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PubMed1975

Interaction of adenine nucleotides with multiple binding sites on beef heart mitochondrial adenosine triphosphatase.

Beef heart mitochondrial ATPase (F1) contained 2 mol of ADP and 1 mol of ATP/mol of enzyme, which resisted removal by Sephadex chromatography with dilute buffers or repeated precipitation with ammonium sulfate. The native enzyme also contained two apparently equivalent binding sites, which participated in readily reversible binding of adenyl-5'-ylimidodiphosphate (AMP-P(NH)P), with a Kd of 1.3 mum. The failure of AMP-P(NH)P to compete effectively with ADP for binding sites on F1 may be related to the failure of the analog to inhibit oxidative phosphorylation. Virtually complete removal of all adenine nucleotides from F1 occurred when the enzyme was chromatographed on columns of Sephadex equilibrated with 50% glycerol. No loss in ATPase activity was observed following removal of nucleotides from the enzyme, which was then capable of binding more than 4 mol of ADP and almost 5 mol of AMP-P(NH)P/mol of protein. Subsequent chromatography on columns of Sephadex equilibrated with dilute buffers containing Mg2+ removed only 1.5 mol of ADP and no AMP-P(NH)P from the enzyme. Reconstitution of F1 with ADP or with almost 5 mol of AMP-P(NH)P resulted in preparations that exhibited an undiminished capacity to restore oxidative phosphorylation in F1-deficient submitochondrial particles.

Adenine Nucleotides↗
PubMed1977

Adenosine triphosphate synthesis by electrochemical proton gradient in vesicles reconstituted from purified adenosine triphosphatase and phospholipids of thermophilic bacterium.

Vesicles were reconstituted from a purified dicyclohexyl-carbodiimide-sensitive ATPase complex (TF0-F1) and phospholipids of a thermophilic bacterium PS3. These vesicles synthesized ATP from ADP and Pi with energy from an electrochemical proton gradient (delta-micronH+) formed by a pH gradient and an electrical potential across their membranes. Maximal ATP synthesis was achieved by incubating the vesicles in malonate at pH 5.5 with valinomycin, and then rapidly transferring them to a solution of pH 8.4 and 150 mM K+. Under these conditons ATP synthesis continued at a decreasing rate for 30 s at 40 degrees. Appreciable formation of ATP (40 to 150 nmol/mg of TF0-F1) occurred at an initial delta-micronH+ above 205 mV and moderate formation at an initial value above 180 mV. ATP hydrolysis by the vesicles produced a delta-micronH+, and the additions of 32Pi and hexokinase to them resulted in 32Pi esterification. Analysis of the time courses of 32Pi esterification and decays of the pH difference and membrane potential, followed using 9-aminoacridine and 8-anilinonaphthalene-1-sulfonate, respectively, as probes, showed a relationship between delta-micronH+ and the rate of ATP synthesis. These results demonstrate that purified TF0-F1 is itself a reversible H+-translocating ATPase of oxidative phosphorylation.

Adenosine Triphosphatases↗
PubMed1985

Energy-dependent dissociation of ATP from high affinity catalytic sites of beef heart mitochondrial adenosine triphosphatase.

Incubation of [gamma-32P]ATP with a molar excess of the membrane-bound form of mitochondrial ATPase (F1) results in binding of the bulk of the radioactive nucleotide in high affinity catalytic sites (Ka = 10(12) M-1). Subsequent initiation of respiration by addition of succinate or NADH is accompanied by a profound decrease in the affinity for ATP. About one-third of the bound radioactive ATP appears to dissociate, that is, the [gamma-32P]ATP becomes accessible to hexokinase. The NADH-stimulated dissociation of [gamma-32P]ATP is energy-dependent since the stimulation is inhibited by uncouplers of oxidative phosphorylation and is prevented by respiratory chain inhibitors. The rate of the energy-dependent dissociation of ATP that occurs in the presence of NADH, ADP, and Pi is commensurate with the measured initial rate of ATP synthesis in NADH-supported oxidative phosphorylation catalyzed by the same submitochondrial particles. Thus, the rate of dissociation of ATP from the high affinity catalytic site of submitochondrial particles meets the criterion of kinetic competency under the conditions of oxidative phosphorylation. These experiments provide evidence in support of the argument that energy conserved during the oxidation of substrates by the respiratory chain can be utilized to reduce the very tight binding of product ATP in high affinity catalytic sites and to promote dissociation of the nucleotide.

