Binding of ouabain to Na+, K+-dependent ATPase during the ATPase reaction. Evidence for a dimer structure of the ATPase.

Na+, K+-dependent ATPase [EC 3.6.1.3] was purified from porcine kidney by the method of Lane et al. [(1973) J. Biol. Chem. 248, 7197-7200] with slight modifications [Yamaguchi, M. & Tonomura, Y., (1979) J. Biochem. 86, 509-523]. The amounts of a phosphorylated intermediate (EP) and ouabain bound to the enzyme during the ATPase reaction were measured in 2.1 mM MgCl2 and various concentrations of NaCl and KCl at pH 7.5 and 20 degrees C. In presence of NaCl and the absence of KCl, the molar ratio of the amounts of EP and bound ouabain was 1 : 2. In the presence of both NaCl and KCl, it was 1 : 1. In both cases, the amount of bound ouabain was equal to that of EP in the absence of ouabain. These findings suggest that the functional unit of the transport ATPase is a dimer.     

Inactive to active transitions of the mitochondrial ATPase complex as controlled by the ATPase inhibitor.

The hydrolytic and phosphorylation activities of the ATPase complex of bovine heart mitochondria are regulated by the ATPase inhibitor of Pullman and Monroy [1]. The inhibiting action of the peptide on ATPase activity can be overcome by a proton-motive force. Submitochondrial particles that contain the inhibitor, either intrinsically or externally added, show a lag that precedes phosphorylation. Particles devoid of the inhibitor, of particles that are in an 'active' state fail to present the lag. Accordingly, the data indicate that, prior to the onset of phosphorylation, the ATPase complex undergoes a transition to an active state through a process that involves the inhibitor. The transition depends on the concentration of ATP, 50 microM ATP giving 50% inhibition of the proton-motive force-induced transition.     

Thermus thermophilus membrane-associated ATPase. Indication of a eubacterial V-type ATPase

An ATPase with Mr of 360,000 was purified from plasma membranes of a thermophilic eubacterium Thermus thermophilus, and was characterized. ATP hydrolytic activity of the purified enzyme was extremely low, 0.07 mumol of Pi released mg-1 min-1, and it was stimulated up to 30-fold by bisulfite. The following properties of the enzyme indicate that it is not a usual F1-ATPase but that it belongs to the V-type ATPase family, another class of ATPases found in membranes of archaebacteria and eukaryotic endomembranes. Among its four kinds of subunits with approximate Mr values of 66,000 (alpha), 55,000 (beta), 30,000 (gamma), and 12,000 (delta), the alpha subunit had a similar molecular size to the catalytic subunits of the V-type ATPases but was significantly larger than the alpha subunit of F1-ATPases. ATP hydrolytic activity was not affected by azide, an inhibitor of F1-ATPases, but was inhibited by nitrate, an inhibitor of the V-type ATPase. N-terminal amino acid sequences determined for the purified alpha and beta subunits showed much higher similarity to those of the V-type ATPases than those of F1-ATPases. Thus the distribution of the V-type ATPase in the prokaryotic kingdom may not be restricted to archaebacteria.     

Inhibition of muscle actomyosin ATPase activity by mitochondrial ATPase inhibitor.

Ascites hepatoma cell line AH-130 was tested for the ability to transport various amino acids and glutathione before and after γ-glutamyl transpeptidase of the cells was affinity-labeled and inactivated by 6-diazo-5-oxo-L-norleucine, a glutamine analog. The rate of uptake of alanine, glycine, leucine and glutamine by the cells remained unchanged after γ-glutamyl transpeptidase was inactivated by this affinity label. This indicated that γ-glutamyl transpeptidase of the cell was not involved in the transport process of these amino acids tested. The uptake of glutathione was also tested before and after affinity labeling the enzyme. The total amount of the radioactivity incorporated into the cells was not significantly affected by the enzyme inactivation. However, the relative amount of incorporated intact glutathione was found to be slightly but significantly increased after membraneous γ-glutamyl transpeptidase was inactivated by the affinity label, while that of component amino acid, glycine, was found to decrease. This indicated that glutathione was taken up by the cell in its intact form as well as in degraded forms into its component amino acids, and γ-glutamyl transpeptidase in the ascites tumor cell AH-130 seemed to be involved in the metabolic process via the latter system.     

