Kinetics of interaction between the H+-translocating component of the mitochondrial ATPase complex and oligomycin or dicyclohexylcarbodiimide

Kinetics of interaction between the H+-translocating component of the mitochondrial ATPase complex and oligomycin or dicyclohexylcarbodiimide were studied in beef heart submitochondrial particles, and the results suggest that the two inhibitors have different binding sites with respect to the membrane and to F1. Oligomycin seems to be bound to a subunit or a part of a subunit in F0, which is localized superficially, and which is influenced by F1, since the presence of F1 considerably lowers the rate of inhibition. The oligomycin binding site further seems to be influenced by the different conformational states of F1 occurring during the catalytic cycle of the enzyme. The binding site of DCCD on F0, on the other hand, seems to be deeply embedded in the membrane and not influenced by F1.     

Topography of a vacuolar-type H+-translocating ATPase: chromaffin-granule membrane ATPase I.

Proteins exposed on the cytoplasmic face of isolated chromaffin granules were labelled by lactoperoxidase-catalysed radioiodination and by non-enzymic biotinylation. Granule membranes were then prepared, and the H+-translocating ATPase isolated by fractionation with Triton X-114. The labelling of individual ATPase subunits was assessed by polyacrylamide-gel electrophoresis, followed by autoradiography or by blotting and decoration with 125I-labelled streptavidin. Subunits of 72, 57 and kDa were strongly labelled, and could be removed from the membrane at pH 11: they are therefore extrinsic proteins. The 120 kDa subunit was also labelled, but it was not solubilized at pH 11. Photolabelling with a hydrophobic probe indicated that this subunit penetrates the bilayer, and enzymic degradation studies showed the presence of N-linked oligosaccharides; this subunit therefore spans the chromaffin-granule membrane. Labelling of the 17 kDa subunit occurred predominantly on the extracytoplasmic (matrix) face of the granule membrane. These results are consistent with this V-type ATPase having a structure that is generally similar to that of mitochondrial (F-type) ATPases, although the attachment of the 120 kDa subunit may be asymmetrical.     

H+-translocating ATPase in Golgi apparatus. Characterization as vacuolar H+-ATPase and its subunit structures

Golgi apparatus was prepared from rat liver, and enzymatic properties and the subunit structure of the H+-ATPase were characterized. GTP (and also ITP) was found to drive H+-transport with about 20% of the initial velocity as that of ATP. Bafilomycin, a specific inhibitor for vacuolar H+-ATPase, inhibited the activity at 2.5 nM. The H+-ATPase was completely inhibited in the cold in the presence of MgATP (5 mM) and NaNO3 (0.1 M). The cold inactivation of the H+-ATPase resulted in release of a set of polypeptides from Golgi membrane, with molecular masses almost identical to that of the hydrophilic sector of chromaffin granule H+-ATPase (72, 57, 41, 34, and 33 kDa). Three of these polypeptides (72, 57, and 34 kDa), cross-reacted with antibodies against the corresponding subunits of the chromaffin granule H+-ATPase. A counterpart of the 39-kDa hydrophobic component of chromaffin granule H+-ATPase was identified in the membrane, but no 115-kDa component was found. Hence, the Golgi H+-ATPase shows typical features of vacuolar H+-ATPase, in relatively low substrate specificity, its response to inhibitors, inactivation by cold treatment in the presence of MgATP, and subunit composition judged by antibody cross-reactivity.     

Isolation of H+ -translocating ATPase in tonoplast of Tradescantia virginiana L. leaf cells

The tonoplast of Tradescantia virginiana L. was prepared from leaf cells and then solubilized with deoxycholate (DOC) and n-octyl-beta-D-glucoside (n-OG). Three major polypeptides (68, 60, 16 kDa) and several other minor components were isolated. These polypeptides were reconstituted in soybean phospholipids (asolectin). The H(+) pump activity was investigated with the reconstituted system as well as with the tonoplast. In both cases, the quinacrine-fluorescence quenching was observed in the presence of ATP-Mg(2+), indicating the H(+) pumping. The H(+) pump activity was inhibited by gramicidin D, a channel-forming ionophore, and by KNO(3), an inhibitor specific to tonoplast-type (V-type) H(+)-ATPase.     

