Crystals of an ATPase fragment of bovine clathrin uncoating ATPase

A 44,000 Mr amino-terminal, clathrin-independent ATPase fragment of the bovine clathrin uncoating ATPase has been crystallized in a form suitable for X-ray diffraction studies. The crystals are orthorhombic, space group P2(1)2(1)2(1), a = 145.3 A, b = 65.0 A, c = 46.9 A, with one protein molecule per asymmetric unit (1 A = 0.1 nm).     

Properties of Plasmamembrane ATPase and Mitochondrial ATPase of Saccharomyces cerevisiae

The properties of mitochondrial ATPase and plasmamembrane ATPase of Saccharomyces cerevisiae are compared. The pH dependence differs considerably. At low pH plasmamembrane ATPase is inactivated. High salt concentrations protect the ATPase against acid inactivation. K⁺ is more effective than Na⁺. The sensitivity of mitochondrial ATPase towards azide, Dio 9 and oligomycin is far greater than found with plasmamembrane ATPase. There are no indications that the membrane ATPase is involved directly either in monovalent cation uptake or in divalent cation uptake. Sr²⁺ and Ca²⁺ do not activate plasmamembrane ATPase and inhibit Mg²⁺-activated ATPase. The substrate specificity of plasmamembrane ATPase is much greater than the substrate specificity of mitochondrial ATPase.     

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.     

牛心线粒体ATP酶抑制蛋白对酶的催化部位构象的影响

以标记在ＡＴＰ酶（Ｆ_１）催化部位的ＴＮＰ－ＡＴＰ为荧光探针,比较测定了Ｆ_１与其抑制蛋白（ＩＦ_１）结合前后的ＴＮＰ－ＡＴＰ荧光光谱、荧光寿命和荧光偏振光谱。结果表明在ＩＦ１的作用下,酶分子催化部位的极性下降,ＴＮＰ－ＡＴＰ分子运动的自由度减小,提示ＩＦ_１引起了Ｆ_１催化部位的构象改变。     

Monoclonal antibody modification of the ATPase activity of Escherichia coli F1 ATPase

Monoclonal antibodies (mAbs) have been made against each of the five subunits of ECF1 (alpha, beta, gamma, delta, and epsilon), and these have been used in topology studies and for examination of the role of individual subunits in the functioning of the enzyme. All of the mAbs obtained reacted with ECF1, while several failed to react with ECF1F0, including three mAbs against the gamma subunit (gamma II, gamma III, and gamma IV), one mAb against delta, and two mAbs against epsilon (epsilon I and epsilon II). These topology data are consistent with the gamma, delta, and epsilon subunits being located at the interface between the F1 and F0 parts of the complex. Two forms of ECF1 were used to study the effects of mAbs on the ATPase activity of the enzyme: ECF1 with the epsilon subunit tightly bound and acting to inhibit activity and ECF1* in which the delta and epsilon subunits had been removed by organic solvent treatment. ECF1* had an ATPase activity under standard conditions of 93 mumol of ATP hydrolyzed min-1 mg-1, cf. an activity of 7.5 units mg-1 for our standard ECF1 preparation and 64 units mg-1 for enzyme in which the epsilon subunit had been removed by trypsin treatment. The protease digestion of ECF1* reduced activity to 64 units mg-1 in a complicated process involving an inhibition of activity by cleavage of the alpha subunit, activation by cleavage of gamma, and inhibition with cleavage of the beta subunit. mAbs to the gamma subunit, gamma II and gamma III, activated ECF1 by 4.4- and 2.4-fold, respectively, by changing the affinity of the enzyme for the epsilon subunit, as evidenced by density gradient centrifugation experiments. The gamma-subunit mAbs did not alter the ATPase activity of ECF1*- or trypsin-treated enzyme. The alpha-subunit mAb (alpha I) activated ECF1 by a factor of 2.5-fold and ECF1F0 by 1.3-fold, but inhibited the ATPase activity of ECF1* by 30%.     

