ATP Phosphohydrolase Research Articles

Lipid composition modulates ATP hydrolysis and calcium phosphate mineral propagation by TNAP-harboring proteoliposomes

Bone biomineralization is mediated by a special class of extracellular vesicles, named matrix vesicles (MVs), released by osteogenic cells. The MV membrane is enriched in sphingomyelin (SM), cholesterol (Chol) and tissue non-specific alkaline phosphatase (TNAP) compared with the parent cells’ plasma membrane. TNAP is an ATP phosphohydrolase bound to cell and MV membranes via a glycosylphosphatidylinositol (GPI) anchor. Previous studies have shown that the lipid microenvironment influences the catalytic activity of enzymes incorporated into lipid bilayers. However, there is a lack of information about how the lipid microenvironment controls the ability of MV membrane-bound enzymes to induce mineral precipitation. Herein, we used TNAP-harboring proteoliposomes made of either pure dimyristoylphosphatidylcholine (DMPC) or DMPC mixed with either Chol, SM or both of them as MV biomimetic systems to evaluate how the composition modulates the lipid microenvironment and, in turn, TNAP incorporation into the lipid bilayer by means of calorimetry. These results were correlated with the proteoliposomes’ catalytic activity and ability to induce the precipitation of amorphous calcium phosphate (ACP) in vitro. DMPC:SM proteoliposomes displayed the highest efficiency of mineral propagation, apparent affinity for ATP and substrate hydrolysis efficiency, which correlated with their highest degree of membrane organization (highest ΔH), among the tested proteoliposomes. Results obtained from turbidimetry and Fourier transformed infrared (FTIR) spectroscopy showed that the tested proteoliposomes induced ACP precipitation with the order DMPC:SM>DMPC:Chol:SM≈DMPC:Chol>DMPC which correlated with the lipid organization and the presence of SM in the proteoliposome membrane. Our study arises important insights regarding the physical properties and role of lipid organization in MV-mediated mineralization.

Age-associated oxidative modifications of mitochondrial α-subunit of F1 ATP synthase from mouse skeletal muscles

The objective of this study was to investigate the pattern of age-associated oxidative post-translational modifications in the skeletal muscles of a mammalian species and to address whether the modifications result in the loss of function of the oxidatively modified protein(s). Accordingly, proteins in the mitochondrial matrix of the hind limb of C57BL/6Nnia mice were examined for modifications by carbonylation—an established marker of oxidative post-translational modifications—by Western blotting using anti-2,4-dinitrophenyl antibodies and tritiated sodium borohydride methods. An age-associated increase in carbonylation of mitochondrial matrix proteins was observed, but not all proteins were equally susceptible. A 55 kDa protein, identified as the α-subunit of the F1 complex of ATP synthase (ATP phosphohydrolase [H+-transporting]), had approximately 17% and 27% higher levels of protein carbonyls in adult and old animals, respectively, in comparison to the young controls as estimated using tritiated sodium borohydride. In addition, an age-associated decline in its activity was observed, with approximately 9% and 28% decrease in the activity in the adult and old animals, respectively, in comparison to young controls. It may be concluded that such oxidative post-translational modifications and the resultant attenuation of the protein activity may contribute to the age-related energy loss and muscular degeneracy.

Partial Purification and Properties of Adenosine Triphosphatase (ATPase) from Liver Fluke Fasciola hepatica

The adenosine triphosphatase (ATP phosphohydrolase, EC 3.6.1.3.;ATPase) is a membrane -bound enzyme which transport protons across the plasma membrane using ATP as an energy source. The adenosine triphosphatase (ATPase ; EC: 3.6.1.3) was extracted from membrane preparations of adult Fasciola hepatica by chloroform treatment and purified by means of ammonium sulphate fractionation, gel filtration on sephadex G-200 and DEAE- Cellulose chromatography. The molecular weight was calculated to be 305.000 dalton by gel filtration. Kinetic experiments demonstrated a biphasic linear lineweaver - burk relationship (km=0.142 and 1.66 mM) thus revealing the existence of two substrate binding enzyme sites. In our study revealed that partial inhibition of Mg²⁺ dependent purified enzyme by oligomycin suggest the absence of mitochondrial ATPase in F. hepatica.

