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PubMed1975

Molecular properties of purified (sodium + potassium)-activated adenosine triphosphatases and their subunits from the rectal gland of Squalus acanthias and the electric organ of Electrophorus electricus.

The chemical properties of two highly purified preparations of (sodium + potassium)-activated adenosine triphosphatase (NaK ATPase) and their subunits have been compared. One preparation is derived from the rectal gland of the spiny dogfish shark, Squalus acanthias and the other preparation is derived from the electric organ of the electric eel, Electrophorus electricus. Ouabain binding and phosphorylation from [gamma-32-P]ATP for both enzymes ranged from 4000 to 4300 pmol per mg of protein. This gives a stoichiometry for ouabain binding and phosphorylation of 1:1 for both enzymes. The molar ratios of catalytic subunit to glycoprotein was 2:1 for both enzymes, suggesting a minimum molecular weight of 250, 000, which agrees with the molecular weight obtained by radiation inactivation. Assuming that only one of the two catalytic subunits is phosphorylated and binds ouabain per (sodium + potassium)-activated adenosine triphosphatase molecule the data on phosphorylation and ouabain binding also give a molecular weight of 250, 000. The data on phosphorylatiion, ouabain binding, subunit composition, and molecular weight based on radiaion inactivation are thus all internally consistent. A technique has been developed for isolation of pure catalytic subunit and glycoprotein in good yields by preparative sodium dodecyl sulfate-polyacrylamide gel electrophoresis. A variety of chemical studies have been carried out with the purified subunits. The amino acid composition of the catalytic subunit was different from that of the glycoprotein, but the amino acid composition of each of the two subunits was essentially the same for both species. However, the NH2-terminal amino acid for the catalytic subunit was alanine for the rectal gland enzyme and serine for the electric organ enzyme, suggesting some differencesin amino acid sequences for the two species. The NH2-terminal amino acid for the glycoprotein was alanine for the two species. The glycoproteins from both species contained the same carbohydrates but in quite differing amounts. The carbohydrates were glucosamine, sialic acid, fucose, galactose, mannose, and glucose. The release of all the sialic acid from the electric organ enzyme and the release of 40% of the sialic acid from the rectal gland enzyme did not affect (sodium + potassium)-activated adenosine triphosphatase activity. Both enzymes contained the following phospholipids, which accounted for 98 to 100% of the total phospholipid phosphorus: sphingomyelin, lecithin, phosphatidylserine, phosphatidylethanolamine, and phosphatidylinositol. With the exception of phosphatidylethanolamine, and phosphatidylinositol. With the exception of phosphatidylserine, the amount of any phospholipid per mg of enzyme as well as the total phospholipid content were quite different for the two enzymes.

Adenosine Triphosphatases↗

PubMed1971

Dinitrophenol-stimulated adenosine triphosphatase activity in extracts of Desulfovibrio gigas.

A dinitrophenol (DNP)-stimulated adenosine triphosphatase (ATPase) has been found in both the soluble and particulate fractions of the anaerobic sulfate-reducing bacterium, Desulfovibrio gigas. As the soluble ATPase was labile to storage, only the particulate enzyme was studied in detail. It was optimally stimulated by DNP at 4 mm, and activity was insensitive to inhibition by ouabain. The ATPase was stimulated by both Ca(2+) and Mg(2+), but the magnitude of the stimulation was dependent upon pH. In the presence of Ca(2+) the optimum pH was 6.5, whereas, in the presence of Mg(2+) the pH optimum was 8.0. However, under optimal conditions the activity was the same with either Mg(2+) or Ca(2+). Both adenosine triphosphate and guanosine triphosphate were hydrolyzed, but activity toward guanosine triphosphate was only one-tenth that observed with adenosine triphosphate.

Adenosine Triphosphatases↗

PubMed1978

Erythrocyte membrane Na+ and K+ activated adenosine triphosphatase in protein-calorie malnutrition.

Total, ouabain insensitive and ouabain sensitive Na+ and K+ adenosine triphosphatase activity in the erythrocyte membrane of protein-calorie malnourished children with marasmus and kwashiorkor was compared with the enzyme activity in apparently healthy children (normals). Na+ and K+ contents of erythrocyte and plasma were also determined in these patients. Specific activity (units per milligram of membrane protein) of ouabain sensitive Na+ and K+ adenosine triphosphatase was significantly higher in erythrocyte membrane preparations from children with kwashiorkor but not from children with marasmus. After 4 to 5 weeks of treatment with diets sufficient in protein and calories the specific activity of the enzyme was lower as compared to that on admission. Erythrocyte and plasma Na+ content (microgram/10(6) cells and microgram per milliliter of plasma) in children with kwashiorkor were not different from those in normal children, however, there was reduction in K+ content of erythrocytes and plasma of these children. After treatment, erythrocyte Na+ and K+ and plasma K+ in children with kwashiorkor increased significantly. In marasmic children erythrocyte Na+ was higher as compared to normal but there were no differences in K+ content of either eyrthrocytes or plasma in these children.

