Proton-translocating Adenosine Triphosphatase Research Articles

PS-B12-4: SOLUBLE (PRO)RENIN RECEPTOR LEVEL INCREASED IN A RAT MODEL OF REDUCED UTERINE PERFUSION PRESSURE

Objective: The (pro)renin receptor [(P)RR)] is a multifunctional protein with roles in angiotensin II-dependent and -independent intracellular cell signaling and as an adaptor protein between the Wnt receptor complex and vacuolar proton-translocating adenosine triphosphatase. The (P)RR is cleaved from the full length (P)RR to generate soluble (P)RR [s(P)RR], reflecting the status of the tissue activity of the (P)RR. We have shown that maternal blood s(P)RR concentrations in early pregnancy are associated with increased blood pressure in late pregnancy, while higher levels of s(P)RR in cord blood in babies are at lower risk of low birth weight. We have also reported that intracellular s(P)RR is increased by hypoxia and interacts with pyruvate dehydrogenase β; subunit (PDHB) to stabilize PDHB protein and pyruvate dehydrogenase (PDH) activity in trophoblast cells, leading to our hypothesis that (P)RR may help maintain oxidative metabolism and efficient energy production during placental ischemia. In this in vivo study, we investigated effects of reduced uterine perfusion pressure (RUPP) on PDH activity and s(P)RR levels in rats. Design and method: RUPP was induced by placing sliver clips around the aorta above the iliac bifurcation and the bilateral uterine arcades in pregnant Sprague Dawley rats on gestational day 14 (RUPP rats). Weights of placentas and infants were measured on gestational day 21. mRNA expressions of (P)RR, hypoxia-inducible factor 1-alpha (HIF1a), and PDHB, and protein levels of HIF1a, (P)RR, s(P)RR, PDHB, lactate, and pyruvate, and activity of PDH in placentas were measured on gestational day 21. Result: Weight of placentas and infants were significantly decreased in RUPP rats when compared with those of sham rats. mRNA and protein levels of HIF1a were significantly higher in RUPP rats than in control sham rats, indicating that ischemia was actually induced by RUPP. mRNA levels of (P)RR and PDHB were not significantly different between the groups. The protein level of (P)RR was not significantly different between the groups while s(P)RR protein level was significantly higher in RUPP rats than in controls. Concentrations of lactate or pyruvate and PDH activity were not significantly different between the groups. Conclusion: PDH activity was not decreased even under the condition of placental ischemia. s(P)RR but not full length (P)RR was increased by placental ischemia. These data suggested that s(P)RR might be increased by placental ischemia to maintain mitochondrial oxidative metabolism in rats.

PS-C22-10: SERUM SOLUBLE (PRO)RENIN RECEPTOR CONCENTRATION AS A PROGNOSTIC FACTOR IN PATIENTS UNDERGOING MAINTENANCE HEMODIALYSIS

Objective: The (pro)renin receptor [(P)RR)] is a multifunctional protein with roles in angiotensin II-dependent and -independent intracellular cell signaling and as an adaptor protein between the Wnt receptor complex and vacuolar proton-translocating adenosine triphosphatase. The (P)RR is cleaved to generate soluble (P)RR [s(P)RR], reflecting the status of the tissue renin-angiotensin system and/or activity of the (P)RR. Patients undergoing hemodialysis (HD) have poor prognosis because of the increased prevalence of cardiovascular and malignant diseases. This study was conducted to investigate whether the s(P)RR level is associated with new onset of cardiovascular events or malignant diseases and prognosis in patients undergoing HD. Design and method: A total of 258 patients undergoing maintenance HD who were enrolled in our cohort study investigating the relationships between serum s(P)RR levels and background factors were prospectively followed up for 60 months. We investigated the relationships between s(P)RR levels and new onset of cardiovascular events or malignant diseases and mortality during the follow-up period. Results: The cumulative incidence of new onset of cardiovascular events (P = 0.009) and cardiovascular deaths (P < 0.001), but not of malignant diseases, was significantly greater in patients with higher serum s(P)RR level (≧ 29.8 ng/ml) than in those with lower s(P)RR level (< 29.8 ng/ml). A high serum s(P)RR level was independently correlated with cardiovascular mortality (95% CI 1.001 - 1.083, P = 0.046). Conclusions: These data showed that the serum s(P)RR level is associated with cardiovascular events and mortality, suggesting that the serum s(P)RR level could be used as a biomarker for selecting patients requiring intensive care.