Lysosomal H+-translocating ATPase has a similar subunit structure to chromaffin granule H+-ATPase complex

Subunit structure of the lysosomal H+-ATPase was investigated using cold inactivation, immunological cross-reactivity with antibodies against individual subunits of the H+-ATPase from chromaffin granules and chemical modification with N,N'-dicyclohexyl[14C]carbodiimide. The lysosomal H+-ATPase was irreversibly inhibited when incubated at 0 degrees C in the presence of chloride or nitrate and MgATP. Inactivation in the cold resulted in the release of several polypeptides (72, 57, 41, 34 and 33 kDa) from the membrane, which had the same electrophoretic mobility as the corresponding subunits of chromaffin granule H+-ATPase. Cross-reactivity of antibodies revealed that the 72, 57 and 34 kDa polypeptides were immunologically identical to the corresponding subunits of chromaffin granule H+-ATPase. Dicyclohexylcarbodiimide, which inhibits proton translocation in the vacuolar ATPase, predominantly labeled two polypeptides of 18 and 15 kDa, which compose the membrane sector of the enzyme. These results suggest that the lysosomal H+-ATPase is a multimeric enzyme, whose subunit structure is similar to the chromaffin granule H+-ATPase. The subunit structure of other vacuolar H+-ATPases, revealed by cold inactivation and immunological cross-reactivity, is also presented.     

Regulatory role of the ATPase inhibitor protein on proton conduction by mitochondrial H+-ATPase complex

This study shows that the natural inhibitor protein of mitochondrial H+-ATPase complex (IF1) inhibits, in addition to the catalytic activity, the proton conductivity of the complex. The inhibition of ATPase activity by IF1 is less effective in the purified F1 than in submitochondrial particles where F1 is bound to F0. No inhibition of H+ conductivity by F0 is observed in F1-depleted particles.     

Catabolite repression of the H(+)-translocating ATPase in Vibrio parahaemolyticus.

Cells of Vibrio parahaemolyticus grown in the presence of glucose showed reduced (by about 40%) oxidative phosphorylation. With this observation as a basis, we examined the effect of glucose on the level of H(+)-translocating ATPase. The addition of glucose to the growth medium reduced the specific activity and the amount of the H(+)-translocating ATPase in membrane vesicles of V. parahaemolyticus. These reductions were reversed by adding cyclic AMP (cAMP) to the growth medium. We cloned some parts of the unc genes encoding subunits of the H(+)-translocating ATPase of V. parahaemolyticus by means of the polymerase chain reaction. Using an amplified DNA fragment, we carried out Northern (RNA) blot analysis and found that glucose reduced the mRNA level of the H(+)-translocating ATPase gene by about 40% and that cAMP restored it. We determined the DNA sequence of the unc promoter region of V. parahaemolyticus and found a consensus sequence for the cAMP receptor protein-cAMP-binding site. Such a sequence was also found in the promoter region of the unc operon of Vibrio alginolyticus but not in its counterpart in Escherichia coli. We observed a similar reduction in the level of ATPase due to glucose in V. alginolyticus. In E. coli, however, reductions in the ATPase and the unc mRNA levels were not observed. Thus, the unc operon is controlled by cAMP-regulated catabolite repression in V. parahaemolyticus and V. alginolyticus but not in E. coli. Catabolite repression of the unc operon in V. parahaemolyticus is not severe.     