Conformational changes of the multifunction p97 AAA ATPase during its ATPase cycle

p97 (also called VCP), a member of the AAA ATPase family, is involved in several cellular processes, including membrane fusion and extraction of proteins from the endoplasmic reticulum for cytoplasmic degradation. We have studied the conformational changes that p97 undergoes during the ATPase cycle by cryo-EM and single-particle analysis. Three-dimensional maps show that the two AAA domains, D1 and D2, as well as the N-domains, experience conformational changes during ATP binding, ATP hydrolysis, P(i) release and ADP release. The N-domain is flexible in most nucleotide states except after ATP hydrolysis. The rings formed by D1 and D2 rotate with respect to each other, and the size of their axial openings fluctuates. Taken together, our results depict the movements that this and possibly other AAA ATPases can undergo during an ATPase cycle.     

Inhibition of mitochondrial F1 ATPase and sarcoplasmic reticulum ATPase by hydrophobic molecules

The hydrophobic nature of the active site of two energy-transducing ATPases was explored by comparing interactions between Pi and each of three hydrophobic drugs in the absence and presence of organic solvents. The drugs tested were the Fe . bathophenanthroline complex and the anticalmodulin drugs, calmidazolium and trifluoperazine. All inhibit the Pi in equilibrium with ATP exchange reaction catalyzed by submitochondrial particles and the ATPase activity of both submitochondrial particles and soluble F1 ATPase. The inhibition by the three drugs is reversed by either raising the Pi concentration or by adding organic solvent (dimethylsulfoxide, ethyleneglycol or methanol) to the medium. The inhibition of the Pi in equilibrium with ATP exchange by trifluoperazine becomes more pronounced when the electrochemical proton gradient formed across the membrane of the submitochondrial particles is decreased by the addition to the medium of the proton ionophore carbonylcyanide p-trifluoromethoxyphenylhydrazone. The ATPase activity and the Ca2+ uptake by sarcoplasmic reticulum vesicles are inhibited by the Fe . bathophenanthroline complex, calmidazolium and trifluoperazine. Phosphorylation of the ATPases by Pi, synthesis of ATP from ADP and Pi and the fast efflux of Ca2+ observed during reversal of the Ca2+ pump are inhibited by the three drugs. The inhibition is reversed by raising the concentration of Pi or dimethylsulfoxide. The three drugs tested appear to compete with Pi for a common binding site on the Ca2+-ATPase. The data presented are interpreted according to the proposal that the catalytic site of an enzyme involved in energy transduction undergoes a hydrophobic-hydrophilic transition during the catalytic cycle.     

Kinetic Characterization of the ATPase and Actin-activated ATPase Activities of Acanthamoeba castellanii Myosin-2

Phosphorylation of Ser-639 in loop-2 of the catalytic motor domain of the heavy chain of Acanthamoeba castellanii myosin-2 and the phosphomimetic mutation S639D have been shown previously to down-regulate the actin-activated ATPase activity of both the full-length myosin and single-headed subfragment-1 (Liu, X., Lee, D. Y., Cai, S., Yu, S., Shu, S., Levine, R. L., and Korn, E. D. (2013) Proc. Natl. Acad. Sci. U.S.A. 110, E23–E32). In the present study we determined the kinetic constants for each step in the myosin and actomyosin ATPase cycles of recombinant wild-type S1 and S1-S639D. The kinetic parameter predominantly affected by the S639D mutation is the actin-activated release of inorganic phosphate from the acto myosin·ADP·P_i complex, which is the rate-limiting step in the steady-state actomyosin ATPase cycle. As consequence of this change, the duty ratio of this conventional myosin decreases. We speculate on the effect of Ser-639 phosphorylation on the processive behavior of myosin-2 filaments.     

Effects of organic solvents and orthophosphate on the ATPase activity of F1 ATPase

The ATPase activity of soluble F1 ATPase of mitochondria is activated by Pi. The concentration of Pi required for half-maximal activation decreases from a value higher than 50 mM to about 1 mM Pi when one of the organic solvents dimethyl sulfoxide (15 to 30%), methanol (7.5 to 15%) or ethylene glycol (10 to 30%) is added to the assay medium. This effect is observed in the presence of MgCl2 but not in the presence of CaCl2.     