Transport ATPase: thimerosal inhibits the Na+ +K+-dependent ATPase activity without diminishing the Na+-dependent ATPase activity.

A guinea pig kidney membrane preparation was incubated with thimerosal and then thoroughly washed. Comparison of the properties of the native and the modified membranes showed that (a) Na⁺+K⁺-dependent activity is substantially inhibited by thimerosal; (b) thimerosal does not diminish Na⁺-dependent ATPase activity; and (c) the thimerosal treated enzyme, like the native enzyme, is phosphorylated in the presence of Na⁺ and ATP, and dephosphorylated upon the addition of K⁺. It is suggested that thimerosal does not affect the binding of ATP to the high-affinity catalytic site, but that it blocks the binding of ATP to a low affinity modifying site the occupation of which is essential for the dissociation of the stable K⁺-dephosphoenzyme and the recycling of the enzyme.     

A kinetic study of the kinesin ATPase.

The mechanism of kinesin ATPase has been investigated by transient state kinetic analysis. The results satisfy the scheme [formula: see text] where T, D, and P(i) refer to nucleotide tri- and diphosphate and inorganic phosphate, respectively. The nucleotide-binding steps were measured by the fluorescence enhancement of mant (2'-(3')-O-(N-methylanthraniloyl)-ATP and mant-ADP. The initial rapid equilibrium binding steps (1) and (6) are followed by isomerizations (k2 = 170 +/- 30 s-1 at 20 degrees C, k-5 greater than 100 s-1). The increase in fluorescence is 20-25% larger for K.T** than K.D*. The rate constant of the hydrolysis step k3 is 6-7 s-1. The fluorescence decreases after formation of K.T** at a rate of 7-10 s-1. This change could occur in step 3 or in step 4 if k4 much greater than k3. The value of k4 is larger than 0.1 s-1. The steady state rate is 0.003 s-1 which agrees with the rate of ADP dissociation (k5). Step 5 is rate limiting in the scheme in agreement with the conclusion of Hackney (Hackney, D. D. (1988) Proc. Natl. Acad. Sci. U.S.A. 85, 6314-6318) that ADP dissociation is the rate-limiting step.     

A convenient method for the ATPase assay.

A new method for the determination of inorganic phosphorus released in ATPase assay has been evaluated. The method is based on the reduction of a phosphomolybdate complex by Elon in a copper acetate buffer. In contrast to current methods, there is no interference by ATP with color development. There is also less or no interference by other compounds usually present in ATPase assay media. The method is simple, sensitive, and reproducible.     

Cross-linking of the sarcoplasmic reticulum ATPase protein.

Treatment of rabbit sarcoplasmic reticulum vesicles with the cross-linking agent, cupric phenanthroline, causes production of high-molecular weight bands on SDS-gel electrophoresis. A plot of log mol wt $\text{vs}$ mobility indicates that the main band produced from the ATPase (mol wt = 10⁵) has a mol wt of 4 × 10⁵ and thus suggests formation of a tetramer. Notably, bands corresponding to dimers, trimers, pentamers, etc., are absent. The bands attributable to calsequestrin and calcium binding protein are unchanged by cupric phenanthroline. With extended treatment, the tetramer itself is polymerized (mol wt>10⁶). Partial disruption of the membranes with deoxycholate or Triton X-100 before cross-linking favors tetramer formation; the presence of sodium dodecyl sulfate, on the other hand, prevents intermolecular cross-linking. Our results suggest that the ATPase is at least partially associated within the membrane as a tetramer.     

Structural conformation of the ciliary ATPase dynein.

The ATPase dynein forms part of a mechanoohemically active complex responsible for the sliding filament mechanism of ciliary and flagellar motion. Extraction of demembranated cilia from the lamellibranch mollusc Unio or the protozoan Tetrahymena by 0.5 m-KCl solubilizes the outer rows of dynein and, as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, releases the A form (Mr 360,000) of dynein into solution. Negative contrast electron microscopy of the solubilized dynein fraction reveals an homogeneous array of 93 Å particles that we identify as the ATPase dynein in its monomeric form. Because of the method of dynein extraction, the conformation of the molecule, and the size and shape of the outer arms in situ, we suggest that monomeric dynein is only one part of a larger, non-covalently joined molecular complex that forms the entire arm. When KCl-extracted axonemes are viewed by negative contrast but prior to fractionation of the dynein, individual arms can be seen that comprise three to four of the 93 Å subunits, thus suggesting that each arm is a multisubunit polymer of dynein or dynein-like molecules.     

