The partial purification of sodium-plus-potassium ion-dependent adenosine triphosphatase from the gills of Anguilla anguilla and its inhibition by orthovanadate.

1. (Na+ +K+)-dependent ATPase was partially purified from eel gills by a procedure in which the microsomal fraction of crude preparations of chloride cells was selectively extracted with sodium dodecyl sulphate. 2. The microsomal specific activity was increased 2-fold during optimal treatment with detergent. 3. The final preparation (56% pure) had a specific activity of 341 mumol of ATP hydrolysed/h per mg of protein and a turnover number of 3560 min-1. The number of ouabain-binding sties equalled the number of sites phosphorylated by ATP. 4. Both sodium orthovanadate and ouabain inhibited the purified preparation more than the microsomal fraction, vanadate being more effective on an equimolar basis than ouabain. 5. Inhibition by orthovanadate was not enhanced at 28 mM-as compared with 1mM-MgCl2 and was not reversed by beta-adrenergic agonists (cf. Josephson & Cantley (1977) Biochemistry 16, 4572--4578). 6. Of various other metallic oxyanions tested only niobate proved an effective inhibitor of the enzyme although this anion was less effective than orthovanadate. 7. Orthovanadate partially inhibited phosphorylation of the enzyme by ATP in the presence of 28 mM-MgCl2.     

Lipid requirement of the membrane sodium-plus-potassium ion-dependent adenosine triphosphatase system.

The dependence of the (Na-++K-+)-dependent ATPase (adenosine triphosphatase) (EC 3.6.1.3) on lipid has been examined in a number of different ways, with the use of various preparations from kidney tissue. The main findings were as follows. (1) The ATPase activities of the preparations examined were closely correlated with their total phospholipid content. (2) Extraction of the ATPase with deoxycholate or Lubrol W, combined with suitable salt-fractionation and washing procedures, removed phospholipid, cholesterol and enzymic activity in parallel; but activity was completely lost before all lipid had been removed. (3) The loss of activity could not be attributed to inhibition by residual detergent. (4) No selective removal of any particular phospholipid class by detergent could be detected. (5) Consistent reactivation of the Lubrol-extracted enzymes was obtained by adding dispersions of exogenous phospholipid, but only some, bearing a net negative charge, such as phosphatidylserine and phosphatidylglycerol, were effective. (6) The degree of reactivation was correlated with the amount of residual activity remaining after lipid depletion. (7) Partial purification of the ATPase, giving a 50-fold increase in specific activity, was not accompanied by selective enhancement of any particular class of phospholipid. We conclude that although the ATPase is dependent on phospholipid, only the reactivation results provide evidence for specificity.     

Differential effects of lipid depletion on membrane sodium-plus-potassium ion-dependent adenosine triphosphatase and potassium ion-dependent phosphatase.

The phospholipid-dependence of the (Na-++K-+)-dependent ATPase (adenosine triphosphatase) (EC 3.6.1.3) and associated K-+-dependent phosphatase activity (EC 3.6.1.7) have been compared. Unlike the (Na-++K-+)-dependent ATPase activities, the K-+-dependent phosphatase activities of a number of different preparations were not closely correlated with their total phospholipid contents. After partial lipid depletion with a single extraction in Lubrol W the residual ATPase and phosphatase activities were correlated, but their magnitudes were quite different: on average only about 5% of the former remained compared with 50% of the latter. A similar differential effect on these activities was found after extraction with deoxycholate. In contrast with the ATPase, consistent restoration of the phosphatase activity of Lubrol-extracted enzymes by added exogenous phospholipids was not observed. We conclude that, although the K-+-dependent phosphatase may be lipid-dependent, the lipid requirement must be different from that of the complete ATPase system, and this difference should help investigations of their relationship.     

The reversible delipidation of a solubilized sodium-plus-potassium ion-dependent adenosine triphosphatase from the salt gland of the spiny dogfish.

A microsomal fraction rich in Na+, K+-ATPase (sodium-plus-potassium ion-dependent adenosine triphosphatase) and the corresponding K+-dependent p-nitrophenyl phosphatase from the rectal salt gland of the spiny dogfish was solubilized by treatment with deoxycholate at high ionic strength. On gel filtration through Sepharose 6B, the ATPase apoenzyme could be separated, in apparently soluble form, from the tissue-fraction phospholipids and was almost free of enzymic activity (2% of the p-nitrophenyl phosphatase activity and 0.2% of the ATPase activity being recovered). On mixing the apoenzyme with an activator consisting of cooked ox brain, a large proportion of the original enzymic activity was obtained. Specific activities of the re-activated enzyme were somewhat higher than in the material before gel filtration: values of 1300-1450 mumol and 250-290 mumol/h per mg of protein were obtained for the hydrolysis of ATP and of p-nitrophenyl phosphate respectively. The activity was inhibitible by ouabain.     