Subunit interaction in the mitochondrial H+-translocating ATPase. Association of the 26,500-dalton atpase binding protein and oligomycin sensitivity conferral protein with F1-ATPase

The rat liver 26,500-dalton ATPase binding protein and beef heart oligomycin sensitivity conferral protein are able to interact with the rat liver Type II ATPase to form discrete complexes. The equilibrium constants for these interactions are similar and each forms a 1:1 complex with the ATPase. The reassociated complex of Type II ATPase and 26,500-dalton ATPase binding protein or of oligomycin sensitivity conferral protein and Type II ATPase has properties similar to that of Type I ATPase. Dimerization of oligomycin sensitivity conferral protein by oxidation with copper phenanthroline chelate abolishes its ability to interact with the Type II ATPase. The isoelectric point and amino acid composition of the 26,500-dalton ATPase binding protein and oligomycin sensitivity conferral protein are similar. The polypeptide patterns produced by cyanogen bromide cleavage indicates a similar but nonidentical pattern to the 26,500-dalton ATPase binding protein and the oligomycin sensitivity conferral protein.     

Subunit interaction in the mitochondrial H+-translocating ATPase. The role of oligomycin sensitivity conferral protein and coupling factor 6 in ATPase binding and Pi-ATP exchange in mitochondrial membranes

Oligomycin sensitivity conferral protein, in the absence of coupling factor 6 (F6), is able to bind the ATPase to mitochondrial membranes with an apparent association constant of 10(6) M-1. The F6-dependent ATPase binding has an apparent association constant 1 to 2 orders of magnitude lower than that obtained with oligomycin sensitivity conferral protein. The oligomycin sensitivity conferral protein-dependent, membrane-bound ATPase activity is sensitive to rutamycin while the F6-dependent, membrane-bound ATPase activity is insensitive to rutamycin. F1-ATPase and Type II ATPase require F6 in addition to oligomycin sensitivity conferral protein and FB to reconstitute 32Pi-ATP exchange activity in silicotungstic acid particles. This F6 requirement for the 32Pi-ATP exchange is not related to the F6 effect on the ATPase binding. The Type I ATPase and therefore the 26,500-dalton subunit associated with it requires F6 and FB to reconstitute 32Pi-ATP exchange activity in silicotungstic acid particles. Oligomycin sensitivity conferral protein can be interchanged with the 26,500-dalton ATPase binding protein in the binding of the ATPase and the 32Pi-ATP exchange.     

Purification and functional properties of the DCCD-reactive proteolipid subunit of the H+-translocating ATPase from Mycobacterium phlei

Interaction of N,N'-dicyclohexylcarbodiimide (DCCD) with ATPase of Mycobacterium phlei membranes results in inactivation of ATPase activity. The rate of inactivation of ATPase was pseudo-first order for the initial 30-65% inactivation over a concentration range of 5-50 microM DCCD. The second-order rate constant of the DCCD-ATPase interaction was k = 8.5 X 10(5) M-1 X min(-1). The correlation between the initial binding of [14C]DCCD and 100% inactivation of ATPase activity shows 1.57 nmol DCCD bound per mg membrane protein. The proteolipid subunit of the F0F1-ATPase complex in membranes of M. phlei with which DCCD covalently reacts to inhibit ATPase was isolated by labeling with [14C]DCCD. The proteolipid was purified from the membrane in free and DCCD-modified form by extraction with chloroform/methanol and subsequent chromatography on Sephadex LH-20. The polypeptide was homogeneous on SDS-acrylamide gel electrophoresis and has an apparent molecular weight of 8000. The purified proteolipid contains phosphatidylinositol (67%), phosphatidylethanolamine (18%) and cardiolipin (8%). Amino acid analysis indicates that glycine, alanine and leucine were present in elevated amounts, resulting in a polarity of 27%. Cysteine and tryptophan were lacking. Butanol-extracted proteolipid mediated the translocation of protons across the bilayer, in K+-loaded reconstituted liposomes, in response to a membrane potential difference induced by valinomycin. The proton translocation was inhibited by DCCD, as measured by the quenching of fluorescence of 9-aminoacridine. Studies show that vanadate inhibits the proton gradient driven by ATP hydrolysis in membrane vesicles of M. phlei by interacting with the proteolipid subunit sector of the F0F1-ATPase complex.     