Studies on the ATPase of Bacillus cereus.

The membrane ATPase (EC 3.6.1.3) of Bacillus cereus was solubilized by a 'shock-wash' process and purified. The non-specific phosphatase contaminant was separated by glycerol density gradient centrifugation. The optimum temperature was 39.5 degrees C and the pH optimum at 7.5. On SDS-polyacrylamide gel electrophoresis two classes of subunits were observed in equal proportions with molecular weights of 70 K and 83 K. The effect of various compounds on the enzymatic activity was studied. The enzyme was insensitive to NaN3, oligomycin and to divalent cations, but was inhibited by citrate and oxalate.     

Action of alkyl cations and the natural ATPase inhibitor from mitochondria on soluble mitochondrial ATPase.

The effect of the natural ATPase inhibitor and octylguanidine on the ATPase activity of soluble oligomycin-insensitive mitochondrial F1 were compared. Both compounds induced a maximal inhibition of 60-80% in various preparation of F1 studied. The inhibition was of the uncompetitive type with respect to MgATP, and the action of the compounds was partially additive. The data suggest that octylguanidine reproduces the action of the natural ATPase inhibitor. Alkylammonium salts also affect the ATPase activity in a similar form. F1 bound to Sepharose-hexylammonium is largely inactive, whilst free hexylammonium at higher concentrations induces only a partial inhibition of the activity. This suggests that the degree of immobilization of F1 is related to the magnitude of inhibition of ATPase activity induced by alkyl cations. The binding of F1 to Sepharose-hexylammonium is prevented by high concentrations of Na+ or K+.     

Sodium-stimulated ATPase in Streptococcus faecalis.

We measured Na+-stimulated ATPase activity in a mutant of Streptococcus faecalis defective in the generation of proton motive force. The activity in membrane vesicles was 62.1 +/- 5.9 nmol of phosphate produced per min per mg of protein when cells were grown on medium containing 0.12 M Na+. Activity decreased as the concentration of Na+ in the growth medium decreased. The decrease in enzyme activity corresponded to the decrease in transport activity for Na+ in both whole cells and membrane vesicles. The effects of pH on both activities were identical. Thus, it is suggested that Na+ movement is mediated by this enzyme. Sodium extrusion and ATPase activity in the wild-type strain were markedly lower than those observed in the mutant strain. Elevated activities of both Na+ extrusion and Na+-stimulated ATPase could be detected in the wild-type strain when cells were grown in the absence of proton motive force. Thus, we propose that the level of ATPase is increased by dissipation of the proton motive force.     

A DNA-dependent ATPase of calf-thymus.

A DNA-dependent ATPase has been purified from calf thymus. The enzyme hydrolyses ATP and dATP in the presence of heat-denatured DNA. It does not hydrolyse the corresponding nucleoside triphosphates of guanine, uridine and cytosine. The Km values for ATP and dATP are both 0.62 mM. The enzyme requires magnesium or manganese ions. Its sedimentation coefficient is about 4.4 S. The catalytic activity is inhibited by N-ethylmaleimide but is not sensitive to novobiocin and nalidixic acid which are potent inhibitors of bacterial DNA gyrase. In some cases, during purification, chromatographically distinct additional DNA-dependent ATPase activities were detected. Limited proteolysis or covalent modification of the enzyme in the tissues, or during the first steps of its extraction, are probably responsible for the appearance of these chromatographically distinct forms.     

Actin-activated ATPase from human erythrocytes.