Photosynthetic-Product-Dependent Activation of Plasma Membrane H+-ATPase and Nitrate Uptake in Arabidopsis Leaves.

Plasma membrane (PM) proton-translocating adenosine triphosphatase (H+-ATPase) is a pivotal enzyme for plant growth and development that acts as a primary transporter and is activated by phosphorylation of the penultimate residue, threonine, at the C-terminus. Small Auxin-Up RNA family proteins maintain the phosphorylation level via inhibiting dephosphorylation of the residue by protein phosphatase 2C-D clade. Photosynthetically active radiation activates PM H+-ATPase via phosphorylation in mesophyll cells of Arabidopsis thaliana, and phosphorylation of PM H+-ATPase depends on photosynthesis and photosynthesis-related sugar supplementation, such as sucrose, fructose and glucose. However, the molecular mechanism and physiological role of photosynthesis-dependent PM H+-ATPase activation are still unknown. Analysis using sugar analogs, such as palatinose, turanose and 2-deoxy glucose, revealed that sucrose metabolites and products of glycolysis such as pyruvate induce phosphorylation of PM H+-ATPase. Transcriptome analysis showed that the novel isoform of the Small Auxin-Up RNA genes, SAUR30, is upregulated in a light- and sucrose-dependent manner. Time-course analyses of sucrose supplementation showed that the phosphorylation level of PM H+-ATPase increased within 10 min, but the expression level of SAUR30 increased later than 10 min. The results suggest that two temporal regulations may participate in the regulation of PM H+-ATPase. Interestingly, a 15NO3- uptake assay in leaves showed that light increases 15NO3- uptake and that increment of 15NO3- uptake depends on PM H+-ATPase activity. The results opened the possibility of the physiological role of photosynthesis-dependent PM H+-ATPase activation in the uptake of NO3-. We speculate that PM H+-ATPase may connect photosynthesis and nitrogen metabolism in leaves.

Serum Soluble (Pro)renin Receptor Level as a Prognostic Factor in Patients Undergoing Maintenance Hemodialysis

Background: The (pro)renin receptor [(P)RR)] is a multifunctional protein with roles in angiotensin II-dependent and -independent intracellular cell signaling and as an adaptor protein between the Wnt receptor complex and vacuolar proton-translocating adenosine triphosphatase. The (P)RR is cleaved to generate soluble (P)RR [s(P)RR], reflecting the status of the tissue renin-angiotensin system and/or activity of the (P)RR. Patients undergoing hemodialysis (HD) have poor prognosis because of the increased prevalence of cardiovascular and malignant diseases. This study was conducted to investigate whether the s(P)RR level is associated with new onset of cardiovascular events or malignant diseases and prognosis in patients undergoing HD. Methods: A total of 258 patients undergoing maintenance HD who were enrolled in our cohort study investigating the relationships between serum s(P)RR levels and background factors were prospectively followed up for 60 months. We investigated the relationships between s(P)RR levels and new onset of cardiovascular events or malignant diseases and mortality during the follow-up period. Results: The cumulative incidence of new onset of cardiovascular events (P = 0.009) and cardiovascular deaths (P < 0.001), but not of malignant diseases, was significantly greater in patients with higher serum s(P)RR level (≥ 29.8 ng/ml) than in those with lower s(P)RR level (< 29.8 ng/ml). A high serum s(P)RR level was independently correlated with cardiovascular mortality (P = 0.046). Conclusions: These data showed that the serum s(P)RR level is associated with cardiovascular events and mortality, suggesting that the serum s(P)RR level could be used as a biomarker for selecting patients requiring intensive care.