Adenosine Triphosphatases↗

PubMed1984

The plasma membrane (Mg2+)-dependent adenosine triphosphatase from the human erythrocyte is not an ion pump.

The plasma membrane (Mg2+)-dependent adenosine triphosphatase [Mg2+)-ATPase) from human erythrocytes has been tested for its ability to transport ions. Using a preparation of inside-out vesicles loaded with the pH-sensitive fluorescence probe 1-hydroxypyrene-3,6,8-trisulfonic acid (HPTS), we have demonstrated the absence of proton movement during (Mg2+)-ATPase activity. From the rate of ATP hydrolysis and the passive proton permeability of these vesicles, an upper limit of 0.03 H+ transported per ATP hydrolyzed was calculated. To verify that proton pumping could be detected in this system, the intravesicular pH was monitored during (Ca2+)-dependent adenosine triphosphatase [Ca2+)-ATPase) activity. Proton efflux associated with (Ca2+)-ATPase activity was observed (in agreement with a recent report of proton pumping by a reconstituted erythrocyte (Ca2+)-ATPase (Niggli, V., Sigel, E., Carafoli, E. (1982) J. Biol. Chem. 257:2350-2356] and was shown to be stimulated by calmodulin. The ability of the (Mg2+)-ATPase to pump 28Mg2+, 35SO2-4 and 86Rb+ was also tested, with the results leading to the conclusion that the human erythrocyte enzyme does not function as an ion transport system.

Adenosine Triphosphatases↗

PubMed1965

Effects of sodium and potassium ions on oxidative phosphorylation in relation to respiratory control by a cell-membrane adenosine triphosphatase.

1. A study has been made of the oxygen consumption of kidney homogenates in relation to the ADP concentration as regulated by the cell-membrane adenosine triphosphatase. Stimulation of this enzymic activity by Na(+) and K(+) caused parallel increases in oxygen consumption and ADP concentration. Similarly, inhibition with ouabain caused a parallel fall. The membrane adenosine triphosphatase concerned in active transport therefore appears to regulate respiration through its control of ADP concentration. 2. The respiration of homogenates and mitochondria was also stimulated by K(+) in a way independent of adenosine-triphosphatase activity. It was shown that K(+) facilitates oxidative phosphorylation and the respiratory response to ADP. A K(+) concentration of 25-50mm was needed for maximum oxidative phosphorylation in the presence of physiological concentration of Na(+). Na(+) counteracted K(+) in the effects on mitochondria. It is concluded that K(+) regulates cellular respiration at two structures, one directly in mitochondria, and the second indirectly through control of ADP production at the cell membrane.

Journal Article↗

PubMed1979

An adenosine triphosphate dependent deoxyribonuclease with adenosine triphosphatase, activity from Bacillus cereus.

An adenosine triphosphate-stimulated deoxyribonuclease was purified to about 4200 fold from Bacillus cereus. The enzyme activity of the crude extract increased by a factor of about 5 after dialysis. One of the low molecular weight inhibitors of the crude extract was found to be inorganic phosphate. During enzyme purification two nucleases were identified. One of them was specific to denatured DNA and the other one degraded both denatured DNA and native DNA. The activity towards native DNA could be increased several times by ATP. Through all steps of purification the ATP-independent DNase always accompanied the ATP-dependent one and the ratio of their activity was found to be constant. The ATP-dependent DNase also possessed ATPase activity stimulated both by native and denatured DNA. The fact that ATPase was stimulated by DNA and went together with ATP-dependent DNase during purification suggests that these functions belong to the same enzyme complex. Maximal activity of ATPase had broader pH, Mg2+ and ATP concentration ranges than that of DNase. Cooperation of the two functions may be limited only to a narrow range of ATP concentration. Km for ATPase was 1.6x10-4 M ATP.

Adenosine Triphosphatases↗

PubMed1981

K+-independent active transport of Na+ by the (Na+ and K+)-stimulated adenosine triphosphatase.

