Identification of the major cytoplasmic regions of the Neurospora crassa plasma membrane H(+)-ATPase using protein chemical techniques

The transmembrane topography of the Neurospora crassa plasma membrane H(+)-ATPase has been investigated using purified, reconstituted components and direct protein chemical techniques. Reconstituted proteoliposomes containing H(+)-ATPase molecules oriented predominantly with their cytoplasmic surface facing outward were treated with trypsin to liberate peptides present on the cytoplasmic surface of the H(+)-ATPase as recently described (Hennessey, J.P., Jr., and Scarborough, G. (1990) J. Biol. Chem. 265, 532-537. The released peptides were then separated from the proteoliposomes by gel filtration chromatography and further purified by high performance liquid chromatography. Fourteen such peptides were identified by NH2-terminal amino acid sequence analysis, directly defining these parts of the molecule as present on the cytoplasmic surface of the membrane. Moreover, this information identified several additional flanking stretches as likely to be cytoplasmically located by virtue of the fact that they are too short to cross the membrane and return. These results and the results of other recent experiments establish 417 residues of the 919 present in the ATPase molecule, at positions 2-100, 186-256, 441-663, and 897-920, as cytoplasmically located. Taken together with the results of our preliminary investigations of the membrane embedded sectors of the ATPase, this information allows the formulation of a reasonably detailed model for the transmembrane topography of the ATPase polypeptide chain.     

Biogenesis of mitochondria: Defective yeast H+-ATPase assembled in the absence of mitochondrial protein synthesis is membrane associated

We have investigated the extent to which the assembly of the cytoplasmically synthesized subunits of the H⁺-ATPase can proceed in a mtDNA-less (rho°) strain of yeast, which is not capable of mitochondrial protein synthesis. Three of the membrane sector proteins of the yeast H⁺-ATPase are synthesized in the mitochondria, and it is important to determine whether the presence of these subunits is essential for the assembly of the imported subunits to the inner mitochondrial membrane. A monoclonal antibody against the cytoplasmically synthesized -subunit of the H⁺-ATPase was used to immunoprecipitate the assembled subunits of the enzyme complex. Our results indicate that the imported subunits of the H⁺-ATPase can be assembled in this mutant, into a defective complex which could be shown to be associated with the mitochondrial membrane by the analysis of the Arrhenius kinetics of the mutant mitochondrial ATPase activity.This paper is No. 61 in the seriesBiogenesis of Mitochondria. For paper No. 60, see Novitskiet al. (1984).     

Cytoplasmic location of amino acids 359-440 of the Neurospora crassa plasma membrane H(+)-ATPase.

The topographic location of the region comprising amino acids 359-440 of the Neurospora crassa plasma membrane H(+)-ATPase has been elucidated using reconstituted proteoliposomes and protein chemical techniques. Proteoliposomes containing H(+)-ATPase molecules oriented predominantly with their cytoplasmic surface facing outward were cleaved with trypsin and the resulting digest was subjected to centrifugation on a glycerol step gradient to separate the released and liposome-bound peptides. The released peptides were recovered in the upper regions of the step gradient, whereas the liposome-bound peptides were recovered near the 40% glycerol interface. The released peptides present in the upper fractions were reduced, 14C-carboxy-methylated, and then separated by high performance liquid chromatography. Two radioactive cysteine-containing peptides with retention times of about 162 and 182 min were identified as H(+)-ATPase peptides comprising residues Leu363-Lys379 and Leu388-Arg414, respectively, by comparison to standards prepared from the purified ATPase. This information thus establishes a cytoplasmic location for residues 359-418 in the H(+)-ATPase polypeptide chain. It also infers a cytoplasmic location for residues 419-440, since this stretch of amino acids is too short to cross the membrane and return between regions known to be cytoplasmically located. These results and the results of other recent experiments establish the topographical location of nearly all of the 919 residues in the H(+)-ATPase molecule.     