Mechanisms of Exogenous Nitric Oxide and 24-Epibrassinolide Alleviating Chlorosis of Peanut Plants Under Iron Deficiency

Iron (Fe) is a crucial transition metal for all living organisms including plants; however, Fe deficiency frequently occurs in plant because only a small portion of Fe is bioavailable in soil in recent years. To cope with Fe deficiency, plants have evolved a wide range of adaptive responses from changes in morphology to altered physiology. To understand the role of nitric oxide (NO) and 24-epibrassinolide (EBR) in alleviating chlorosis induced by Fe deficiency in peanut (Arachis hypogaea L.) plants, we determined the concentration of chlorophylls, the activation, uptake, and translocation of Fe, the activities of key enzymes, such as ferric-chelate reductase (FCR), proton-translocating adenosine triphosphatase (H+-ATPase), and antioxidant enzymes, and the accumulation of reactive oxygen species (ROS) and malondialdehyde (MDA) of peanut plants under Fe sufficiency (100 pmol L−1 ethylenediaminetetraacetic acid (EDTA)-Fe) and Fe deficiency (0 µmol L−1 EDTA-Fe). We also investigated the production of NO in peanut plants subjected to Fe deficiency with foliar application of sodium nitroprusside (SNP), a donor of NO, and/or EBR. The results showed that Fe deficiency resulted in severe chlorosis and oxidative stress, significantly decreased the concentration of chlorophylls and active Fe, and significantly increased NO production. Foliar application of NO and/or EBR increased the activity of antioxidant enzymes, superoxide dismutase, peroxidase, and catalase, and decreased the ROS and MDA concentrations, thus enhancing the resistance of plants to oxidative stress. Application of NO also significantly increased Fe translocation from the roots to the shoots and enhanced the transfer of Fe from the cell wall fraction to the cell organelle and soluble fractions. Consequently, the concentrations of available Fe and chlorophylls in the leaves were elevated. Furthermore, the activities of H+-ATPase and FCR were enhanced in the Fe-deficient plants. Simultaneously, there was a significant increase in NO production, especially in the plants that received NO, regardless of Fe supply. These suggest that NO or EBR, and, especially, their combination are effective in alleviating plant chlorosis induced by Fe deficiency.

Stomatal Blue Light Response Is Present in Early Vascular Plants.

Light is a major environmental factor required for stomatal opening. Blue light (BL) induces stomatal opening in higher plants as a signal under the photosynthetic active radiation. The stomatal BL response is not present in the fern species of Polypodiopsida. The acquisition of a stomatal BL response might provide competitive advantages in both the uptake of CO2 and prevention of water loss with the ability to rapidly open and close stomata. We surveyed the stomatal opening in response to strong red light (RL) and weak BL under the RL with gas exchange technique in a diverse selection of plant species from euphyllophytes, including spermatophytes and monilophytes, to lycophytes. We showed the presence of RL-induced stomatal opening in most of these species and found that the BL responses operated in all euphyllophytes except Polypodiopsida. We also confirmed that the stomatal opening in lycophytes, the early vascular plants, is driven by plasma membrane proton-translocating adenosine triphosphatase and K(+) accumulation in guard cells, which is the same mechanism operating in stomata of angiosperms. These results suggest that the early vascular plants respond to both RL and BL and actively regulate stomatal aperture. We also found three plant species that absolutely require BL for both stomatal opening and photosynthetic CO2 fixation, including a gymnosperm, C. revoluta, and the ferns Equisetum hyemale and Psilotum nudum.

A Calcium-Independent Activation of the Arabidopsis SOS2-Like Protein Kinase24 by Its Interacting SOS3-Like Calcium Binding Protein1