Adenosine diphosphate binding to sodium-plus-potassium ion-dependent adenosine triphosphatase. The role of lipid in the nucleotide-potassium ion interplay.

Delipidated dogfish rectal-gland Na++K+-ATPase (Na++K+-dependent adenosine triphosphatase), almost devoid of hydrolytic activity, is able to bind about 2nmol of ADP/mg of protein. The "affinity" of delipidated enzyme for ADP is not affected by K+ in concentrations that greatly decrease the "affinity" of native Na++K+-ATPase. The K+-sensitivity of the ADP binding is in part restored by relipidation with dioleoyl phosphatidylcholine.     

The binding of nucleotides and bivalent cations to the calcium-and-magnesium ion-dependent adenosine triphosphatase from rabbit muscle sarcoplasmic reticulum.

The binding of MgATP to purified Ca2+Mg2+-dependent adenosine triphosphatase from rabbit muscle sarcoplasmic reticulum was studied by using a flow-dialysis method. Phosphoryl-enzyme formation and catalytic activity were also measured, and all three processes demonstrated negative co-operativity, with half-saturation of all three parameters at a MgATP concentration of 40-50muM, and a Hill coefficient (h) of 0.8. The variation of the binding constant with with pH was measured and showed tighter binding of MgATP with increasing pH over the range 6.8-8.5. Binding parameters for ATP analogues were also measured. The binding of Ca2+ in the presence and absence of ATP analogues gave half saturation at a Ca2+ concentration of 1.2-1.3muM. Hill plots of Ca2+-binding data gave a slope of 0.8. These results show that the binding of MgATP and Ca2+ can occur in a random manner, with neither substrate influencing the affinity of the enzyme for the other.     

The nature and properties of the inducible sodium-plus-potassium ion-dependent adenosine triphosphatase in the gills of eels (Anguilla anguilla) adapted to fresh water and sea water

1. Gill tissue from eels adapted to fresh water or to sea water was disrupted in 0.32m-sucrose containing 0.1% (w/v) sodium deoxycholate and the subcellular distribution of (Na(+)+K(+))-dependent adenosine triphosphatase was determined. 2. About 70% of the recovered enzyme was in a fraction sedimenting between 225000g(av.)-min and 6000000g(av.)-min; the specific activities of enzymes from tissues of freshwater and seawater eels were 16 and 51 mumol of phosphate/h per mg of protein respectively. 3. The enzymes from gills of freshwater and seawater eels were indistinguishable on the basis of a number of parameters. These included phosphorylation by [gamma-(32)P]ATP, the binding of [(3)H]ouabain, the extent to which bound [(3)H]ouabain was displaced by increasing concentrations of KCl and pH optima. 4. Electrophoresis on polyacrylamide gels in sodium dodecyl sulphate showed that enzyme preparations from both sources had an identical number of protein components. 5. The higher specific activity of (Na(+)+K(+))-dependent adenosine triphosphatase from tissue of seawater eels was accompanied by increased amounts of two protein components. One of these proteins retained (32)P after treatment of the enzyme with [gamma-(32)P]ATP and had mol.wt. 97000; the other component was a glycoprotein with mol.wt. approx. 46000. 6. The results are discussed in terms of the nature of the transepithelial NaCl pumps in the gills of freshwater and seawater fish.     

The magnesium ion-dependent adenosine triphosphatase of myosin. Two-step processes of adenosine triphosphate association and adenosine diphosphate dissociation

The kinetics of protein-fluorescence change when rabbit skeletal myosin subfragment 1 is mixed with ATP or adenosine 5'-(3-thiotriphosphate) in the presence of Mg(2+) are incompatible with a simple bimolecular association process. A substrate-induced conformation change with DeltaG(0)<-24kJ.mol(-1) (i.e. DeltaG(0) could be more negative) at pH8 and 21 degrees C is proposed as the additional step in the binding of ATP. The postulated binding mechanism is M+ATPright harpoon over left harpoonM.ATPright harpoon over left harpoonM*.ATP, where the association constant for the first step, K(1), is 4.5x10(3)m(-1) at I 0.14m and the rate of isomerization is 400s(-1). In the presence of Mg(2+), ADP binds in a similar fashion to ATP, the rate of the conformation change also being 400s(-1), but with DeltaG(0) for that process being -14kJ.mol(-1). The effect of increasing ionic strength is to decrease K(1), the kinetics of the conformation change being essentially unaltered. Alternative schemes involving a two-step binding process for ATP to subfragment 1 are possible. These are not excluded by the experimental results, although they are perhaps less likely because they imply uncharacteristically slow bimolecular association rate constants.     

