Functional role of ecto-ATPase activity  in goldfish hepatocytes

Extracellular [γ-32P]ATP added to a suspension of goldfish hepatocytes can be hydrolyzed to ADP plus γ-32Pidue to the presence of an ecto-ATPase located in the plasma membrane. Ecto-ATPase activity was a hyperbolic function of ATP concentration ([ATP]), with apparent maximal activity of 8.3 ± 0.4 nmol Pi ⋅ (106cells)−1 ⋅ min−1and substrate concentration at which a half-maximal hydrolysis rate is obtained of 667 ± 123 μM. Ecto-ATPase activity was inhibited 70% by suramin but was insensitive to inhibitors of transport ATPases. Addition of 5 μM [α-32P]ATP to the hepatocyte suspension induced the extracellular release of α-32Pi[8.2 pmol ⋅ (106cells)−1 ⋅ min−1] and adenosine, suggesting the presence of other ectonucleotidase(s). Exposure of cell suspensions to 5 μM [2,8-3H]ATP resulted in uptake of [2,8-3H]adenosine at 7.9 pmol ⋅ (106cells)−1 ⋅ min−1. Addition of low micromolar [ATP] strongly increased cytosolic free Ca2+([Formula: see text]). This effect could be partially mimicked by adenosine 5′- O-(3-thiotriphosphate), a nonhydrolyzable analog of ATP. The blockage of both glycolysis and oxidative phosphorylation led to a sixfold increase of[Formula: see text] and an 80% decrease of intracellular ATP, but ecto-ATPase activity was insensitive to these metabolic changes. Ecto-ATPase activity represents the first step leading to the complete hydrolysis of extracellular ATP, which allows 1) termination of the action of ATP on specific purinoceptors and 2) the resulting adenosine to be taken up by the cells.

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Dissociation of clathrin coats coupled to the hydrolysis of ATP: role of an uncoating ATPase.

The Journal of Cell Biology ◽

10.1083/jcb.99.2.734 ◽

1984 ◽

Vol 99 (2) ◽

pp. 734-741 ◽

Cited By ~ 104

Author(s):

W A Braell ◽

D M Schlossman ◽

S L Schmid ◽

J E Rothman

Keyword(s):

Atpase Activity ◽

Atp Hydrolysis ◽

Low Ph ◽

Coated Vesicles ◽

Magnesium Concentration ◽

Cross Linking ◽

Dependent Atpase ◽

Event Triggering ◽

Hydrolysis Of

ATP hydrolysis was used to power the enzymatic release of clathrin from coated vesicles. The 70,000-mol-wt protein, purified on the basis of its ATP-dependent ability to disassemble clathrin cages, was found to possess a clathrin-dependent ATPase activity. Hydrolysis was specific for ATP; neither dATP nor other ribonucleotide triphosphates would either substitute for ATP or inhibit the hydrolysis of ATP in the presence of clathrin cages. The ATPase activity is elicited by clathrin in the form of assembled cages, but not by clathrin trimers, the product of cage disassembly. The 70,000-mol-wt polypeptide, but not clathrin, was labeled by ATP in photochemical cross-linking, indicating that the hydrolytic site for ATP resides on the uncoating protein. Conditions of low pH or high magnesium concentration uncouple ATP hydrolysis from clathrin release, as ATP is hydrolyzed although essentially no clathrin is released. This suggests that the recognition event triggering clathrin-dependent ATP hydrolysis occurs in the absence of clathrin release, and presumably precedes such release.

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Actin filaments stimulate the Na(+)-K(+)-ATPase

AJP Renal Physiology ◽

10.1152/ajprenal.1995.269.5.f637 ◽

1995 ◽

Vol 269 (5) ◽

pp. F637-F643 ◽

Cited By ~ 8

Author(s):

H. F. Cantiello

Keyword(s):