The salt stress-induced SALT-OVERLY-SENSITIVE (SOS) pathway in Arabidopsis (Arabidopsis thaliana) involves the perception of a calcium signal by the SOS3 and SOS3-like CALCIUM-BINDING PROTEIN8 (SCaBP8) calcium sensors, which then interact with and activate the SOS2 protein kinase, forming a complex at the plasma membrane that activates the SOS1 Na⁺/H⁺ exchanger. It has recently been reported that phosphorylation of SCaBP proteins by SOS2-like protein kinases (PKSs) stabilizes the interaction between the two proteins as part of a regulatory mechanism that was thought to be common to all SCaBP and PKS proteins. Here, we report the calcium-independent activation of PKS24 by SCaBP1 and show that activation is dependent on interaction of PKS24 with the C-terminal tail of SCaBP1. However, unlike what has been found for other PKS-SCaBP pairs, multiple amino acids in SCaBP1 are phosphorylated by PKS24, and this phosphorylation is dependent on the interaction of the proteins through the PKS24 FISL motif and on the efficient activation of PKS24 by the C-terminal tail of SCaBP1. In addition, we show that Thr-211 and Thr-212, which are not common phosphorylation sites in the conserved PFPF motif found in most SCaBP proteins, are important for this activation. Finally, we also found that SCaBP1-regulated PKS24 kinase activity is important for inactivating the Arabidopsis plasma membrane proton-translocating adenosine triphosphatase. Together, these results suggest the existence of a novel SCaBP-PKS regulatory mechanism in plants.

The Function of Vacuolar ATPase (V-ATPase) a Subunit Isoforms in Invasiveness of MCF10a and MCF10CA1a Human Breast Cancer Cells

The vacuolar H(+) ATPases (V-ATPases) are ATP-driven proton pumps that transport protons across both intracellular and plasma membranes. Previous studies have implicated V-ATPases in the invasiveness of various cancer cell lines. In this study, we evaluated the role of V-ATPases in the invasiveness of two closely matched human breast cancer lines. MCF10a cells are a non-invasive, immortalized breast epithelial cell line, and MCF10CA1a cells are a highly invasive, H-Ras-transformed derivative of MCF10a cells selected for their metastatic potential. Using an in vitro Matrigel assay, MCF10CA1a cells showed a much higher invasion than the parental MCF10a cells. Moreover, this increased invasion was completely sensitive to the specific V-ATPase inhibitor concanamycin. MCF10CA1a cells expressed much higher levels of both a1 and a3 subunit isoforms relative to the parental line. Isoforms of subunit a are responsible for subcellular localization of V-ATPases, with a3 and a4 targeting V-ATPases to the plasma membrane of specialized cells. Knockdown of either a3 alone or a3 and a4 together using isoform-specific siRNAs inhibited invasion by MCF10CA1a cells. Importantly, overexpression of a3 but not the other a subunit isoforms greatly increased the invasiveness of the parental MCF10a cells. Similarly, overexpression of a3 significantly increased expression of V-ATPases at the plasma membrane. These studies suggest that breast tumor cells employ particular a subunit isoforms to target V-ATPases to the plasma membrane, where they function in tumor cell invasion.

A vacuolar-type proton (H +) translocating ATPase α subunit encoded by the Hc- vha- 6 gene of Haemonchus contortus

In the present study, a full-length cDNA (designated Hc- vha- 6) inferred to encode an α subunit of a vacuolar-type proton translocating adenosine triphosphatase (V-ATPase) was isolated from the parasitic nematode Haemonchus contortus, and characterized. The transcript for Hc- vha- 6 was detected in all developmental stages and both sexes of H. contortus. Elements, including two TATA box (TATAA), two inverted CAAT box (ATTGG), five E box (CANNTG) and six GATA as well as five inverse GATA (TTATC) transcription factor motifs, were identified in the non-coding region upstream of Hc- vha- 6. The open reading frame (ORF) of 2601 nucleotides encoded a protein ( Hc-VHA-6) of 866 amino acids and a molecular weight of ∼98.7 kDa. Comparison with a published protein sequence for a homologue (VPH1P) from yeast showed that Hc-VHA-6 had nine transmembrane domains and the 14 essential amino acid residues associated with enzyme activity, assembly, intracellular and/or membrane targeting. Phylogenetic analyses of selected amino acid sequence data revealed Hc-VHA-6 to be most closely related to VHA-6 of Caenorhabditis elegans. A predictive network analysis inferred that vha- 6 interacts with at least seven other genes encoding V-ATPase subunits and a small Rab GTPase. This study provides the first insight into a V-ATPase of parasitic nematodes and a sound basis for future functional genomic work.