The (Na+ and K+)-stimulated adenosine triphosphatase (Na+,K+)-ATPase) from canine kidney reconstituted into phospholipid vesicles showed an ATP-dependent, ouabain-inhibited uptake of 22Na+ in the absence of added K+. This transport occurred against a Na+ concentration gradient, was not affected by increasing the K+ concentration to 10 microM (four times the endogenous level), and could not be explained in terms of Na+in in equilibrium Na+out exchange. K+-independent transport occurred with a stoichiometry of 0.5 mol of Na+ per mol of ATP hydrolyzed as compared with 2.9 mol of Na+ per mol of ATP for K+-dependent transport.

Animals↗

PubMed1978

Viral adenosine triphosphatase.

The catalytic and immunological properties of an adenosine triphosphatase from different types of virus have been studied. The avian myeloblastosis virus has been found to be specialized in holding this enzyme in a highly active state as compared to other virus with respect to their host cell enzyme. Catalytically myeloblastosis virus and Rous virus ATPase behave alike, while that of the Reo virus is significantly different.

Adenosine Triphosphatases↗

PubMed1976

Synaptosomal adenosine triphosphatase (ATPase) inhibition by organophosphates.

Chicken spinal cord adenosine triphosphatases (both Na+, K+ stimulated and ouabain insensitive) were inhibited by tri-o-tolyl phosphate (TOTP, a neurotoxic organophosphate which is not a cholinesterase inhibitor) and mevinphos (a non-neurotoxic compound but inhibitor of cholinesterases). The inhibition was concentration and time dependent, with an initial rapid drop in activity followed by a gradual exponential decline.

Adenosine Triphosphatases↗

PubMed1981

A modification of the adenosine triphosphatase method to demonstrate epidermal Langerhans cells.

Epidermal Langerhans cells are most commonly demonstrated by utilizing their adenosine triphosphatase reactivity. Although this is one of the best methods available to the light microscopist, it is a capricious technique which does not always permit optimal demonstration of this cell population. Prolonged fixation in cacodylate formalin and incubation in 1.32 X 10-3 molar adenosine triphosphatase permits reliable reproduction and good definition of these dendritic cells.

Adenosine Triphosphatases↗

PubMed1977

Inhibition of renal adenosine triphosphatase by cadmium.

The effects of CdCl2 on adenosine triphosphatase (ATPase) were studied microsomal fractions or tissue homogenates of outer cortex, inner cortex and outer medulla of dog kidney. Cd was found to be an inhibitor of Na+ +K+ Atpase with 150 value of 2.1 to 3.2 X 10(-4) M regardless of type or source of the enzyme preparation tested. Mg++ ATPase was about 10-fold less sensitive to inhibition by Cd than Na+ +K+ ATPase. The inhibition of microsomal NA+ +K+ ATPase from outer medulla was characterized by irreversible kinetics. The inhibitory effect was not altered by varying Na+ or K+ concentrations, but was decreased by disodium ethylenediaminetetracetic acid (EDTA). EDTA was more effective in preventing than in reversing the inhibition. Na+ +K+ ATPase from kidneys of several other mammalian species showed a similar sensitivity to Cd.

Adenosine Triphosphatases↗

PubMed1979

Effects of acute and continuous morphine administration on catecholamine-sensitive adenosine triphosphatase in mouse brain.

The catecholamine-stimulated adenosine triphosphatase (ATP-ase) activities in mouse brain synaptosomes were inhibited by morphine both in vitro and in vivo. Morphine up to 10(-3) M had no effect on basal ATPase activities but at 10(-4) M significantly inhibited dopamine-sensitive ATPase activities in vitro. The morphine effect was antagonized by an opiate antagonist, naloxone. The catecholamine-sensitive ATPase activities were also inhibited by acute administration of morphine. The inhibition was dose-dependent. However, naloxone partially antagonized the morphine inhibition of depamine-sensitive ATPase activity but not norepinephrine-sensitive ATPase activity. A significant decrease in the sensitivity of synaptosomal ATPase to catecholamines was observed in mice rendered tolerant by morphine pellet implantation. The Na+,K+-ATPase was more affected by morphine as compared to Mg++-ATPase activity. The dopamine-sensitive Na+,K+-ATPase activity was restored by 50% in precipitated withdrawal mouse brain synaptosomes. Norepinephrine-sensitive ATPase activity was also restored partially in precipitated withdrawal animals. These results suggest that in mouse brain synaptosomes morphine may be interacting with ATPase at or near the catecholamine-active sites.

Adenosine Triphosphatases↗

PubMed1980

Subcellular localization and properties of adenosine triphosphatase in human polymorphonuclear leucocytes.