Crystal Structure of Baculovirus RNA Triphosphatase Complexed with Phosphate

Baculovirus RNA 5'-triphosphatase (BVP) exemplifies a family of RNA-specific cysteine phosphatases that includes the RNA triphosphatase domains of metazoan and plant mRNA capping enzymes. Here we report the crystal structure of BVP in a phosphate-bound state at 1.5 A resolution. BVP adopts the characteristic cysteine-phosphatase alpha/beta fold and binds two phosphate ions in the active site region, one of which is proposed to mimic the phosphate of the product complex after hydrolysis of the covalent phosphoenzyme intermediate. The crystal structure highlights the role of backbone amides and side chains of the P-loop motif (118)HCTHGXNRT(126) in binding the cleavable phosphate and stabilizing the transition state. Comparison of the BVP structure to the apoenzyme of mammalian RNA triphosphatase reveals a concerted movement of the Arg-125 side chain (to engage the phosphate directly) and closure of an associated surface loop over the phosphate in the active site. The structure highlights a direct catalytic role of Asn-124, which is the signature P-loop residue of the RNA triphosphatase family and a likely determinant of the specificity of BVP for hydrolysis of phosphoanhydride linkages.

Chemiosmotic ATP synthesis in photosynthetically inactive chromoplasts from Narcissus pseudonarcissus L. linked to a redox pathway potentially also involved in carotene desaturation.

Mature chromoplasts from daffodil (Narcissus pseudonarcissus) flowers, although devoid of thylakoid structures, contain immunologically detectable alpha-subunits of ATP-synthase (H(+)-transporting ATP phosphohydrolase; EC 3.6.3.14). To show the presence of the entire functional protein complex, chromoplast membrane proteins were solubilized and reconstituted in phosphatidylcholine liposomes. The membranes were energized by an acid-base transition in the presence of a K(+)/valinomycin diffusion potential, and the initial rate of ATP synthesis was measured with a luciferin/luciferase assay. In addition, by demonstrating NADPH-dependent ATP synthesis, we show that an NAD(P)H-dependent respiratory redox pathway in chromoplasts, previously identified as an important constituent of the carotene desaturation system, proceeds concomitant with membrane energization.

A COCCOLITHOPHORID CALCIFYING VESICLE WITH A VACUOLAR‐TYPE ATPASE PROTON PUMP: CLONING AND IMMUNOLOCALIZATION OF THE V0 SUBUNIT c

Coccolithophorid calcification is subcellular. It relies on a single Golgi apparatus to produce coccoliths consisting of an organic baseplate and calcite. The calcification reaction is initiated in a calcifying vesicle derived from the trans‐most Golgi. We have cloned a subunit of a V (vacuolar)‐ATPase (EC 3.6.1.3., ATP phosphohydrolase) from a Pleurochrysis (Haptophyceae) cDNA library of transcripts expressed during calcification. Degenerate PCR primers were developed after alignment of the higher plant V‐ATPase subunit c genes to identify conserved consensus sequences. The library was screened with a homologous probe obtained by PCR. The cloned gene is found as a single copy on the P. cartarae (strain 136) genome and includes a 495‐base pair open reading frame encoding a 164 amino acid polypeptide and deduced molecular mass of 16.2 kDa. Its deduced amino acid sequence shows a close relationship to subunit c of the Vo domain of the vacuolar proton‐pumping ATPase of higher plants. An in vitro‐synthesized oligopeptide corresponding to the L2 extramembrane domain was used for rabbit immunization. Affinity‐purified antiserum detected a polypeptide band with an apparent molecular mass of 24 kDa in immunoblots of highly enriched coccolith vesicle membranes. Immunofluoresence microscopy showed antibody specificity for the membranes of isolated coccolith vesicles. This work provides support for the existence of an authentic, vacuolar‐type, proton‐pumping ATPase on coccolith vesicle membranes in a calcifying coccolithophorid.

An HPLC method for the assay of starch synthase

A method is presented for the assay of starch synthase activity in plant extracts which does not require the use of radioactive substrates. The method is based on the quantification by HPLC of phosphorylated nucleosides generated in the reaction mixture during incorporation of ADP glucose (ADPGlc) into polymeric glucan. Estimation of starch synthase activity based on the rate of ADP accumulation in the medium or on the incorporation of glycosyl residues of ADP [U- 14C]Glc into polymeric produced comparable results in extracts of tobacco, spinach or potato leaves. With potato tuber extracts AMP accumulated in the medium instead of ADP. This is ascribed to the activity of apyrase (EC 3.6.1.5), a fluoride-insensitive ATP phosphohydrolase which removes g-phosphoryl groups of ATP and ADP and it is particularly abundant in potato tubers. In this case, the rate of AMP accumulation in the reaction mixture well correlated with the rate of ADP [U- 14C]Glc incorporation into polymeric glucan. Thus, the HPLC method presented for the assay of starch synthase activity is also suitable for tissues containing apyrase activity.