Adenosine Triphosphatases↗
PubMed1988

Transient-kinetic studies of the adenosine triphosphatase activity of scallop heavy meromyosin.

Fluorescence stopped-flow experiments were performed to elucidate the elementary steps of the ATPase mechanism of scallop heavy meromyosin in the presence and in the absence of Ca2+. ATP binding and hydrolysis, as monitored by the change in tryptophan fluorescence, appear to be Ca2+-insensitive, whereas both Pi release and ADP release are markedly suppressed in the absence of Ca2+. Rate constants for Pi release are 0.2 s-1 and 0.002 s-1 and for ADP release are 6 s-1 and 0.01 s-1 in the presence and in the absence of Ca2+ respectively. Ca2+ binding to the specific site of the regulatory domain is rapid and its release occurs at 25 s-1, consistent with the time scale of a twitch of the striated adductor muscle. Nucleotide binding is a multi-step process requiring a minimum of three states. In such a model Ca2+ controls the rate of conformational changes at the active site in both the forward and the reverse direction, leading to a large dependence of the rate of nucleotide release, but a lesser effect on the overall equilibrium position. The kinetic trapping of nucleotides and Pi at the active site, in the absence of Ca2+, appears to be a fundamental step in suppressing the interaction of the myosin head with the thin filaments in relaxed molluscan muscle.

Adenosine Diphosphate↗
PubMed1976

Essential arginyl residues in mitochondrial adenosine triphosphatase.

Treatment of either beef heart or rat liver mitochondrial ATPase with the arginine reagent, 2,3-butanedione, resulted in enzyme inactivation. The reaction followed pseudo-first order kinetics until 90 to 95% of the enzyme had been inactivated, and prolonged incubation with butanedione resulted in complete inactivation. When the modification reaction was performed in the presence of ATP, the rate of inactivation was significantly decreased. The kinetics of inactivation indicates that the reaction of 1 molecule of reagent per active site of beef heart mitochondrial ATPase is necessary for inactivation. The loss of ATPase activity was also observed when submitochondrial particles were treated with butanedione. Studies with beef heart mitochondrial ATPase indicated that the inactivation was not due to enzyme dissociation into subunits. Kinetic studies with partially inactivated enzyme demonstrated that the Km values of ITP and of ATP in the presence of HCO3-were similar to the same constants for the control enzyme. When ATP was used as the substrate in the absence of anion activator, the partially inactivated enzyme still exhibited negative cooperativity. Inactivation was also observed when beef heart mitochondrial ATPase was treated with another arginine reagent, phenylglyoxal. The loss of ATPase activity was analyzed in terms of [14C]phenylglyoxal incorporation. From the present studies it is concluded that arginyl residues play an essential role in mitochondrial ATPase, probably at the hydrolytic site.

Adenosine Diphosphate↗
PubMed1980

Functional groups at the catalytic site of F1 adenosine triphosphatase.

The protection of F1 ATPase by inorganic phosphate, ADP, ATP, and magnesium ion against inactivation by 1-fluoro-2,4-dinitrobenzene, 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole, and 1-(ethoxycarbonyl)-2-ethoxy-1,2-dihydroquinoline, respectively, has been investigated. Dissociation equilibrium constants and rate constants for the labeling reactions have been deduced from a quantitative treatment of the kinetic data. Comparison of these dissociation constants with each other and with the corresponding literature values indicates that the essential Tyr, Arg, Lys, and Glu or Asp residues are indeed located at the catalytic site of the enzyme. Examination of the rate constants for the labeling reactions in the presence of excess inorganic phosphate, ADP, ATP, or magnesium ion, respectively, suggests that the essential phenol and amino groups are located nearer to the bound inorganic phosphate or the gamma-phosphate group than to the alpha- or beta-phosphate group of the bound ATP, that the essential guanidinium group is located nearer to the alpha- or beta-phosphate group than to the gamma-phosphate group of the bound ATP or the bound inorganic phosphate, and that the essential carboxylate group is located slightly farther away but complexed with magnesium ion which it shares with the bound inorganic phosphate. A mechanism consistent with these topographical relationships is proposed for the catalytic hydrolysis and synthesis of ATP.