Direct evidence for the cytoplasmic location of the NH2- and COOH-terminal ends of the Neurospora crassa plasma membrane H+-ATPase

Reconstituted proteoliposomes containing Neurospora plasma membrane H+-ATPase molecules oriented predominantly with their cytoplasmic portion facing outward have been used to determine the location of the NH2 and COOH termini of the H+-ATPase relative to the lipid bilayer. Treatment of the proteoliposomes with trypsin in the presence of the H+-ATPase ligands Mg2+, ATP, and vanadate produces approximately 97-, 95-, and 88-kDa truncated forms of the H+-ATPase similar to those already known to result from cleavage at Lys24, Lys36, and Arg73 at the NH2-terminal end of the molecule. These results establish that the NH2-terminal end of the H+-ATPase polypeptide chain is located on the cytoplasmic side of the membrane. Treatment of the same proteoliposome preparation with trypsin in the absence of ligands releases approximately 50 water-soluble peptides from the proteoliposomes. Separation of the released peptides by high performance liquid chromatography and spectral analysis of the purified peptides identified only a few peptides with the properties expected of a COOH-terminal, tryptic undecapeptide with the sequence SLEDFVVSLQR, and NH2-terminal amino acid sequence analysis identified this peptide among the possible candidates. Quantitative considerations indicate that this peptide must have come from H+-ATPase molecules oriented with their cytoplasmic portion facing outward, and could not have originated from a minor population of H+-ATPase molecules of reverse orientation. These results directly establish that the COOH-terminal end of the H+-ATPase is also located on the cytoplasmic side of the membrane. These findings are important for elucidating the topography of the membrane-bound H+-ATPase and are possibly relevant to the topography of other aspartyl-phosphoryl-enzyme intermediate ATPases as well.     

Functional Identification of H<Superscript>+</Superscript>-ATPase and Na<Superscript>+</Superscript>/H<Superscript>+</Superscript> Antiporter in the Plasma Membrane Isolated from the Root Cells of Salt-Accumulating Halophyte <Emphasis Type="Italic">Suaeda altissima</Emphasis>

A membrane fraction enriched in plasma membrane (PM) vesicles was isolated from the root cells of a salt-accumulating halophyte Suaeda altissima (L.) Pall. by means of centrifugation in discontinuous sucrose density gradient. The PM vesicles were capable of generating ΔpH at their membrane and the transmembrane electric potential difference (Δψ). These quantities were measured with optical probes, acridine orange and oxonol VI, sensitive to ΔpH and Δψ, respectively. The ATP-dependent generation of ΔpH was sensitive to vanadate, an inhibitor of P-type ATPases. The results contain evidence for the functioning of H⁺-ATPase in the PM of the root cells of S. altissima. The addition of Na⁺ and Li⁺ ions to the outer medium resulted in dissipation of ΔpH preformed by the H⁺-ATPase, which indicates the presence in PM of the functionally active Na⁺/H⁺ antiporter. The results are discussed with regard to involvement of the Na⁺/H⁺ antiporter and the PM H⁺-ATPase in loading Na⁺ ions into the xylem of S. altissima roots.     

V H-ATPase along the yeast secretory pathway: Energization of the ER and Golgi membranes

H⁺ transport driven by V H⁺-ATPase was found in membrane fractions enriched with ER/PM and Golgi/Golgi-like membranes of Saccharomyces cerevisiae efficiently purified in sucrose density gradient from the vacuolar membranes according to the determination of the respective markers including vacuolar Ca²⁺-ATPase, Pmc1::HA. Purification of ER from PM by a removal of PM modified with concanavalin A reduced H⁺ transport activity of P H⁺-ATPase by more than 75% while that of V H⁺-ATPase remained unchanged. ER H⁺ ATPase exhibits higher resistance to bafilomycin (I₅₀ = 38.4 nM) than Golgi and vacuole pumps (I₅₀ = 0.18 nM). The ratio between a coupling efficiency of the pumps in ER, membranes heavier than ER, vacuoles and Golgi is 1.0, 2.1, 8.5 and 14 with the highest coupling in the Golgi. The comparative analysis of the initial velocities of H⁺ transport mediated by V H⁺-ATPases in the ER, Golgi and vacuole membrane vesicles, and immunoreactivity of the catalytic subunit A and regulatory subunit B further supported the conclusion that V H⁺-ATPase is the intrinsic enzyme of the yeast ER and Golgi and likely presented by distinct forms and/or selectively regulated.     

Nitric oxide enhances salt tolerance in maize seedlings through increasing activities of proton-pump and Na+/H+ antiport in the tonoplast