A respiratory-driven and an artificially driven ATP synthesis in mutants of Vibrio parahaemolyticus lacking H+-translocating ATPase

Mutants of Vibrio parahaemolyticus lacking the H+-translocating ATPase were isolated to evaluate both the role of this enzyme and the possibility of the involvement of other cation-translocating ATPase in the energy transduction in this organism. Dicyclohexylcarbodiimide-sensitive ATPase activity which represents the H+-translocating ATPase was not detected either in the membrane vesicles or in the cytosol of the mutants. Three major subunits, alpha, beta and gamma, of the H+-translocating ATPase were missing in the membranes of the mutants. Although ATP was synthesized in wild type cells when an artificial H+ gradient was imposed, little ATP was synthesized in the mutants. However, we observed a large ATP synthesis driven by the respiration not only in the wild type but also in the mutants. The respiratory-driven ATP synthesis in wild type was inhibited by an H+ conductor, carbonylcyanide m-chlorophenylhydrazone, by about 50%. On the other hand, the ATP synthesis in the mutants was not affected by the H+ conductor. Since this organism possesses a respiratory Na+ pump, Na+-coupled ATP synthesis might take place. In fact, we observed some ATP synthesis driven by an artificially imposed Na+ gradient both in the wild type and the mutant.     

Essential arginyl residues in the H+-translocating ATPase of plasma membrane from the yeast Schizosaccharomyces pombe

The H+-translocating adenosine-5'-triphosphatase (ATPase) purified from the yeast Schizosaccharomyces pombe is inactivated upon incubation with the arginine modifier 2,3-butanedione. The inactivation of the enzyme is maximal at pH values above 8.5. The modified enzyme is reactivated when incubated in the absence of borate after removal of 2,3-butanedione. The extent of inactivation is half maximal at 10 mM 2,3-butanedione for an incubation of 30 min at 30 degrees C at pH 7.0. Under the same conditions, the time-dependence of inactivation is biphasic in a semi-logarithmic plot with half-lives of 10.9 min and 65.9 min. Incubation with 2,3-butanedione lowering markedly the maximal rate of ATPase activity does not modify the Km for MgATP. These data suggest that two classes of arginyl residues play essential role in the plasma membrane ATPase activity. Magnesium adenosine 5'-triphosphate (MgATP) and magnesium adenosine 5'-diphosphate (MgADP), the specific substrate and product, protect partially against enzyme inactivation by 2,3-butanedione. Free ATP or MgGTP which are not enzyme substrates do not protect. Free magnesium, another effector of enzyme activity, exhibits partial protection at magnesium concentrations up to 0.5 mM, while increased inactivation is observed at higher Mg2+ concentrations. These protections indicate either the existence of at least one reactive arginyl in the substrate binding site or a general change of enzyme conformation induced by MgATP, MgADP or free magnesium.     

Cation transport by gastric H+:K+ ATPase.

A vesicular microsomal fraction isolated from hog fundic mucosa demonstrates the capacity to take up equal amounts of RB+ and Cl-. The amount of the Rb+ uptake is sensitive to the extravesicular osmolarity, and rate of uptake is sensitive to temperature. 86Rb+ efflux is dependent upon the cation composition of the diluting solution. ATP, but not beta-gamma methylene ATP, induces a reversible efflux of 86Rb+ from loaded vesicles, and this is dependent upon a functional K+-ATPase. The ATP induced efflux is not affected by CCCP (carbonyl cyanide m-chlorophenylhydrazone) or TCS (tetrachlorosalicylanilide) nor by lipid soluble ions or valinomycin. Nigericin inhibits the efflux by 40%. Uptake of the lipid soluble ion 14C-SCN- has been demonstrated and is enhanced by ATP only in the presence of valinomycin. The results are consistent with a neutral or isopotential exchange of H+ for Rb+ mediated by K+-ATPase.     