A Comprehensive Analysis of the 14-3-3 Interactome in Barley Leaves Using a Complementary Proteomics and Two-Hybrid Approach

This study describes the identification of over 150 target proteins of the five 14-3-3 isoforms in 7-d-old barley (Hordeum vulgare) cv Himalaya seedlings using yeast two-hybrid screens complemented with 14-3-3 protein affinity purification and tandem mass spectrometry. Independent experiments for a subset of genes confirmed the yeast two-hybrid interactions, demonstrating a low false positive identification rate. These combined approaches resulted in the identification of more than 150 putative targets; 15% were previously reported to be 14-3-3 interactors, including, for example, Serpin, RF2A, WPK4 kinase, P-type proton-translocating adenosine triphosphatase, EF1A, glutamine synthetase, and invertases. The affinity purification resulted in 30 interactors, of which 44% function in metabolism, while the yeast two-hybrid screens identified 132 different proteins, with 35% of the proteins involved in signal transduction. A number of proteins have a well-described function in hormonal signaling, such as the auxin transport protein PIN1 and NPH3 and components of the brassinosteroid pathway, such as the receptor kinase BAK1 (OsPERK1) and BRI1-kinase domain-interacting protein 129. However, 14-3-3 interactions with these signal mediators have not been confirmed in the affinity purification. Confirmations of the 14-3-3 interaction with the three ABF-like transcription factors are shown using far western analysis. Also, a REPRESSION OF SHOOT GROWTH ortholog named RF2A was identified; these transcription factors play important roles in the abscisic acid and gibberellin pathways, respectively. We speculate that 14-3-3 proteins have a role in cross talk between these hormonal pathways. The specificity and complementary nature of both the affinity purification and the yeast two-hybrid approaches is discussed.

Mutational Analysis of the Stator Subunit E of the Yeast V-ATPase

Subunit E is a component of the peripheral stalk(s) that couples membrane and peripheral subunits of the V-ATPase complex. In order to elucidate the function of subunit E, site-directed mutations were performed at the amino terminus and carboxyl terminus. Except for S78A and D233A/T202A, which exhibited V(1)V(o) assembly defects, the function of subunit E was resistant to mutations. Most mutations complemented the growth phenotype of vma4Delta mutants, including T6A and D233A, which only had 25% of the wild-type ATPase activity. Residues Ser-78 and Thr-202 were essential for V(1)V(o) assembly and function. The mutation S78A destabilized subunit E and prevented assembly of V(1) subunits at the membranes. Mutant T202A membranes exhibited 2-fold increased V(max) and about 2-fold less of V(1)V(o) assembly; the mutation increased the specific activity of V(1)V(o) by enhancing the k(cat) of the enzyme 4-fold. Reduced levels of V(1)V(o) and V(o) complexes at T202A membranes suggest that the balance between V(1)V(o) and V(o) was not perturbed; instead, cells adjusted the amount of assembled V-ATPase complexes in order to compensate for the enhanced activity. These results indicated communication between subunit E and the catalytic sites at the A(3)B(3) hexamer and suggest potential regulatory roles for the carboxyl end of subunit E. At the carboxyl end, alanine substitution of Asp-233 significantly reduced ATP hydrolysis, although the truncation 229-233Delta and the point mutation K230A did not affect assembly and activity. The implication of these results for the topology and functions of subunit E within the V-ATPase complex are discussed.

Reminiscences of Leon A. Heppel

Reminiscences of Leon A. Heppel

Different roles of proteolipids and 70-kDa subunits of V-ATPase in growth and death of cultured human cells.