Calcium ion-dependent p-nitrophenyl phosphate phosphatase activity and calcium ion-dependent adenosine triphosphatase activity from human erythrocyte membranes

In the presence of ATP and of Mg(2+), human erythrocyte membranes show a phosphatase activity towards p-nitrophenyl phosphate which is activated by low concentrations of Ca(2+). The effect of Ca(2+) is strongly enhanced if either K(+) or Na(+) is also present. Activation of the p-nitrophenyl phosphate phosphatase by Ca(2+) reaches a half-maximum at about 8mum-Ca(2+) and is apparent only when the ion has access to the inner surface of the cell membrane. Ca(2+)-dependent phosphatase activity can only be observed if ATP is at the inner surface of the cell membrane, and the presence of ATP seems to be absolutely necessary, since either its removal or its replacement by other nucleoside triphosphates abolishes the activating effect of Ca(2+). The properties of the (ATP+Ca(2+))-dependent phosphatase are very similar to those of the Ca(2+)-dependent ATPase (adenosine triphosphatase), also present in erythrocyte membranes, which probably is involved in Ca(2+) transport in erythrocytes. The similarities suggest that both activities may be properties of the same molecular system. This view is further supported by the fact that p-nitrophenyl phosphate inhibits to a similar extent Ca(2+)-dependent ATPase activity and ATP-dependent Ca(2+) extrusion from erythrocytes.     

The cerebral sodium-plus-potassium ion-stimulated adenosine triphosphatase of bovine brain and its microsomal matrix

1. Adenosine triphosphatase activities of dispersions prepared from bovine cerebral cortex that had been frozen, were greater than those of dispersions prepared from fresh tissue. The subcellular distribution of components of the dispersion was not altered by freezing the tissue and a microsomal fraction enriched in Na(+)+K(+)-stimulated adenosine triphosphatase activity was prepared. 2. The bovine cerebral microsomes were further treated with a 2m-sodium iodide reagent to obtain a particulate preparation with minimal Na(+)+K(+)-independent adenosine triphosphatase activity. Na(+)+K(+)-stimulated activity was increased by the sodium iodide treatment and this preparation was shown to be enriched in lipid constituents. 3. Density-gradient centrifugation of the sodium iodide treated preparation gave three main subfractions each containing approximately equal amounts of phospholipid and protein. Further exposure of the sodium iodide-treated preparation to the 2m-sodium iodide reagent altered the distribution of protein and phospholipid among the fractions obtained by density-gradient centrifugation. Dissociation of phospholipids from protein in the sodium iodide-treated preparation was brought about also by high concentrations of arginine. Concentrated solutions of arginine and sodium thiocyanate brought about dissociation of phospholipids from protein of the microsomal preparation. 4. Many amino acids were found to inhibit Na(+)+K(+)-stimulated adenosine triphosphatase activity when present in high concentrations. The inhibition was complex but resulted, in part at least, from diminished affinity for ATP and Na(+) in the presence of the amino acids. 5. A non-ionic detergent, Lubrol W, solubilized up to 40% of the enzyme activity of the sodium iodide-treated preparation together with 30% of the protein and phospholipid in the preparation. Protein was released from the sodium iodide-treated preparation by pancreatic elastase but Na(+)+K(+)-stimulated adenosine triphosphatase activity of the residue was diminished. Ultrasonic treatment of the sodium iodide-treated preparation failed to release a significant proportion of Na(+)+K(+)-stimulated adenosine triphosphatase activity into a form not deposited by ultracentrifugation.     

Calcium ion-dependent adenosine triphosphatase activity and plasma-membrane phosphorylation in the human neutrophil.

A plasma-membrane fraction was isolated from a post-nuclear extract of human neutrophils by centrifugation through a linear sucrose density gradient. This fraction exhibited a Ca2+-dependent adenosine triphosphatase (ATPase) activity that could be differentiated from mitochondrial or myosin ATPase and from plasma-membrane Mg2+-dependent ATPase. When assayed in the presence of [gamma-32P]ATP, the Ca2+-dependent ATPase reaction resulted in the formation of an acid-resistant hydroxylamine-sensitive bond between the gamma-[32P] phosphate group and a membrane protein subunit with an apparent mol.wt. of 135000. Half-maximal activating effect of Ca2+ was found at 82nM and 0.18 microM for the ATPase and the formation of the 32P-membrane complex respectively. Generation of the phosphorylated product attained the steady state at 0 degrees C by about 30s, and was rapidly reversed by ADP. These results suggest that the Ca2+-activated ATPase reaction occurs through the formation of a phosphoprotein intermediate, similar to that described for some Ca2+-dependent ATPase enzymes associated with Ca2+ transport. The possibility thus exists that the neutrophil Ca2+-dependent ATPase catalyses a process of Ca2+ extrusion from the cell, thereby participating in the regulation of several Ca2+-dependent neutrophil functions.