Sites of aspecific ATP-phosphohydrolase activity in the branchial region of chick embryos, with particular emphasis on the myogenic cell population.

The distribution pattern of unspecific ATP-phosphohydrolase (ATP-PH) activity was studied in serial sections through the branchial region of chick embryos from HH stage 14 to 27. A selective reaction was found in the prospective myogenic mesoderm of voluntary muscles of the first three branchial arches. This property initially characterized uncondensed myogenic cell populations in the mandibular and hyoid arches and appeared later on in the third arch. In each case, the positive premuscular mesoderm was closely apposed to the lateral wall of the corresponding aortic arch. Myogenic primordia retained a strong ATP-PH activity up to stage 27 when they began to segregate into smaller premuscular units. Discrete changes observed in the mandibular and hyoid blastemas suggested that non reacting cells probably of neural crest origin invade their proximal core and contribute to the subsequent pathfinding of muscular nerves. Other selective sites of ATP-PH reactivity were detected in the odd-numbered rhombomeres, rhombencephalic floorplate, notochord, arterial endothelial cells, prospective dermis and perichondrium.

Rotational mobility of Ca 2+-ATPase of sarcoplasmic reticulum in viscous media

The rotational diffusion of Ca 2+-ATPase [Ca 2+,Mg 2+-activated ATP phosphohydrolase E.C. 3.6.1.38] was studied in native sarcoplasmic reticulum membrane by saturation transfer ESR spectroscopy after covalent labelling of intramembranous sulfhydryl groups with nitroxyl derivative of maleimide (5-MSL) as a function of sucrose and glycerol in the suspending medium. The relative enzymatic activity of sarcoplasmic reticulum was followed by increasing the viscosity of the aqueous phase. The ATP hydrolysing activity of the enzyme decreased differently on adding sucrose and glycerol. In the case of sucrose the reciprocal of power dependence of viscosity was observed, whereas for glycerol an exponential decay law was obtained, indicating solvent-protein interaction. On increasing the viscosity of the aqueous phase by either sucrose or glycerol, no changes were observed in the intramembranous viscosity as measured using intercalated spin-labelled stearic acid (16-SASL). The effective rotational correlation time of the protein was measured, as a mobility parameter, using saturation transfer ESR spectroscopy and found to be increased linearly with the viscosity of the sucrose containing medium and for the extramembranous size a height of 6.8 nm was obtained, indicating that approx. 82% of the volume of Ca 2+-ATPase protein is external to the sarcoplasmic reticulum. The addition of glycerol probably promoted protein–protein interaction, as indicated by the larger changes in rotational diffusion and non-linear viscosity dependence.

Adjustment of K' to varying pH and pMg for the creatine kinase, adenylate kinase and ATP hydrolysis equilibria permitting quantitative bioenergetic assessment.

Physiologists and biochemists frequently ignore the importance of adjusting equilibrium constants to the ionic conditions of the cell prior to calculating a number of bioenergetic and kinetic parameters. The present study examines the effect of pH and free magnesium levels (free [Mg2+]) on the apparent equilibrium constants (K') of creatine kinase (ATP: creatine N-phosphotransferase; EC 2.7.3.2), adenylate kinase (ATP:AMP phosphotransferase; EC 2.7.4.3) and adenosinetriphosphatase (ATP phosphohydrolase; EC 3.6.1.3) reactions. We show how K' can be calculated using the equilibrium constant of a specified chemical reaction (Kref) and the appropriate acid-dissociation and Mg(2+)-binding constants at an ionic strength (I) of 0.25 mol l-1 and 38 degrees C. Substituting the experimentally determined intracellular pH and free [Mg2+] into the equation containing a known Kref and two variables, pH and free [Mg2+], enables K' to be calculated at the experimental ionic conditions. Knowledge of K' permits calculation of cytosolic phosphorylation ratio ([ATP]/[ADP][Pi]), cytosolic free [ADP], free [AMP], standard transformed Gibbs energy of formation (delta fG' degrees ATP) and the transformed Gibbs energy of the system (delta fG' ATP) for the biological system. Such information is vital for the quantification of organ and tissue bioenergetics under physiological and pathophysiological conditions.