Energetics of the calcium-transporting ATPase

A thermodynamic cycle for catalysis of calcium transport by the sarcoplasmic reticulum ATPase is described, based on equilibrium constants for the microscopic steps of the reaction shown in Equation 1 under a single set of experimental (formula; see text) conditions (pH 7.0, 25 degrees C, 100 mM KCl, 5 mM MgSO4): KCa = 5.9 X 10(-12) M2, K alpha ATP = 15 microM, Kint = 0.47, K alpha ADP = 0.73 mM, K'int = 1.7, K"Ca = 2.2 X 10(-6) M2, and Kp = 37 mM. The value of K"Ca was calculated by difference, from the free energy of hydrolysis of ATP. The spontaneous formation of an acylphosphate from Pi and E is made possible by the expression of 12.5 kcal mol-1 of noncovalent binding energy in E-P. Only 1.9 kcal mol-1 of binding energy is expressed in E X Pi. There is a mutual destabilization of bound phosphate and calcium in E-P X Ca2, with delta GD = 7.6 kcal mol-1, that permits transfer of phosphate to ADP and transfer of calcium to a concentrated calcium pool inside the vesicle. It is suggested that the ordered kinetic mechanism for the dissociation of E-P X Ca2, with phosphate transfer to ADP before calcium dissociation outside and phosphate transfer to water after calcium dissociation inside, preserves the Gibbs energies of these ligands and makes a major contribution to the coupling in the transport process. A lag (approximately 5 ms) before the appearance of E-P after mixing E and Pi at pH 6 is diminished by ATP and by increased [Pi]. This suggests that ATP accelerates the binding of Pi. The weak inhibition by ATP of E-P formation at equilibrium also suggests that ATP and phosphate can bind simultaneously to the enzyme at pH 6. Rate constants are greater than or equal to 115 s-1 for all the steps in the reaction sequence to form E-32P X Ca2 from E-P, Ca2+ and [32P]ATP at pH 7. E-P X Ca2 decomposes with kappa = 17 s-1, which shows that it is a kinetically competent intermediate. The value of kappa decreases to 4 s-1 if the intermediate is formed in the presence of 2 mM Ca2+. This decrease and inhibition of turnover by greater than 0.1 mM Ca2+ may result from slow decomposition of E-P X Ca3.     

A plasmid-encoded anion-translocating ATPase

An anion-translocating ATPase has been identified as the product of the arsenical resistance operon of resistance plasmid R773. When expressed in Escherichia coli this ATP-driven oxyanion pump catalyzes extrusion of the oxyanions arsenite, antimonite and arsenate. Maintenance of a low intracellular concentration of oxyanion produces resistance to the toxic agents. The pump is composed of two polypeptides, the products of the arsA and arsB genes. This two-subunit enzyme produces resistance to arsenite and antimonite. A third gene, arsC, expands the substrate specificity to allow for arsenate pumping and resistance.     

Plasma membrane ATPase of sugarbeet

A membrane fraction enriched with magnesium-dependent ATPase activity was isolated from sugarbeet (Beta vulgaris L.) taproot by a combination of differential centrifugation, extraction with KI and sucrose density gradient centrifugation. This activity was inhibited by vanadate, N,N′-dicyclohexylcarbodiimide and diethylstilbestrol, but was insensitive to molybdate, azide, oligomycin, ouabain, and nitrate, suggesting enrichment in plasma membrane ATPase. The enzyme was substrate specific for ATP, had a pH optimum of 7.0, but showed little stimulation by 50 mM KCl. The sugarbeet ATPase preparation contained endogenous protein kinase activity which could be reduced by extraction of the membranes with 0.1% (w/v) sodium deoxycholate. Reduction of protein kinase activity allowed the demonstration of a rapidly turning over phosphorylated intermediate on a M_r 105000 polypeptide, most likely representing the catalytic subunit of the ATPase. Phosphorylation was magnesium dependent, sensitive to diethylstilbestrol and vanadate but insensitive to oligomycin and azide. Neither the ATPase activity nor phosphoenzyme level were affected by combinations of sodium and potassium in the assay. These results argue against the presence of a synergistically stimulated NaK-ATPase at the plasma membrane of sugarbeet.