4-Chloro-7-nitrobenzofurazan↗
PubMed1979

Evidence for the occurrence of an ecto-(adenosine triphosphatase) in rat epididymal spermatozoa.

Intact spermatozoa from rat cauda epididymis possess a Mg2+-dependent ATPase activity that hydrolyses externally added [gamma-32P]ATP. The ATPase reaction was linear with time for approx. 6 min and there was no detectable uptake of ATP by these cells. The ATPase activity of the whole spermatozoa was not due to leakage of the intracellular enzymic activity, contamination of the broken cells or any possible cell damage during incubation and isolation of spermatozoa. The activity of the enzyme was strongly inhibited (approx. 85%) by p-chloromercuribenzenesulphonic acid (50 microM) or the diazonium salt of sulphanilic acid (50 microM), which are believed not to enter the cells, whereas ouabain (0.5 mM), NaF (10 mM), NaN3 (2.5 mM) and oligomycin (5 microM) had no appreciable effect on the activity of the spermatozoal APTase. There was little loss of ATPase activity from the cells when washed with 0.5 mM-EDTA and an iso-osmotic or hyperosmotic medium. These data are consistent with the view that the observed ATPase activity is located on the external surface of spermatozoa. The sperm ecto-ATPase activity is resistant to the action of proteinases (50 micrograms/ml), namely trypsin, chymotrypsin and Pronase. Studies with various unlabelled phosphate esters indicate that the sperm ecto-ATPase is not a non-specific phosphatase and it has high degree of substrate specificity for ATP.

4-Chloromercuribenzenesulfonate↗
PubMed1986

Intracellular pH regulation and proton transport by rabbit renal medullary collecting duct cells. Role of plasma membrane proton adenosine triphosphatase.

Proton secretion in the renal medullary collecting duct is thought to occur via a luminal proton-ATPase. In order to determine what mechanism(s) participate in proton transport across medullary collecting duct (MCD) cells membranes, intracellular pH (pHi) regulation and proton extrusion rates were measured in freshly prepared suspensions of rabbit outer MCD cells. Cells were separated by protease digestion and purified by Ficoll gradient centrifugation. pHi was estimated fluorometrically using the entrapped intracytoplasmic pH indicator, 6-carboxyfluorescein. Proton extrusion rates were measured using a pH stat. The resting pHi of MCD cells was 7.19 +/- 0.05 (SE) in a nonbicarbonate medium of pH 7.30. When cells were acidified by exposure to acetate salts or by abrupt withdrawal of ammonium chloride, they exhibited pHi recovery to the resting pHi over a 5-min time-course. Depletion of greater than 95% of cellular ATP content by poisoning with KCN in the absence of glucose inhibited pHi recovery. ATP depletion inhibited proton extrusion from MCD cells. Treatment with N-ethylmaleimide also inhibited pHi recovery. In addition, cellular ATP content was dependent on transmembrane pH gradients, suggesting that proton extrusion stimulated ATP hydrolysis. Neither removal of extracellular sodium nor addition of amiloride inhibited pHi recovery. These results provide direct evidence that a plasma membrane proton-ATPase, but not a Na+/H+ exchanger, plays a role in proton transport and pHi regulation in rabbit MCD.

Acetates↗
PubMed1976

Steady-state studies of the actin-activated adenosine triphosphatase activity of myosin.