Sequence Analysis ◽

Atpase Activity ◽

Actin Filaments ◽

Functional Role ◽

Atp Hydrolysis ◽

Rat Kidney ◽

Alpha Subunit ◽

Actin Binding ◽

Binding Domains

In this report, the functional role of actin on Na(+)-K(+)-adenosinetriphosphatase (Na(+)-K(+)-ATPase) activity was explored. The Na(+)- and K(+)-dependent, ouabain-sensitive ATP hydrolysis mediated by rat kidney Na(+)-K(+)-ATPase increased by 74% in the presence of previously unpolymerized actin (24 microM), whereas addition of polymerized actin was without effect. Addition of actin was associated instead with an increase in the affinity of the Na(+)-K(+)-ATPase for Na+ but not other enzymatic substates. A maximal stimulatory effect (296%) was observed either at an Na(+)-K(+)-ATPase:actin ratio of 1:50,000 or at lower ratios (1:625) by shifting from the E2 (K+ selective) to the E1 (Na+ selective) conformation of the enzyme. Immunoblotting of actin to the purified Na(+)-K(+)-ATPase suggested that this interaction may be linked to binding of actin to the enzyme, which was further supported by sequence analysis indicating putative actin-binding domains in the alpha-subunit of the enzyme. The interaction between actin and the Na(+)-K(+)-ATPase may imply a novel functional role of the cytoskeleton in the control of ion transport.

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An ATP-inhibited soluble 5'-nucleotidase of rat kidney

AJP Renal Physiology ◽

10.1152/ajprenal.1988.254.2.f191 ◽

1988 ◽

Vol 254 (2) ◽

pp. F191-F195

Author(s):

M. Le Hir ◽

U. C. Dubach

Keyword(s):

High Speed ◽

Catalytic Properties ◽

Divalent Cations ◽

Rat Kidney ◽

Maximal Activity ◽

Rate Of Hydrolysis ◽

Membrane Bound ◽

Ph Range ◽

Hydrolysis Of

Hydrolysis of 5'-AMP by 5'-nucleotidase is a possible source of adenosine in the kidney. A renal membrane-bound ecto-5'-nucleotidase has been previously described. The present study deals with the catalytic properties of a 5'-AMP phosphohydrolase partially purified from high-speed supernatants of rat kidney homogenates. It exhibits phosphatase activity toward 5'-AMP, 5'-IMP, and 5'-GMP, but not toward 2'- and 3'-AMP and corresponds therefore to a 5'-nucleotidase. The hydrolysis of 5'-AMP by the soluble 5'-nucleotidase requires divalent cations. Maximal activity is reached with 10 microM of either Mn2+ or Co2+, whereas half-maximal activity is obtained with approximately 400 microM Mg2+. The soluble 5'-nucleotidase exhibits Michaelis-Menten kinetics with a Km of 9.5 microM for 5'-AMP. In the presence of 1 mM of free Mg2+, physiological concentrations of ATP provoke an increase of the Km for 5'-AMP and a decrease of Vmax. An increase of the pH of 0.4 units in the pH range 6.4-7.4 roughly doubles the rate of hydrolysis of 5'-AMP. The effects of ATP and of the pH are compatible with a role of the renal soluble 5'-nucleotidase in the hydrolysis of 5'-AMP and in the production of adenosine during hypoxia.

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The basal Mg2+-dependent ATPase activity is not part of the (H++K+)-transporting ATPase reaction cycle

Biochemical Journal ◽

10.1042/bj2670565 ◽

1990 ◽

Vol 267 (3) ◽

pp. 565-572 ◽

Cited By ~ 4

Author(s):

H T W M Van der Hijden ◽

S Kramer-Schmitt ◽

E Grell ◽

J J H H M de Pont

Keyword(s):

Gastric Mucosa ◽

Atpase Activity ◽

Hydrolytic Activity ◽

Reaction Cycle ◽

Dependent Atpase ◽

Atpase Reaction ◽

Phosphorylation Reaction ◽

Rate Of Hydrolysis ◽

Hydrolysis Of

Purified gastric (H(+)+K+)-transporting ATPase [(H(+)+K+)-ATPase] from the parietal cells always contains a certain amount of basal Mg2(+)-dependent ATPase (Mg2(+)-ATPase) activity. lin-Benzo-ATP (the prefix lin refers to the linear disposition of the pyrimidine, benzene and imidazole rings in the ‘stretched-out’ version of the adenine nucleus), an ATP analogue with a benzene ring formally inserted between the two rings composing the adenosine moiety, is an interesting substrate not only because of its fluorescent behaviour, but also because of its geometric properties. lin-Benzo-ATP was used in the present study to elucidate the possible role of the basal Mg2(+)-ATPase activity in the gastric (H(+)+K+)-ATPase preparation. With lin-benzo-ATP the enzyme can be phosphorylated such that a conventional phosphoenzyme intermediate is formed. The rate of the phosphorylation reaction, however, is so low that this reaction with subsequent dephosphorylation cannot account for the much higher rate of hydrolysis of lin-benzo-ATP by the enzyme. This apparent kinetic discrepancy indicates that lin-benzo-ATP is not a substrate for the (H(+)+K+)-ATPase reaction cycle. This idea was further supported by the finding that lin-benzo-ATP was unable to catalyse H+ uptake by gastric-mucosa vesicles. The breakdown of lin-benzo-ATP by the (H(+)+K+)-ATPase preparation must be due to a hydrolytic activity which is not involved in the ion-transporting reaction cycle of the (H(+)+K+)-ATPase itself. Comparison of the basal Mg2(+)-ATPase activity (with ATP as substrate) with the hydrolytic activity of (H(+)+K+)-ATPase using lin-benzo-ATP as substrate and the effect of the inhibitors omeprazole and SCH 28080 support the notion that lin-benzo-ATP is not hydrolysed by the (H(+)+K+)-ATPase, but by the basal Mg2(+)-ATPase, and that the activity of the latter enzyme is not involved in the (H(+)+K+)-transporting reaction cycle (according to the Albers-Post formalism) of (H(+)+K+)-ATPase.