Na+-translocating membrane pyrophosphatases are widespread in the microbial world and evolutionarily precede H+-translocating pyrophosphatases

Membrane pyrophosphatases (PPases), divided into K(+)-dependent and K(+)-independent subfamilies, were believed to pump H(+) across cell membranes until a recent demonstration that some K(+)-dependent PPases function as Na(+) pumps. Here, we have expressed seven evolutionarily important putative PPases in Escherichia coli and estimated their hydrolytic, Na(+) transport, and H(+) transport activities as well as their K(+) and Na(+) requirements in inner membrane vesicles. Four of these enzymes (from Anaerostipes caccae, Chlorobium limicola, Clostridium tetani, and Desulfuromonas acetoxidans) were identified as K(+)-dependent Na(+) transporters. Phylogenetic analysis led to the identification of a monophyletic clade comprising characterized and predicted Na(+)-transporting PPases (Na(+)-PPases) within the K(+)-dependent subfamily. H(+)-transporting PPases (H(+)-PPases) are more heterogeneous and form at least three independent clades in both subfamilies. These results suggest that rather than being a curious rarity, Na(+)-PPases predominantly constitute the K(+)-dependent subfamily. Furthermore, Na(+)-PPases possibly preceded H(+)-PPases in evolution, and transition from Na(+) to H(+) transport may have occurred in several independent enzyme lineages. Site-directed mutagenesis studies facilitated the identification of a specific Glu residue that appears to be central in the transport mechanism. This residue is located in the cytoplasm-membrane interface of transmembrane helix 6 in Na(+)-PPases but shifted to within the membrane or helix 5 in H(+)-PPases. These results contribute to the prediction of the transport specificity and K(+) dependence for a particular membrane PPase sequence based on its position in the phylogenetic tree, identity of residues in the K(+) dependence signature, and position of the membrane-located Glu residue.     

Chloride-ion stimulation of the tonoplast H+-translocating ATPase from Hevea brasiliensis (rubber tree) latex. A dual mechanism.

The effect of Cl- and other anions on the tonoplast H+-translocating ATPase (H+-ATPase) from Hevea brasiliensis (rubber tree) latex was investigated. Cl- and other anions stimulated the ATPase activity of tightly sealed vesicles prepared from Hevea tonoplast, with the following decreasing order of effectiveness: Cl- greater than Br- greater than SO4(2-) greater than NO3-. As indicated by the changes of the protonmotive potential difference, anion stimulation of tonoplast H+-ATPase was caused in part by the ability of these anions to dissipate the electrical potential. This interpretation assumes not a channelling of these anions against a membrane potential, negative-inside, but a modification of the permeability of these ions through the tonoplast membrane. In addition, Cl- and the other anions stimulated the ATPase activity solubilized from the tonoplast membrane. Consequently, the tonoplast H+-pumping ATPase can be considered as an anion-stimulated enzyme. These results are discussed in relation to various models described in the literature for the microsomal H+-ATPase systems claimed as tonoplast entities.     

Kinetics of inhibition and binding of dicyclohexylcarbodiimide to the 82,000-dalton mitochondrial K+/H+ antiporter

Inhibition of K+/H+ antiport by N,N'-dicyclohexylcarbodiimide in Mg2+ depleted mitochondria follows first order kinetics, exhibiting a half-time of 13 min when mitochondria are incubated with 50 nmol/mg inhibitor at 0 degrees C. 14C radiolabeled N,N'-dicyclohexylcarbodiimide binds to the 82,000-dalton protein, and the second order rate constant for binding is found to be approximately the same as the second order rate constant for inhibition. These findings provide additional confirmation of the identification of this porter with the 82,000-dalton protein and permit us to estimate that rat liver mitochondria contain about 8 pmol/mg of K+/H+ antiporter with a turnover number of 700 s-1. The K+/H+ antiporter of rat liver mitochondria is protected from N,N'-dicyclohexylcarbodiimide inhibition and binding by quinine and by endogenous Mg2+. An 82,000-dalton, [14C]N,N'-dicyclohexylcarbodiimide-binding protein is also observed in rat liver submitochondrial particles, establishing this as an integral protein of the inner membrane. Submitochondrial particles, presumed to be inverted in membrane orientation, are protected from radiolabeling by external Mg2+, supporting the contention that the Mg2+ binding site is localized to the matrix side of the K+/H+ antiporter.