Reconstituted actomyosin (ATP phosphohydrolase, EC 3.6.1.3) (0.400 mg F-actin/mg myosin) in 10.0 muM ATP loses 96% of its specific ATPase activity when its reaction concentration is decreased from 42.0 mug/ml down to 0.700 mug/ml. The loss of specific activity at the very low enzyme concentrations is prevented by the addition of more F-actin to 17.6 mug/ml. It is concluded that at low actomyosin concentrations the complex dissociates into free myosin with a very low specific ATPase activity and free F-actin with no ATPase. The dissociation of the essential low molecular weight subunits of myosin from the heavy chains at very low actomyosin concentrations may be a contributing factor. Actomyosin has its maximum specific activity at pH 7.8-8.2. The Km for ATP is 9.4 muM, which is at least 20-fold greater than myosin's Km for ATP. The actin-activated ATPase of myosin follows hyperbolic kinetics with varying F-actin concentrations. The Km values for F-actin are 0.110 muM (4.95 mug/ml) at pH 7.4 and 0.241 muM (10.8 mug/ml) at pH 7.8. The actin-activated maximum turnover numbers for myosin are 9.3 s-1 at pH 7.4 and 11.6 s-1 at pH 7.8. The actomyosin ATPase is inhibited by KCl. This KCl inhibition is not competitive with respect to F-actin, and it is not a simple form of non-competitive inhibition.

Actins↗
PubMed1986

Comparison between calcium transport and adenosine triphosphatase activity in membrane vesicles derived from rabbit kidney proximal tubules.

Characteristics of Ca2+ uptake were studied in a vesicular preparation of proximal tubule plasma membranes from rabbit kidney and compared with the properties of both membrane-bound and solubilized Ca2+-ATPase activities. Calcium uptake required both ATP and MgCl2 and revealed two kinetic components with respect to Ca2+ concentration requirements, one with a high affinity for Ca2+ (1.8 microM), operative in the range of cytosolic Ca2+ activity, and one with a low affinity for Ca2+ (250 microM) which may become active only at abnormally high cytosolic Ca2+ concentrations. The high- and low-affinity components were stimulated to similar extents by phosphate, and required similar concentrations of ATP (0.6 mM) for half-maximal activity. The amount of membrane-bound phosphoenzyme formed from ATP in the presence of Ca2+ was the same regardless of whether only one or both sites were saturated, suggesting that occupancy of the second Ca2+ binding site accelerates the enzyme turnover. Inhibition of Ca2+ transport by Na+ was reversed by the addition of ouabain or an ATP-regenerating system, indicating that this inhibitory effect of Na+ on Ca2+ uptake may be due to the accumulation of ADP in the medium as a result of Na+ pump activity. Low concentrations of carbonyl cyanide p-trifluoromethoxyphenylhydrazone and valinomycin (2.5 and 1 microM, respectively) were without effect on Ca2+ uptake in the presence of phosphate, whereas higher concentrations of the ionophores (200 and 100 microM, respectively) reduced uptake by 60% or more. The calmodulin antagonist 48/80 also reduced Ca2+ uptake with half-maximal effectiveness at 100 micrograms/ml. None of these drugs affected either ATPase activity or the EGTA-induced Ca2+ efflux from preloaded vesicles. The Ca2+ dependence of ATP hydrolysis by the membrane-bound enzyme preparation was similar to that observed for Ca2+ uptake by the vesicles. However, with solubilized enzyme, concentrations of Ca2+ similar to that found in the plasma reduced Ca2+-stimulated ATP hydrolysis to one-half of its maximal rate. This indicates that peritubular Ca2+ may play a role in the regulation of Ca2+ transport across the tubular epithelium. ATP could not be replaced by ITP as a substrate for Ca2+ uptake, and the (Ca2+ + Mg2+)ITPase activity of soluble enzyme was 25-fold lower than in the presence of ATP. This is an indication that the active Ca2+ pumping mechanism in proximal tubules is critically dependent on the nucleoside moiety of the substrate.