The vacuolar-type proton-translocating adenosine triphosphatase (V-ATPase) plays important roles in cell growth and tumour progression. V-ATPase is composed of two distinct structures, a hydrophilic catalytic cytosolic sector (V(1)) and a hydrophobic transmembrane sector (V(0)). The V(1) sector is composed of 5-8 different subunits with the structure A(3)B(3)C(1)D(1)E(1)F(1)G(1)H(1). The V0 sector is composed of 5 different subunits with the structure 1161381191166. The over-expression of 16-kDa proteolipid subunit of V-ATPase in the perinuclear region of the human adventitial fibroblasts promotes phenotypic modulation that contributes to neointimal formation and medial thickening. A relationship between oncogenicity and the expression of the 16-kDa proteolipid has also been suggested in human pancreatic carcinoma tissue. We found that the mRNA levels of the 16-kDa proteolipid but not of the 70-kDa subunit of V-ATPase in human myofibroblasts were more abundant in serum-containing medium (MF(+) cells) than serum-free medium (MF(-) cells). In HeLa cells, the levels of mRNA and protein of the 16-kDa, 21-kDa or 70-kDa were clearly suppressed when the corresponding anti-sense oligonucleotides were administered to the culture medium. The growth rate and viability (mostly due to necrosis) of HeLa cells were reduced markedly by the 16-kDa and 21-kDa anti-sense, but little by the 70-kDa anti-sense, and not at all by any sense oligonucleotides. The localization of 16-kDa/21-kDa proteolipid subunits was different from that of the 70-kDa subunit in HeLa cells. These results suggest that the 16-kDa and 21-kDa proteolipid subunits of the V0 sector play crucial roles in growth and death of cultured human cells. Our results may provide new insights into the mechanism and therapeutic implications for vessel wall hyperplasia and tumorigenesis.

Yeast V-ATPase Complexes Containing Different Isoforms of the 100-kDa a-subunit Differ in Coupling Efficiency and in VivoDissociation

The 100 kDa a-subunit of the yeast vacuolar (H(+))-ATPase (V-ATPase) is encoded by two genes, VPH1 and STV1. These genes encode unique isoforms of the a-subunit that have previously been shown to reside in different intracellular compartments in yeast. Vph1p localizes to the central vacuole, whereas Stv1p is present in some other compartment, possibly the Golgi or endosomes. To compare the properties of V-ATPases containing Vph1p or Stv1p, Stv1p was expressed at higher than normal levels in a strain disrupted in both genes, under which conditions V-ATPase complexes containing Stv1p appear in the vacuole. Complexes containing Stv1p showed lower assembly with the peripheral V(1) domain than did complexes containing Vph1p. When corrected for this lower degree of assembly, however, V-ATPase complexes containing Vph1p and Stv1p had similar kinetic properties. Both exhibited a K(m) for ATP of about 250 microm, and both showed resistance to sodium azide and vanadate and sensitivity to nanomolar concentrations of concanamycin A. Stv1p-containing complexes, however, showed a 4-5-fold lower ratio of proton transport to ATP hydrolysis than Vph1p-containing complexes. We also compared the ability of V-ATPase complexes containing Vph1p or Stv1p to undergo in vivo dissociation in response to glucose depletion. Vph1p-containing complexes present in the vacuole showed dissociation in response to glucose depletion, whereas Stv1p-containing complexes present in their normal intracellular location (Golgi/endosomes) did not. Upon overexpression of Stv1p, Stv1p-containing complexes present in the vacuole showed glucose-dependent dissociation. Blocking delivery of Vph1p-containing complexes to the vacuole in vps21Delta and vps27Delta strains caused partial inhibition of glucose-dependent dissociation. These results suggest that dissociation of the V-ATPase complex in vivo is controlled both by the cellular environment and by the 100-kDa a-subunit isoform present in the complex.

The plasma membrane proton-translocating ATPase.

Living cells require membranes and membrane transporters for the maintenance of life. After decades of biochemical scrutiny, the structures and molecular mechanisms by which membrane transporters catalyze transmembrane solute movements are beginning to be understood. The plasma membrane proton-translocating adenosine triphosphatase (ATPase) is an archetype of the P-type ATPase family of membrane transporters, which are important in a wide variety of cellular processes. The H+-ATPase has been crystallized and its structure determined to a resolution of 8 angstrom in the membrane plane. When considered together with the large body of biochemical information that has been accumulated for this transporter, and for enzymes in general, this new structural information is providing tantalizing insights regarding the molecular mechanism of active ion transport catalyzed by this enzyme.

