrane preparations. It seems that the enzyme(s) involved in ATP hydrolysis by M. laidlawii membranes is strongly bound to the membrane subunits, which would account for the failure to purify the enzyme by protein fractionation techniques. The adenosine triphosphatase activity of mycoplasma membranes resembles in its properties that of similar enzymes studied in bacteria. The mycoplasma enzyme(s) seems to differ from the adenosine triphosphatase associated with ion transport in mammalian cell membranes and from mitochondrial adenosine triphosphatase.
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PMID:Adenosine triphosphatase activity of mycoplasma membranes. 422 19

When samples of pea tendril tissue were incubated in the Wachstein-Meisel medium for the demonstration of adenosine triphosphatases, deposits of lead reaction product were localized between the membranes of the chloroplast envelope. The presence of Mg(2+) was necessary for adenosine triphosphatase activity, and Ca(2+) could not substitute for this requirement. Varying the pH of incubation to 5.5 or 9.4 inhibited enzyme activity, as did the addition of p-chloromercuribenzoic acid or N-ethylmaleimide. The adenosine triphosphatase was apparently inactivated or degraded when the plants were grown in the dark for 24 hours prior to incubation. The enzyme was substrate-specific for adenosine triphosphate; no reaction was obtained with adenosine diphosphate, uridine triphosphate, inosine triphosphate, p-nitrophenyl phosphate, and sodium beta-glycerophosphate. Sites of nonspecific depositions of lead are described. The adenosine triphosphatase on the chloroplast envelope may be involved in the light-induced contraction of this organelle.
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PMID:Localization of adenosine triphosphatase activity on the chloroplast envelope in tendrils of Pisum sativum. 424 3

Transient kinetic studies of Mg(2+)-dependent heavy-meromyosin ATPase (adenosine triphosphatase) were done by monitoring the release of both ADP and P(i) into the reaction medium by using linked assay systems. The release of P(i) was monitored by its quantitative transfer to ADP, with concomitant reduction of NAD(+) in the presence of d-glyceraldehyde 3-phosphate, d-glyceraldehyde 3-phosphate dehydrogenase and phosphoglycerate kinase. The dissociation rates of the products, ADP and P(i), from heavy meromyosin were shown to be faster than the rate-controlling process, which occurs after the initial bond cleavage of ATP. The chromophoric ATP analogue, 6-mercapto-9-beta-d-ribofuranosylpurine 5'-triphosphate (thioATP) was used as a substrate and spectral changes associated with a single turnover of heavy meromyosin could be assigned to elementary processes of the mechanism. It was shown that the dissociation rate of thioADP was not the rate-controlling process of the thioATPase, whose catalytic-centre activity was 7.6 times that of the ATPase at pH8. The dissociation rate of ADP from heavy meromyosin was measured by using thioATP as displacing agent and was found to be 2.3s(-1), which is about 50 times the catalytic-centre activity of the ATPase at pH8. Transient kinetic studies with chromophoric adenosine phosphate analogues have general application for kinases and ATPases both in characterizing the chemical states of the intermediates and in delineating the elementary processes of the enzyme mechanism.
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PMID:Elementary processes of the magnesium ion-dependent adenosine triphosphatase activity of heavy meromyosin. A transient kinetic approach to the study of kinases and adenosine triphosphatases and a colorimetric inorganic phosphate assay in situ. 426 38

A mixture of purified muscle glycolytic enzymes was reconstituted and the mixture shown to behave in a fashion analogous to that occurring in vivo. Glycolysis leads to ATP production in muscle and results in the phosphorylation of creatine. The extent of this phosphorylation by anaerobic glycolysis was shown to depend to a small extent on the relative proportions of available P(i) and creatine initially, but more importantly on the first step in glycolysis, in this case the enzyme phosphorylase. With less than 0.1% of the phosphorylase in the a form, only about one-third of the creatine was phosphorylated in 30min, whereas with 4% or more of phosphorylase a, 90% of the creatine was phosphorylated within this time. Inclusion of an adenosine triphosphatase decreased the steady-state concentration of phosphocreatine in the system. Calculations of the theoretical concentrations of ADP and AMP showed that phosphorylase b was almost inactive even in the presence of 9mum-AMP, because of ATP inhibition. With phosphorylase a present, glycolysis was able to continue at least until the calculated concentration of MgADP(-) was only 7mum, and AMP in the sub-mumolar range. The relation of these values to measured concentrations of nucleotides and to phosphorylase a percentages in intact muscle is discussed.
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PMID:Studies with a reconstituted muscle glycolytic system. The rate and extent of creatine phosphorylation by anaerobic glycolysis. 426 7