Adenosine Triphosphatases↗
PubMed1982

Detection of the conformational change in the catalytic site of adenosine triphosphatase from beef liver mitochondria by affinity labeling with the dialdehyde derivative of ethenoadenosine triphosphate.

Beef liver mitochondrial F1ATPase was inactivated by the 2',3'-dialdehyde derivative of ethenoATP (epsilon ATP) in a pseudo-first order reaction. The kinetics of protection of the enzyme against inactivation by various nucleoside triphosphates (NTPs) revealed that the dial-epsilon ATP was bound to the catalytic site as an affinity label. Certain anions (sulfate or bicarbonate) were ineffective for protection. In the early phase of the reaction, inactivation was due to the binding of 1 mol dial-epsilon ATP per mol enzyme. In this phase, dial-epsilon ATP bound exclusively to the subunit beta of the enzyme, indicating that the catalytic site is in this subunit. The fluorescence of the ethenoadenosine moiety, bound exclusively to the subunit beta of the enzyme, was measured as a conformational probe of the catalytic site region. Addition of ATP or CTP to the labeled enzyme resulted in a decrease in the fluorescence intensity. GTP and other NTPs were less effective than ATP or CTP. The anions (sulfate of bicarbonate) suppressed the ability of ATP to decrease the fluorescence in a competitive manner. Quantitative analysis of these fluorescence changes suggested that they might originate from the binding of the NTP to the regulatory site of the enzyme. These findings are in good agreement with the two-site model proposed by us (Wakagi, T. & Ohta, T. (1981) J. Biochem. 89, 1205) which was deduced from the steady state kinetics of the NTPase reactions catalyzed by the F1ATPase.

Adenosine Triphosphatases↗
PubMed1980

Steady-state magnesium ion-adenosine triphosphatase activities of myosin and its subfragment 1 derivative.

The Mg2+-ATPase activity of myosin and its subfragment 1 (ATP phosphohydrolase, EC 3.6.1.3) always followed normal Michaelis-Menten kinetics for ATP concentrations less than 10 microM. The average Km values at pH 7.4 and 25 degrees C are 0.33 +/- 0.04 microM for myosin and 0.43 +/- 0.11 microM for subfragment 1. At low salt concentration myosin yields a second hyperbolic increase in Mg2+-ATPase activity as the ATP rises from 10.2 microM to 153 microM: V doubles with a Km of 11 +/- 5 microM. This second low-salt-dependent increase in Mg2+-ATPase activity occurred between pH 6.8 and pH 8.7. It was not affected by the presence of 0.10 M EGTA to remove Ca2+ contamination. Solubilization of the catalytic sites by assaying myosin for ATPase activity in the presence of 0.60 M NaCl or by conversion of myosin to subfragment 1 eliminated the secondary hyperbolic increase. Subfragment 1 has a significantly different pH-activity curve from that of myosin. Subfragment 1 has an activity peak at pH 6.0, a rising activity as the pH goes from 8.7 to 9.8, and a deep activity valley between pH 6.8 and pH 8.4. Myosin has a very shallow trough of activity at pH 6.8 to 8.4, and in 1.0 mM ATP its activity drops as the pH decreases from 6.8 to 6.0. NaCl is a noncompetitive inhibitor of the Mg2+-ATPase activity of myosin and subfragment 1. Myosin has a greater affinity for NaCl (Ki = 0.101 +/- 0.004 M) than does subfragment 1 (Ki = 0.194 +/- 0.009 M).

Adenosine Triphosphatases↗
PubMed1981

Implications of the existence of two states of beef liver mitochondrial adenosine triphosphatase as revealed by kinetic studies.