LOCALIZATION OF CA2+‐STIMULATED ATPASE IN THE COCCOLITH‐PRODUCING COMPARTMENT OF CELLS OF PLEUROCHRYSIS SP. (PRYMNESIOPHYCEAE)1

ABSTRACTCell homogenates of Pleurochrysis sp. (CCMP299) were fractionated by means of sucrose gradients. Ca2+‐stimulated ATPase (EC 3.6.1.3., ATP phosphohydrolase) was associated primarily with the plasma membrane, Golgi, and high density (1.21 g·cm−3) membranous structures. Ca2+‐stimulated ATPase was highly enriched in the latter. Based on treatments with Triton X‐100 and NBD ceramide, we conclude that the high‐density structures were membrane‐delimited organelles. These vesicle‐like organelles contained complex polysaccharides, a high concentration of calcium, and, upon microscopic examination, structures resembling coccoliths. These findings are consistent with observations on the known composition of coccoliths and the presumed mineralizing function of the sub‐cellular coccolith‐producing compartment. The high‐density vesicles were linked to the Golgi by means of colchicine‐sensitive materials, presumably microtubules. These data and prior ultrastructural observations by other investigators indicating vectorial assembly and secretion suggest that the subcellular movement of the newly formed coccoliths may be directed and/or powered by colchicine‐sensitive cytoskeletal elements. We interpret the data to mean that the high‐density vesicles represent the coccolith‐producing compartment previously observed by others in electron micrographs.

Influence of Poly(ethylene glycol) and Aqueous Viscosity on the Rotational Diffusion of Membranous Na,K-ATPase

The Na,K-ATPase [ATP phosphohydrolase (Na+/K(+)-transporting), E.C. 3.6.1.37] in native membranes from the salt gland of Squalus acanthias has been spin-labeled covalently with a chloromercuri nitroxide derivative, and the rotational diffusion of the protein has been studied, as a function of the concentration of glycerol or poly(ethylene glycol) in the suspending medium, by means of saturation-transfer electron spin resonance spectroscopy. The effective rotational correlation time of the protein increases linearly with the viscosity of the aqueous glycerol medium, with a gradient whose value indicates that ca. 50-70% of the volume of the Na,K-ATPase protein is external to the membrane. The effective rotational correlation times of the protein in poly(ethylene glycol) solutions are considerably greater than those in glycerol solutions of the same viscosity and increase nonlinearly with the viscosity of the suspending medium, indicating that increasing concentrations of poly(ethylene glycol) induce aggregation of the integral proteins within the membrane. The value reached at 50% poly(ethylene glycol) corresponds to a degree of aggregation of the proteins between 2 and 5 depending on whether the ethylene glycol polymer is excluded from the membrane surface region. The results are discussed with respect to hydration forces and poly(ethylene glycol)-induced cell fusion.

Acetylcholinesterase and Na+,K(+)-ATPase activities in different regions of rat brain during insulin-induced hypoglycemia.

The activities of acetylcholinesterase (acetylcholine acetylhydrolase, EC 3.1.1.7), responsible for hydrolysis of acetylcholine and Na+,K(+)-ATPase (Mg(2+)-dependent ATP phosphohydrolase, EC 3.6.1.3), which plays a crucial role in neurotransmission, were determined in four brain regions after 1, 2, and 3 h of insulin administration. Significant decrease in the acetylcholinesterase and Na+,K(+)-ATPase activities was observed in the soluble and total particulate fractions from cerebral hemispheres, cerebellum, brain stem, and diencephalon + basal ganglia after 1, 2, and 3 h of insulin-induced hypoglycemia. Blood glucose level decreased significantly after 1 h of insulin administration and remained at low level for 2 h thereafter, whereas, the protein content in different subcellular fractions from four brain regions did not show any significant change under this physiological stress.

Local translational diffusion rates of membranous Na+,K(+)-ATPase measured by saturation transfer ESR spectroscopy.