Evidence is presented that the myosin subfragment-1-ADP complex, generated by the addition of Mg(2+) and ADP to subfragment 1, is an intermediate within the myosin Mg(2+)-dependent adenosine triphosphatase (ATPase) turnover cycle. The existence of this species as a steady-state intermediate at pH8 and 5 degrees C is demonstrated by fluorescence measurements, but its concentration becomes too low to measure at 21 degrees C. This arises because there is a marked temperature-dependence on the rate of the process controlling ADP dissociation from subfragment 1 (rate=1.4s(-1) at 21 degrees C, 0.07s(-1) at 5 degrees C). In the ATPase pathway this reaction is in series with a relatively temperature-insensitive process, namely an isomerization of the subfragment-1-product complex (rate=0.055s(-1) at 21 degrees C, 0.036s(-1) at 5 degrees C). By means of studies on the P(i) inhibition of nucleotide-association rates, a myosin subfragment-1-P(i) complex was characterized with a dissociation equilibrium constant of 1.5mm. P(i) appears to bind more weakly to the myosin subfragment-1-ADP complex. The studies indicate that P(i) dissociates from subfragment 1 at a rate greater than 40s(-1), and substantiates the existence of a myosin-product isomerization before product release in the elementary processes of the Mg(2+)-dependent ATPase. In this ATPase mechanism Mg(2+) associates as a complex with ATP and is released as a complex with ADP. In 0.1m-KCl at pH8 1.0mol of H(+) is released/mol of subfragment 1 concomitant with the myosin-product isomerization or P(i) dissociation, and 0.23 mol of H(+) is released/mol of subfragment when ATP binds to the protein, but 0.23 mol of H(+) is taken up again from the medium when ADP dissociates. Within experimental sensitivity no H(+) is released into the medium in the step involving ATP cleavage.
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PMID:The characterization of myosin-product complexes and of product-release steps during the magnesium ion-dependent adenosine triphosphatase reaction. 428 53

1. Homogenates of neural lobes of bovine pituitary glands were fractionated by differential and density-gradient ultracentrifugation and the distribution of adenosine triphosphatase (ATPase) activity was studied. It was shown that all the activity was membrane-bound. 2. On the basis of ionic requirements the ATPase activity was grouped into three categories: (a) Mg(2+)-dependent, (b) Ca(2+)-dependent and (c) Mg(2+)+Na(+)+K(+)-dependent (ouabain-sensitive) ATPases. The activity in the absence of bivalent cations was negligible. The ratio between the activities of the three ATPases varied between the different subcellular fractions. 3. Preincubation of the subcellular fractions with deoxycholate increased the activity of the Mg(2+)+Na(+)+K(+)-dependent enzyme, whereas the Mg(2+)- and Ca(2+)-activated ATPases were either unaffected or slightly inhibited. Triton X-100 solubilized the Mg(2+)- and Ca(2+)-ATPases; however, the activity of the Mg(2+)+Na(+)+K(+)-ATPase was abolished by the concentration of Triton X-100 used. 4. All the subfractions displayed unspecific nucleotide triphosphatase activity towards GTP, ITP and UTP. These substrates inhibited the hydrolysis of ATP by all three ATPases. ADP also inhibited the ATPases. 5. Polyacrylamide-gel electrophoresis of extracts containing the Mg(2+)- and Ca(2+)-dependent ATPase activity solubilized by Triton X-100 revealed the presence of two enzymes; one activated by either Mg(2+) or Ca(2+) and the other activated only by Ca(2+). 6. In sucrose density gradients the distribution of vasopressin was different from that of all three types of ATPases. It is therefore suggested that the neurosecretory granules do not possess ATPase activity.
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PMID:Adenosine triphosphatase activity in the neural lobe of the bovine pituitary gland. 428 6


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