The results of studies on the initial velocity in hydrolysis reactions of ATP and other nucleoside triphosphates with beef liver mitochondrial ATPase can be summarized as follows. 1. Double reciprocal plots of substrate concentration vs. initial velocity were linear for all the nucleoside triphosphates tested except for ATP, which showed a negative cooperativity. 2. Bicarbonate ion increased the rate of ATP hydrolysis and diminished its negative cooperativity, whereas hydrolysis of other nucleoside triphosphates was only slightly affected by the anion. 3. An excess of nucleoside triphosphate apparently inhibited its own hydrolysis for all kinds of nucleoside triphosphates tested, whereas an excess of magnesium ion apparently inhibited only ATP hydrolysis. 4. Inhibition of ATPase activity with an excess of magnesium ion was no longer observed when the reaction was carried out at low temperature (10 degrees C) or in the presence of sulfate. Under these conditions, the kinetics of ATP hydrolysis were apparently of simple Michaelis-Menten type. These observations suggest the existence of two states ("A" and "N") of beef liver mitochondrial ATPase. The state "A" is characterized by phenomena specifically affecting ATP hydrolysis, such as the inhibition by excess magnesium ion, and the negatively cooperative profile in the dose-response curve of ATP. In the state "N" proceed the hydrolysis reactions of other nucleoside triphosphates and of ATP under limited conditions. The two states ("A" and "N") can be related to an enzyme model with a catalytic site and a regulatory site. Computer stimulation revealed that such a model could account well for the experimental data.

Adenosine Triphosphatases↗
PubMed1988

Pre-steady-state studies of the adenosine triphosphatase activity of coupled submitochondrial particles. Regulation by ADP.

ATPase activities were measured in 10 mM MgCl2, 5 mM ATP, 1 mM ADP, and 1 microM FCCP with submitochondrial particles from bovine heart that had been stimulated by delta mu H+-forming substrates and with particles whose natural inhibitor protein was partially removed by heating. The activities were not linear with time. With both particles, the rate of ATP hydrolysis in the 7-fold greater than that in the steady state. Pre-steady-state and steady-state kinetic studies showed that the decrease of ATPase activity was due to the binding of ADP in a high-affinity site of the enzyme (K0.5 of 10 microM). Inhibition of ATP hydrolysis was accompanied by the binding of approximately 1 mol of ADP/mol of particulate F1; 10 microM ADP gave half-maximal binding. ADP could be replaced by IDP, but with an affinity 50-fold lower (K0.5 of 0.5 mM). Maximal inhibition by ADP and IDP was achieved in less than 5 s. Inhibition was enhanced by uncouplers. Even in the presence of pyruvate kinase and phosphoenolpyruvate, the rates of hydrolysis were about 2.5-fold higher in the first seconds of reaction than in the steady state. This decrease of ATPase activity also correlated with the binding of nearly 1 mol of ADP/mol of F1. This inhibitory ADP remained bound to the enzyme after several thousand turnovers. Apparently, it is possible to observe maximal rates of hydrolysis only in the first few catalytic cycles of the enzyme.

Adenosine Diphosphate↗
PubMed1974

Separation at neutral pH value of 32P-labelled proteins in a membrane preparation from ox brain. Partial characterization of component of the sodium-plus-potassium ion-activited adenosine triphosphatase.

1. Ox brain microsomal fractions were labelled with [(32)P]ATP in the presence of Na(+) and the reaction was stopped with sodium dodecyl sulphate. The Na(+)-dependent bound phosphate was isolated on Sephadex G-25 and by acetone precipitation. The bound phosphate isolated under these neutral conditions was labile to hydroxylamine and gave the same pH profile of hydrolysis as that isolated by precipitation with strong acids. 2. When membrane protein was labelled with [(32)P]ATP, solubilized with sodium dodecyl sulphate and fractionated on Sepharose 6B, the Na(+)-dependent label emerged in a peak corresponding to protein of molecular weight 570000-580000. On fractionation of this protein peak on polyacrylamide gels containing detergent and urea, the Na(+)-dependent label occurred in a single band corresponding to a protein of molecular weight 102000. 3. Fractionation on Sepharose 6B of protein labelled with [(32)P]ATP in the absence of Na(+) revealed three labelled peaks, one of which corresponded in position to the Na(+)-dependent label. Electrophoresis of this peak material on polyacrylamide gels showed that most of the label occurred in two fast-running bands. Cyclic AMP stimulated the labelling in these two bands, but had no effect on the labelling of the band corresponding in position to the Na(+)-dependent label. 4. Di-isopropyl [(32)P]phosphorofluoridate also labelled the band corresponding to the Na(+)-dependent label on gel electrophoresis. The labelling of this band by the reagent was inhibited by 50-60% by 3mm-ATP, but there was no evidence to suggest that the group labelled is normally phosphorylated by ATP.