Cysteine Scanning Mutagenesis of the Noncatalytic Nucleotide Binding Site of the Yeast V-ATPase

To investigate residues involved in the formation of the noncatalytic nucleotide binding sites of the vacuolar proton-translocating adenosine triphosphatase (V-ATPase), cysteine scanning mutagenesis of the VMA2 gene that encodes the B subunit in yeast was performed. Replacement of the single endogenous cysteine residue at position 188 gave rise to a Cys-less form of the B subunit (Vma2p) which had near wild-type levels of activity and which was used in the construction of 16 single cysteine-containing mutants. The ability of adenine nucleotides to prevent reaction of the introduced cysteine residues with the sulfhydryl reagent 3-(N-maleimidopropionyl)biocytin (biotin-maleimide) was evaluated by Western blot. Biotin-maleimide labeling of the purified V-ATPase from the wild-type and the mutants S152C, L178C, N181C, A184C, and T279C was reduced after reaction with the nucleotide analog 3'-O-(4-benzoyl)benzoyladenosine 5'-triphosphate (BzATP). These results suggest the proximity of these residues to the nucleotide binding site on the B subunit. In addition, we have examined the level of endogenous nucleotide bound to the wild-type V-ATPase and to a mutant (the A subunit mutant R483Q) which is postulated to be altered at the noncatalytic site and which displays a marked nonlinearity in ATP hydrolysis (MacLeod, K. J., Vasilyeva, E., Baleja, J. D., and Forgac, M. (1998) J. Biol. Chem. 273, 150-156). The R483Q mutant contained 2.6 mol of ATP/mol of V-ATPase compared with the wild-type enzyme, which contained 0.8 mol of ATP/mol of V-ATPase. These results suggest that binding of additional ATP to the noncatalytic sites may modulate the catalytic activity of the enzyme.

The effect of metabolic acidosis and alkalosis on the H +-ATPase of rat cerebral microvessels

To determine the role of the proton translocating adenosine triphosphatase (H +-ATPase) of the blood-brain barrier, the density of the 31 Kd subunit of the vacuolar type H +-ATPase was quantitated in isolated rat cerebral microvessels with immunoblotting techniques. To establish the tissue specificity of the findings, synaptosomal membranes were also studied. Metabolic acidosis was induced with 1.5% ammonium chloride in drinking water for five days. Metabolic alkalosis was induced with 2.35% NaHCO 3 in drinking water and daily injections of 10 mg Kg furosemide intraperitoneally for 5 days. The quantity of the 31 Kd subunit (in arbitrary units) in cerebral microvessels was significantly increased in acidosis (3.98 ± 0.45) (p < 0.05) and was significantly decreased in metabolic alkalosis (0.49 ± 0.16) (p < 0.00) compared to controls (1.77 ± 0.73). In synaptosomal membranes, metabolic alkalosis was associated with significant decrease in the quantity of the 31 Kd subunit-H +-ATPase (0.62 ± 0.12 vs 0.92 ± 0.01) p < 0.05. The increase in the 31 Kd subunit in synaptosomal membranes with acidosis did not reach statistical significance. It is concluded that the quantity of vacuolar H +-ATPase in the blood-brain barrier is modulated by blood H + or HCO 3 − content. These changes may be relevant to the physiology of the acid-base balance in the central nervous system.

VMA12 Encodes a Yeast Endoplasmic Reticulum Protein Required for Vacuolar H+-ATPase Assembly

The Saccharomyces cerevisiae vacuolar membrane proton-translocating ATPase (V-ATPase) can be divided into a peripheral membrane complex (V1) containing at least eight polypeptides of 69, 60, 54, 42, 32, 27, 14, and 13 kDa, and an integral membrane complex (V0) containing at least five polypeptides of 100, 36, 23, 17, and 16 kDa. Other yeast genes have been identified that are required for V-ATPase assembly but whose protein products do not co-purify with the enzyme complex. One such gene, VMA12, encodes a 25-kDa protein (Vma12p) that is predicted to contain two membrane-spanning domains. Biochemical analysis has revealed that Vma12p behaves as an integral membrane protein with both the N and C termini oriented toward the cytosol, and this protein immunolocalizes to the endoplasmic reticulum (ER). In cells lacking Vma12p (vma12Delta), the 100-kDa subunit of the V0 complex (which contains six to eight putative membrane-spanning domains) was rapidly degraded (t1/2 approximately 30 min). Protease protection assays revealed that the 100-kDa subunit was inserted/translocated correctly into the ER membrane of vma12Delta cells. These data indicate that Vma12p functions in the ER after the insertion of V0 subunits into the ER membrane. We propose that Vma12p functions directly in the assembly of the V0 subunits into a complex in the ER, and that assembly is required for the stability of the V0 subunits and their transport as a complex out of this compartment.