Diffusion-controlled Heisenberg spin exchange between spin-labeled Na+,K(+)-ATPase [ATP phosphohydrolase (Na+/K(+)-transporting), EC 3.6.1.37] proteins has been studied by saturation transfer ESR spectroscopy in reconstituted membranes. Na+,K(+)-ATPase from the salt gland of Squalus acanthias was solubilized in a polyoxyethylene ether detergent, octa(ethylene glycol) dodecyl monoether. Part of the solubilized enzyme was covalently spin-labeled with a nitroxide derivative of indanedione and recombined with various proportions of the unlabeled enzyme while the native lipid/protein ratio was maintained. Purified membranes were then reconstituted from the various samples by precipitation with divalent ions. The reciprocal integrated intensities of the saturation transfer ESR spectra were found to increase linearly with the fraction of protein that was spin-labeled, and the gradient of the concentration dependence increased with increasing temperature over the range 4 degrees-25 degrees C. Comparison with theoretical analyses of the effects of weak Heisenberg spin exchange [Marsh, D. & Horváth, L. I. (1992) J. Magn. Reson. 97, 13-26] suggests that the effects on the saturation transfer ESR intensity are attributable to short-range diffusional collisions between the spin-labeled protein molecules. The effective value of the local translational diffusion coefficient is 1.8-2.9 microns2.s-1 at 15 degrees C, depending on the diffusion model used, which is much larger than the values obtained for the long-range diffusion coefficient in cells by photobleaching techniques. The temperature dependence of the translational diffusion is larger than expected but correlates with the anomalous temperature dependence of the rotational diffusion observed in the same system.

Ontogeny of the sodium pump in embryos of rockfish of the genusSebastes

The purpose of the present investigation was to determine at what stages of embryonic development the sodium pump (Na+,K+-activated ATP phosphohydrolase, EC 3.6.1.3) appears and whether the ontogeny of the sodium pump plays a role in the matrotrophic viviparity ofSebastes. Early larval stages (stages 1–14) had embryonic tissues nearly devoid of Na, K-ATPase activity. After epiboly, tissues from embryos taken from both species,S. schlegeli andS. taczanowskii, had significant levels of enzymatic activity coincident with the appearance of neuronal tissue in the head fold (stages 15–20). Maximal levels of specific activities of the enzyme were reached with the onset of the vascular circulation and the opening of the buccal cavity together with maturation of the midgut and hindgut regions of the intestinal tract (stages 26 and 28). Ouabain, a specific inhibitor of Na, K-ATPase, was used to measure survival of embryos placed in different seawater concentrations under in vitro conditions. The most sensitive stages to external ouabain were 26–30. These findings support the hypothesis that the intestinal tract is functional prior to gestation and could be transporting important nutritive material found in maternal ovarian fluid.

Ca2+ transport activities of inside-out vesicles prepared from density-separated erythrocytes from rat and human

The plasma membrane Ca2+ ATPase in erythrocytes is vital for the maintenance of intracellular Ca2+ levels. Since the cytoplasmic Ca2+ concentration is elevated in older erythrocytes, the properties of the Ca2+ transport ATPase were examined during cell aging using inside-out vesicles (IOVs) prepared from density-separated, young (less dense, Ey) and old (more dense, Eo) rat and human erythrocytes. The transport of Ca2+ and the coupled hydrolysis of ATP were measured using radiolabeled substrates. The calmodulin-independent Ca2+ transport activity (Ey, 38.8 vs. Eo, 23.3 nmols/min/mg IOV protein) and the Ca2+ dependent ATP phosphohydrolase activity (Ey, 53.5 vs. Eo, 48.8 nmols/min/mg protein) were greater in IOVs prepared from younger (less dense) rat erythrocytes. The calmodulin-independent Ca2+ transport activity in IOVs from human erythrocytes was 12.9 nmols/min/mg IOV protein for Ey and 10.7 nmols/min/mg IOV protein for Eo. Inside-out vesicles from older (more dense) cells exhibited a lower pumping efficiency as determined by the calculated stoichiometry, molecule of Ca2+ transported per molecule of ATP hydrolyzed (rat: Ey, 0.74 vs. Eo, 0.49; human: Ey, 1.22 vs. Eo, 0.77). The enzymatic activity of rat and human Ey IOVs was labile. The Ca2+ transport activity in Ey but not Eo IOVs rapidly declined during cold storage (4 degrees C). The decrease in Ca2+ transport activity during aging may accentuate the age-related decline in several erythrocytic properties.

A nervous system-specific isotype of the beta subunit of Na+,K(+)-ATPase expressed during early development of Xenopus laevis.

We have previously described the isolation of several genes expressed exclusively in the nervous system of adult Xenopus laevis and activated in the embryo shortly after neural induction. The sequence of one of these cDNAs, 24-15, identifies the corresponding protein as an isotype of the beta subunit of Na+,K(+)-ATPase [ATP phosphohydrolase (Na+/K(+)-transporting); EC 3.6.1.37]. This form is distinct from the previously described beta 1 subunit of Xenopus, and the protein sequence comparison suggests that it is not the frog homolog of the mammalian beta 2 subunit; therefore, we refer to the 24-15 protein as the beta 3 subunit of Na+,K(+)-ATPase of Xenopus. Antisera directed against beta 3-subunit fusion protein detected a protein in adult brain extracts with the size and properties expected for a Na+,K(+)-ATPase beta subunit. In Xenopus the beta 1 and beta 3 subunits are expressed as maternal mRNAs at similar levels; during embryogenesis rapid accumulation of beta 3 mRNA begins at stage 14 (early neurula), and the rapid accumulation of beta 1 mRNA begins at stage 23/24. In situ hybridization of antisense RNA probes to tadpole brain sections indicates that beta 3 subunit is expressed throughout the developing brain. We suggest that beta 3 is a major Na+,K(+)-ATPase beta subunit present during early nervous system development in the frog.