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Determination of Na-K-ATPase activity in single segments of the mammalian nephron

AJP Renal Physiology ◽

10.1152/ajprenal.1979.237.2.f105 ◽

1979 ◽

Vol 237 (2) ◽

pp. F105-F113 ◽

Cited By ~ 9

Author(s):

A. Doucet ◽

A. I. Katz ◽

F. Morel

Keyword(s):

Enzyme Activity ◽

Atpase Activity ◽

Inorganic Phosphate ◽

Rapid Freezing ◽

Phosphate Release ◽

Large Numbers ◽

Hydrolysis Of ◽

Single Tubules

A micromethod for the determination of Na-K-ATPase in discrete segments of nephrons from rabbit, rat, and mouse kidneys is described. To facilitate tubule microdissection, the kidneys were perfused with collagenase after it had been verified that collagenase had no effect on ATPase activity. Individual tubule segments were dissected under stereomicroscopic observation, exposed to a hypotonic environment followed by rapid freezing, and incubated in 1 microliter assay medium. Enzyme activity was determined by direct measurement of labeled inorganic phosphate release by the hydrolysis of [gamma-32P]ATP and was expressed as a function of tubule length. This method is technically simple enough to permit simultaneous measurement of the enzyme in large numbers of tubules and sufficiently sensitive to determine its activity in each region of the nephron. Correlation of Na-K-ATPase activity in single tubules with functional measurements obtained in the corresponding segment of the nephron with the perfused tubule or micropuncture techniques should help define the role of this enzyme in tubular ion transport.

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Comparative Studies on the ATP-Binding Sites in Ca2+-ATPase and (Na+ + K+)-ATPase by the Use of ATP-Analogues

Zeitschrift für Naturforschung C ◽

10.1515/znc-1982-7-820 ◽

1982 ◽

Vol 37 (7-8) ◽

pp. 692-705 ◽

Cited By ~ 2

Keyword(s):

Atpase Activity ◽

Binding Sites ◽

Sulfhydryl Groups ◽

Atp Binding ◽

Stable Form ◽

Atp Binding Site ◽

Atp Analogues ◽

Transport Atpases ◽

High Concentrations ◽

Hydrolysis Of

Abstract The effects of ATP-analogues on Ca2+-ATPase and (Na+ + K+)-ATPase have been studied. The participation of sulfhydryl groups in the recognition of ATP by both transport ATPases is indicat ed by the fact, that the disulfide of thioinosine triphosphate inactivates both enzymes. The reactivity of rapidly and slowly reacting sulfhydryl groups in the ATP binding sites of both enzymes is altered by the presence of transport substrates. At least in (Na+ + K+)-ATPase Na+ and Mg2+ appear to alter the structure of the ATP binding site, which conclusion is fortified by the fact, that the photoinactivation of the enzyme by 3′-O-[3-(2-nitro-4-azidophenyl)-propionyl]-ATP need Mg2+. Chromium(III)ATP, a MgATP analogue, inactivated both transport ATPases by the formation of a stable chromo-phosphointermediate. In the case of Ca2+-ATPase this was concomited by the occlusion of Ca2+ in a stable form. No occlusion of Na+ was observable so far in the (Na++ K+)-ATPase. Contrary to the expectation of the Albers-Post-scheme the hydrolysis of the phosphointermediate formed from chromium(III)ATP was protected by K+, but activated by high concentrations of Na+. Consequently, despite of the inhibition of (Na+ + K+)-ATPase activity chromium(III)-ATP supported the Na+ - Na+ -exchange reaction in everted red bood cells.

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