Magnesium-dependent adenosine triphosphatase (Mg2+-ATPase) activities wee studied in human neutrophilic polymorphonuclear leucocytes. Kinetic studies on whole leucocyte homogenates produced curvilinear kinetics suggesting the presence of at least two forms of Mg2+-ATPase. Neutrophils were homogenized in isotonic sucrose and, after low-speed centrifugation, the supernatant was subjected to analytical subcellular fractionation. Gradient fractions were assayed for Mg2+-ATPase and for principal organelle marker enzymes. Mg2+-ATPase was distributed between the plasma membrane, mitochondrial and cytosol fractions. Kinetic and inhibitor studies on Mg2+-ATPase from each localization indicated the presence of three forms of the enzyme. The plasma membrane and mitochondrial activities had a Km value of 0.2 mmol/l for ATP, whilst the Km for the cytosolic enzyme was 1.8 mmol/l. Inhibitor studies showed further differences between the three enzymes. Neutrophils were isolated from control subjects, patients with chronic granulocytic leukaemia and patients in the third trimester of pregnancy. The specific activities (mUnits/mg protein) of Mg2+-ATPase, in contrast to those of alkaline phosphatase, were similar in all three patient groups. This result, together with the fractionation experiments and inhibitor studies, strongly suggest that the ATPase is not attributable to neutrophil alkaline phosphatase.

Adenosine Triphosphatases↗

PubMed1973

Energy-transducing adenosine triphosphatase from Escherichia coli: purification, properties, and inhibition by antibody.

The membrane adenosine triphosphatase (E.C. 3.6.1.3) from Escherichia coli has been solubilized with Triton X-100 and purified to near homogeneity. The purified enzyme has a sedimentation coefficient of 12.9S in a sucrose gradient, corresponding to a molecular weight of about 360,000. On electrophoresis in gels containing sodium dodecyl sulfate, it dissociates into subunits with apparent molecular weights of 60,000, 56,000, 35,000, and 13,000. The purified enzyme loses activity and breaks down into subunits when stored in the cold. Guanosine 5'-triphosphate and inosine 5'-triphosphate are alternative substrates. Ca(2+) and, to a small extent, Co(2+) or Ni(2+) will substitute for Mg(2+) in the reaction. The K(m) for Mg-adenosine triphosphate of the membrane-bound enzyme is 0.23 mM, and for the pure enzyme it is 0.29 mM. Azide is a noncompetitive inhibitor of both the membrane-bound enzyme and the pure enzyme. P(i) is a noncompetitive inhibitor of the solubilized enzyme. An antibody to the purified enzyme was obtained from rabbits. The antibody inhibits the solubilized enzyme and virtually all of the adenosine triphosphate hydrolysis by membranes from cells grown aerobically or anaerobically. The antibody also inhibits the adenosine triphosphate-stimulated pyridine nucleotide transhydrogenase (E.C. 1.6.1.1) of the E. coli membrane.

Adenosine Triphosphatases↗

PubMed1979

Purification and properties of the adenosine triphosphatase released from the liver mitochondrial membrane by chloroform.

1. Soluble ATPase (adenosine triphosphatase) activity is released when rat liver submitochondrial particles are shaken with chloroform, provided that ATP or glycerol is present in the suspending medium. The extraction is very rapid and appears to be complete. 2. The ATPase of the chloroform extract is about 50% pure and can be readily purified to a specific activity of 60-70mumol/min per mg of protein by (NH(4))(2)SO(4) fractionation and column chromatography on Sephadex G-200. 3. The particulate and soluble ATPases have many similar properties, including their K(m) values for ATP, activation by various metal ions, hydrolytic activity with other nucleotides and stimulation by bicarbonate ions. 4. Unlike the particulate enzyme, the soluble enzyme is cold-labile and insensitive to oligomycin. 5. The molecular weight indicated by the mobility of the soluble ATPase on Sepharose 6B is 360000. 6. The soluble ATPase combines very readily with liver submitochondrial particles depleted of ATPase by salt extraction, and oligomycin-sensitivity is restored. Very little recombination of the enzyme occurs with chloroform-extracted particles. 7. The soluble enzyme contains orcinol-reactive material, suggesting that it may be a glycoprotein. The carbohydrate content was estimated to be 1-2% by weight. 8. It is concluded that the liver ATPase obtained by the chloroform extraction method of Beechey, Hubbard, Linnett, Mitchell & Munn [(1975) Biochem. J.148, 533-537] is similar to other preparations described previously and that this method is superior in simplicity and speed.