A fibrillar protein complex, possessing ouabain-insensitive Ca2+-ATPase activity was isolated from human erythrocyte membranes by using a low ionic strength extraction procedure. Mg2+-ATPase activity was revealed upon addition of rabbit skeletal muscle actin, thus demonstrating the presence of a myosin-like protein in the crude extract of the erythrocyte membrane. Upon sodium dodecylsulfate gel electrophoresis, the extract showed mainly the doublet of subunit molecular weight bands of 230 000 and 210 000, and more than 10 faster moving bands. Gel filtration of the erythrocyte membrane extract on Sepharose 4B furnished 4 fractions. Fraction I, containing the doublet and 80 000, 60 000 and 46 000 subunit molecular weight bands was 5-fold purified with respect to Ca2+-ATPase activity, but was devoid of actin-activated Mg2+-ATPase activity. Fraction II, containing only the doublet, was devoid of Ca2+ and actin-activated Mg2+-ATPase activity. The 210 000 subunit molecular weight protein could be phosphorylated in the presence of Mg2+ in the crude extract and Fraction I but not in Fraction II.     

Structure of the vacuolar ATPase by electron microscopy.

The structure of the vacuolar ATPase from bovine brain clathrin-coated vesicles has been determined by electron microscopy of negatively stained, detergent-solubilized enzyme molecules. Preparations of both lipid-containing and delipidated enzyme have been analyzed. The complex is organized in two major domains, a V(1) and V(0), with overall dimensions of 28 x 14 x 14 nm. The V(1) is a more or less spherical molecule with a central cavity. The V(0) has the shape of a flattened sphere or doughnut with a radius of about 100 A. The V(1) and V(0) are joined by a 60-A long and 40-A wide central stalk, consisting of several individual protein densities. Two kinds of smaller densities are visible at the top periphery of the V(1), and one of these seems to extend all the way down to the stalk domain in some averages. Images of both the lipid-containing and the delipidated complex show a 30-50-kDa protein density on the lumenal side of the complex, opposite the central stalk, centered in the ring of c subunits. A large trans-membrane mass, probably the C-terminal domain of the 100-kDa subunit a, is seen at the periphery of the c subunit ring in some projections. This large mass has both a lumenal and a cytosolic domain, and it is the cytosolic domain that interacts with the central stalk. Two to three additional protein densities can be seen in the V(1)-V(0) interface, all connected to the central stalk. Overall, the structure of the V-ATPase is similar to the structure of the related F(1)F(0)-ATP synthase, confirming their common origin.     

ATPase activities in peroxisome-proliferating yeast.

Preliminary studies on yeast peroxisomes have suggested that the membrane of these organelles may contain a proton-pumping ATPase. It has been reported that peroxisome-associated activity is similar to the F0-F1 mitochondrial type ATPase in its sensitivity to azide at pH 9.0, but characteristics of the plasma membrane type ATPase are also evident in peroxisomal preparations in that they exhibit pH 6.5 activity that is sensitive to vanadate. A comparative study of the prominent organellar ATPase activities was undertaken as a probe into the existence of an enzyme that is unique to the peroxisome, and biochemical properties of yeast mitochondrial, plasma membrane, together with peroxisomally-associated H(+)-ATPases are presented. Enzyme marker analysis of sucrose gradient fractions revealed a high degree of correlation between the amount of azide-sensitive pH 9.0 ATPase activity and that of the mitochondrial membrane marker, cytochrome c oxidase, in peroxisomal preparations. Purified mitochondrial and peroxisomally-associated activities were highly sensitive to the presence of sodium azide, N,N' -dicyclohexylcarbodiimide (DCCD) and venturicidin when measured at pH 9.0. Comparisons of peroxisomal activities with those of the purified plasma membrane at pH 6.0 in the presence of azide showed similar sensitivity profiles with respect to inhibitors of yeast plasma membrane ATPases such as vanadate and p-chloromercuriphenyl-sulfonic acid (CMP). Purified peroxisomal membranes, furthermore, reacted with antibody to the mitochondrial F1 subunit (as revealed by Western blot analysis), and [35S] methionine-labeled, glucose-grown cells processed with unlabeled methanol-grown cells, yielded sucrose gradient fractions that were radioactive in bands that were also recognized by F1 antibody. Isolated fractions in these experiments had similar ratios of cpm:pH 9.0 ATPase activities, suggesting that this activity is mitochondrial in origin. The data presented for the characteristics of the peroxisomally-associated activity strongly suggest that the majority of the ATPase activity found in peroxisomal preparations is derived from other organelles.