A monoclonal antibody against a native conformation of the porcine renal Na +/K +-ATPase α-subunit protein

A monoclonal antibody (mAb50c) against the native porcine renal Na +/K +-transporting adenosinetriphosphatase (EC 3.6.1.37, ATP phosphohydrolase) (Na +/K +-ATPase) was characterized. The antibody could be classified as a conformation-dependent antibody, since it did not bind to Na +/K +-ATPase denatured by detergent and its binding was affected by the normal conformational changes of the enzyme induced by ligands. The binding was the greatest in the presence of Na +, ATP or Mg 2+ (E1 form), slightly less in the presence of K + (E2K form) and the least when the enzyme was phosphorylated, especially in the actively hydrolyzing form in the presence of Na +, Mg 2+ and ATP. The antibody inhibited both the Na +,K +-ATPase activity and the K +-dependent p-nitrophenylphosphatase activity by 25%, but it had no effect on Na +-dependent ATPase activity. The antibody partially inhibited the fluorescence changes of the enzyme labeled with 5′-isothiocyanatofluorescein after the addition of orthophosphate and Mg 2+, and after the addition of ouabain. Proteolytic studies suggest that a part of the epitope is located on the cytoplasmic surface of the N-terminal half of the α-subunit.

Reaction of thiol groups of gizzard myosin heavy chains with 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole

Chicken gizzard myosin treated with 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole (NBD-Cl) resulted in a 65% inhibition of the K +-ATPase (myosin ATP phosphohydrolase (actin translocating), EC 3.6.1.32) activity and 3.5 mol of the reagent was bound per 4.7 × 10 5 g protein. The labeling was limited to the heavy chain region and none of the light chains were lost. MgATP had no effect on the inactivation or labeling pattern. Thiolysis of NBD-myosin with dithiothreitol restored the K +-ATPase activity and concurrently, 1 mol of the NBD group was removed from the heavy chain region. Cysteine residues were modified in NBD-myosin at sites other than the active site when the enzyme activity was inhibited. There was a difference in the extent of NBD-Cl modification of gizzard myosin at 0.6 m KCl (6 S elongated state) when compared to that at 0.15 m KCl (10 S folded state). This was also seen in the heavy meromyosin-like chymotryptic fragments and tryptic fragments of NBD-myosin. The reagent NBD-Cl can detect changes in the conformation of gizzard myosin by way of its reaction with thiol groups of the heavy chain region.

The essential carboxyl group in subunit c of the F1F0 ATP synthase can be moved and H(+)-translocating function retained.

The proteolipid subunit c of F1F0-type H(+)-transporting ATP synthases [ATP phosphohydrolase (H(+)-transporting), EC 3.6.1.34] contains a conserved Asp/Glu residue that is thought to function in H+ translocation. To test the importance of the position of this residue in the Escherichia coli enzyme, we used oligonucleotide-directed mutagenesis to move the carboxyl side chain from position 61 to position 58, 60, or 62. Mutant cells with these changes were incapable of growth via oxidative phosphorylation on succinate. An Asp-61----Glu mutant grew on succinate but at 50% the efficiency of wild type. Hence, even minor changes in the position of the carboxyl group can significantly reduce function. In a second approach, slow-growing revertants to an Asp-61----Gly mutant were isolated. In one such revertant, Ala-24 was changed to Asp, while the original Asp-61----Gly mutation remained unchanged. The Asp-24-Gly-61 double mutant grew on succinate at 60% the efficiency of wild type. Hence the essential carboxyl group of subunit c can function when anchored at either position 24 or position 61, and this supports the idea that these residues may neighbor each other when subunit c is folded in the membrane. The rate of ATP-driven H+ translocation by mutant membrane vesicles was estimated by the quenching of 9-amino-6-chloro-2-methoxyacridine fluorescence and corresponded to actual H+ pumping rates less than 25% that of wild type.