Adenosine Triphosphatases↗

PubMed1971

Oxidative phosphorylation in Escherichia coli K12. Mutations affecting magnesium ion- or calcium ion-stimulated adenosine triphosphatase.

1. Two mutants of Escherichia coli K 12 were isolated which, although able to grow on glucose, are unable to grow with succinate or d-lactate as the sole source of carbon. 2. Genetic mapping of these mutants showed that they both contain a mutation in a gene (designated uncA) mapping at about minute 73.5 on the E. coli chromosome. 3. The uncA(-) alleles were transferred by bacteriophage-mediated transduction into another strain of E. coli and the transductants compared with the parent strain to determine the nature of the biochemical lesion in the mutants. 4. The mutants gave low aerobic growth yields when grown on limiting concentrations of glucose, but oxidase activities in membranes from both the mutants and the normal strain were similar. 5. Measurement of P/O ratios with d-lactate as substrate indicated that a mutation in the uncA gene causes uncoupling of phosphorylation associated with electron transport. 6. Determination of the Mg(2+),Ca(2+)-stimulated adenosine triphosphatase activities in the mutant and normal strains indicated that the uncA gene is probably the structural gene for Mg(2+),Ca(2+)-stimulated adenosine triphosphatase. 7. Mg(2+),Ca(2+)-stimulated adenosine triphosphatase therefore appears to be essential for oxidative phosphorylation in E. coli.

Adenosine Triphosphatases↗

PubMed1978

A thermodynamic analysis of the interaction between the mitochondrial coupling adenosine triphosphatase and its naturally occurring inhibitor protein.

1. The naturally occurring ATPase (adenosine triphosphatase)-inhibitor protein, from bovine heart mitochondria, was obtained as a single pure protein. It was not identical with any of the five subunits (alpha-epsilon) of the isolated ATPase, and appeared to be a single polypeptide chain. 2. The inhibitor combined with the ATPase in a 1:1 molar ratio, producing a completely inhibited ATPase molecule. The affinity of the ATPase for its inhibitor is high; the K(d) is of the order of 10(-8)m. 3. The enthalpy of the ATPase-inhibitor complex-formation is positive, the value of K(d) decreasing as the temperature is raised. This suggests that the forces involved are largely hydrophobic in nature. 4. Hydrolysis of a nucleoside triphosphate promoted formation of the ATPase-inhibitor complex, although the equilibrium position was almost unaffected by the rate of hydrolysis. At low salt concentration, less than 200 turnovers of the ATPase suffice for the ATPase to combine with the inhibitor protein. At higher salt concentrations, a larger number of turnovers is required. It is suggested that the inhibitor binds to a form of the ATPase that is produced transiently during hydrolysis. 5. In the presence of 75mm-K(2)SO(4), the rates of association and dissociation are slow enough to allow their kinetics to be studied. Association is first-order in inhibitor concentration, but fractional order in ATPase concentration. Dissociation is first-order in ATPase-inhibitor complex concentration. The temperature coefficients of the ;on' and ;off' processes were also measured. 6. A simple kinetic model for the ATPase-inhibitor interaction is proposed that can be extended to take into account release of inhibitor protein under energized conditions on the membrane. 7. The isolated ATPase is inhibited by preincubation with Mg(2+), reversible by subsequent addition of EDTA, and by ADP, reversible by subsequent addition of ATP. These effects are not found on the membrane-bound ATPase. The mechanism of these effects is discussed.

Adenosine Triphosphatases↗

PubMed1979

Adenosine triphosphatase localization in the branchial heart appendage of Sepia officinalis L. (Cephalopoda).

Sodium- and potassium-dependent adenosine triphosphatase (Na+--K+-ATPase) has been demonstrated in the branchial heart appendage (pericardial gland) of Sepia officinalis L. by biochemical, cytochemical and autoradiographical methods. The biochemical data indicate the presence of Na+--K+-ATPase, judging from the potassium dependency and, with some restrictions, the inhibition by ouabain. Cytochemically and autoradiographically, the enzyme could be localized on the cytoplasmic surfaces of the lateral plasma membranes and the basal membrane infoldings (basal labyrinth) of the folded epithelium of the branchial heart appendage. The pdocytes of the peripheral zone of the organ reacted negatively. In addition to the Na+--K+-ATPase, a magnesium-activated adenosine triphosphatase (Mg2+-ATPase) was demonstrated in the folded epithelium, localized mainly in the mitochondria but also at the brush border and in the apical intercellular space, whereas a bicarbonate-stimulated ATPase (HCO-3-ATPase) was present only in the mitochondria.

Adenosine Triphosphatases↗