Adenosine Triphosphatases↗
PubMed1976

Removal of "tightly bound" nucleotides from soluble mitochondrial adenosine triphosphatase (F1).

Soluble mitochondrial ATPase (F1) from beef heart prepared in this laboratory contained approximately 1.8 mol of ADP and 0 mol of ATP/mol of F1 which were not removed by repeated precipitation of the enzyme with ammonium sulfate solution or by gel filtration in low ionic strength buffer containing EDTA. This enzyme had full coupling activity. Treatment of the enzyme with trypsin (5 mug/mg of F1 for 3 min) reduced the "tightly bound" ADP to zero, abolished coupling activity, but had no effect on the ATPase activity, stability, or membrane-binding capability of the F1. When the trypsin concentration was varied between 0 and 5 mug/mg of F1, tightly bound ADP was removed to varying degrees, and a correlation was seen between amount of residual tightly bound ADP and residual coupling activity. Gel filtration of the native F1 in high ionic strength buffer containing EDTA also caused complete loss of tightly bound ADP and coupling ability, whereas ATPase activity, stability, and membrane-binding capability were retained. The ADP-depleted F1 preparations were unable to rebind normal amounts of ADP or any ATP in simple reloading experiments. The results strongly suggest that tightly bound ADP is required for ATP synthesis and for energy-coupled ATP hydrolysis on F1. The results also suggest that ATP synthesis and energy-linked ATP hydrolysis rather than involving one nucleotide binding site on F1, involve a series or "cluster" of sites. The ATP hydrolysis site may represent one component of this cluster. The results show that nonenergy-coupled ATP hydrolysis on F1 can occur in the absence of tightly bound ADP or ATP.

Adenosine Diphosphate↗
PubMed1995

Detection of different adenosine triphosphatases in human placental brush border membranes.

The microvillous membrane of human placental syncytiotrophoblast cells contains a high ATPase activity. The purpose of this study was to characterize this activity and to investigate the presence of vacuolar type H+ ATPase in this membrane. Intact brush border membrane vesicles strongly hydrolyzed ATP, reflecting the presence of ATPase on the external side of the membrane. The ATPase activity was entirely Mg2+ dependent and increased with pH. At pH 7.5, Vmax was 31.0 +/- 1.7 mumol/mg/20 min and Km 0.18 +/- 0.03 mM ATP. Hydrolysis of ATP was not influenced by the presence of bicarbonate or alkaline phosphatase inhibitors, but at pH 8 it decreased by half following addition of 100 microM dicyclohexylcarbodiimide (DCCD). At pH 7.5, 1 mM N-ethylmaleimide (NEM) depressed this activity by less than 5%. Opening the membrane vesicles with 0.1% desoxycholate (DOC) or Triton-X neither revealed any additional ATPase activity nor altered the low sensitivity to NEM. Treatment of these membranes with 1% cholate decreased the ATPase activity by more than 70% and did not enhance the sensitivity of ATP hydrolysis to NEM. 10(-7) M Bafilomycin, which reduced by 56 +/- 9% the ATPase activity in dog kidney brush border membranes treated with 0.1% DOC, had no effect on placental brush border membranes subjected to the same procedure. Finally, neither immunocytochemical staining using monoclonal antibody to the M(r) 31000 subunit of V-type H+ ATPase, nor electron microscopic examination detected the presence of H(+) ATPase in placental membranes. In conclusion, the placental brush border membrane is the site of a strong "ecto" ATPase activity which is partially DCCD sensitive.(ABSTRACT TRUNCATED AT 250 WORDS)

Adenosine Triphosphatases↗