Chromaffin granule membrane glycoprotein IV is identical with Ac45, a membrane-integral subunit of the granule's H +-ATPase

Glycoprotein IV of bovine adrenal chromaffin granule membranes was purified by membrane fractionation with Triton X-114 and lectin affinity chromatography. An antiserum raised against this protein recognized the same component as one directed against subunit Ac45 of the proton-translocating adenosine triphosphatase in the granule membrane. Amino acid sequencing confirmed that glycoprotein IV and Ac45 are identical proteins, and also showed that they are derived from a larger precursor by removal of a 246-amino acid N-terminal sequence. Enzymatic deglycosylation indicated an apparent polypeptide molecular mass of 29 kDa of the mature Ac45/glycoprotein IV. Blue Native electrophoresis confirmed that this protein is a component of the membrane sector of the V-ATPase.

Analysis of nucleotide binding by a vacuolar proton-translocating adenosine triphosphatase.

The vacuolar-type proton-translocatine adenosine triphosphatase from bovine adrenal secretory granules (chromaffin granules) was purified and reconstituted into proteoliposomes. The binding of nucleotides to the enzyme was studied by quantifying their effects on the rate of inactivation by N-ethylmaleimide (MalNEt) of ATP-dependent proton translocation, and by direct measurement of the binding of [3H]MgADP. The results of these experiments are consistent with a model of the enzyme that had been developed as a result of kinetic experiments, the features of which are that the enzyme exists in two states, each containing three nucleotide-binding sites on catalytic subunits, and that nucleoside diphosphates regulate the enzyme by binding with high affinity to a single site in the inactive T state of the enzyme. Under the conditions of the experiments, MalNEt inactivated the ATPase in a pseudo-first order reaction. Rate constants of inactivation were reduced in the presence of MgADP, MgIDP and free ADP; the kinetics of protection suggested that the two conformational states of the enzyme were inactivated at different rates and also confirmed the existence of two different types of binding site for MgADP. Low nucleotide concentrations afforded partial protection from MalNEt; this was ascribed to binding of nucleotide to the regulatory site causing a shift in the conformational equilibrium towards the T state, which was more slowly inactivated than the unliganded R state of the enzyme. At higher nucleotide concentrations, binding at the catalytic site afforded complete protection from MalNEt. Protection by MgADP[S] and magnesium 2'- and 3'-O-[4-benzoylbenzoyl]adenosine 5'-triphosphate showed simpler kinetics but was also consistent with previously reported kinetic results. Analysis of subunit labelling with [3H]MalNEt showed that the three 72-kDa (catalytic) subunits were alkylated by MalNEt with similar rate constants, consistent with a symmetrical arrangement of the catalytic subunits, in contrast to the situation in F-type ATPases. Analysis of the binding of [3H]MgADP also confirmed the results of kinetic experiments. MgADP was shown to bind to the enzyme with an apparent dissociation constant of about 66 nM; assuming that the nucleotide binds only to the T-state, the true dissociation constant is < 1 nM. Using Blue Native polyacrylamide gel electrophoresis to separate the holo-ATPase from the membrane sector, the stoichiometry of binding was calculated to be 0.6 mol/mol enzyme, confirming the existence of a single regulatory site for MgADP. However, binding of MgADP to the enzyme was much slower than could be accounted for by the measured dissociation constants, suggesting that it is rate limited by a step such as a protein conformational change. Treatment designed to remove endogenous nucleotide had no effect on the rate or extent of binding of MgADP.