Nitric oxide (NO), an endogenous signaling molecule in animals and plants, mediates responses to abiotic and biotic stresses. Our previous work demonstrated that 100 μM sodium nitroprusside (SNP, an NO donor) treatment of maize seedlings increased K⁺ accumulation in roots, leaves and sheathes, while decreasing Na⁺ accumulation (Zhang et al. in J Plant Physiol Mol Biol 30:455–459, 2004b). Here we investigate how NO regulates Na⁺, K⁺ ion homeostasis in maize. Pre-treatment with 100 μM SNP for 2 days improved later growth of maize plants under 100 mM NaCl stress, as indicated by increased dry matter accumulation, increased chlorophyll content, and decreased membrane leakage from leaf cells. An NO scavenger, methylene blue (MB-1), blocked the effect of SNP. These results indicated that SNP-derived NO enhanced maize tolerance to salt stress. Further analysis showed that NaCl induced a transient increase in the NO level in maize leaves. Both NO and NaCl treatment stimulated vacuolar H⁺-ATPase and H⁺-PPase activities, resulting in increased H⁺-translocation and Na⁺/H⁺ exchange. NaCl-induced H⁺-ATPase and H⁺-PPase activities were diminished by MB-1. 1-Butanol, an inhibitor of phosphatidic acid (PA) production by phospholipase D (PLD), reduced NaCl- and NO-induced H⁺-ATPase activation. In contrast, applied PA stimulated H⁺-ATPase activity. These results suggest that NO acts as a signal molecule in the NaCl response by increasing the activities of vacuolar H⁺-ATPase and H⁺-PPase, which provide the driving force for Na⁺/H⁺ exchange. PLD and PA play an important role in this process.     

Application of three-dimensional molecular hydrophobicity potential to the analysis of spatial organization of membrane domains in proteins: I. Hydrophobic properties of transmembrane segments of Na+, K+-ATPase

A new computer-aided molecular modeling approach based on the concept of three-dimensional (3D) molecular hydrophobicity potential has been developed to calculate the spatial organization of intramembrane domains in proteins. The method has been tested by calculating the arrangement of membrane-spanning segments in the photoreaction center ofRhodopseudomonas viridis and comparing the results obtained with those derived from the X-ray data. We have applied this computational procedure to the analysis of interhelical packing in membrane moiety of Na⁺, K⁺-ATPase. The work consists of three parts. In Part I, 3D distributions of electrostatic and molecular hydrophobicity potentials on the surfaces of transmembrane helical peptides were computed and visualized. The hydrophobic and electrostatic properties of helices are discussed from the point of view of their possible arrangement within the protein molecule. Interlocation of helical segments connected with short extramembrane loops found by means of optimization of their hydrophobic/hydrophilic contacts is considered in Part II. The most probable 3D model of packing of helical peptides in the membrane domain of Na⁺, K⁺-ATPase is discussed in the final part of the work.     

Application of three-dimensional molecular hydrophobicity potential to the analysis of spatial organization of membrane domains in proteins: I. Hydrophobic properties of transmembrane segments of Na+, K+-ATPase

A new computer-aided molecular modeling approach based on the concept of three-dimensional (3D) molecular hydrophobicity potential has been developed to calculate the spatial organization of intramembrane domains in proteins. The method has been tested by calculating the arrangement of membrane-spanning segments in the photoreaction center ofRhodopseudomonas viridis and comparing the results obtained with those derived from the X-ray data. We have applied this computational procedure to the analysis of interhelical packing in membrane moiety of Na⁺, K⁺-ATPase. The work consists of three parts. In Part I, 3D distributions of electrostatic and molecular hydrophobicity potentials on the surfaces of transmembrane helical peptides were computed and visualized. The hydrophobic and electrostatic properties of helices are discussed from the point of view of their possible arrangement within the protein molecule. Interlocation of helical segments connected with short extramembrane loops found by means of optimization of their hydrophobic/hydrophilic contacts is considered in Part II. The most probable 3D model of packing of helical peptides in the membrane domain of Na⁺, K⁺-ATPase is discussed in the final part of the work.     

Ultracytochemical study of Ca++-ATPase and K+-NPPase activities in retinal photoreceptors of the guinea pig

Summary Ca⁺⁺-ATPase activity was demonstrated histochemically at light- and electron-microscopic levels in inner and outer segments of retinal photoreceptor cells of the guinea pig with the use of a newly developed one-step lead-citrate method (Ando et al. 1981). The localization of ouabain-sensitive, K⁺-dependent p-nitrophenylphosphatase (K⁺-NPPase) activity, which represents the second dephosphorylative step of the Na⁺-K⁺-ATPase system, was studied by use of the one-step method newly adapted for ultracytochemistry (Mayahara et al. 1980). In retinal photoreceptor cells fixed for 15 min in 2% paraformaldehyde the electron-dense Ca⁺⁺-ATPase reaction product accumulated significantly on the inner membranes of the mitochondria but not on the plasmalemma or other cytoplasmic elements of the inner segments. The membranes of the outer segments remained unstained except the membrane arrays in close apposition to the retinal pigment epithelium. The cytochemical reaction was Ca⁺⁺- and substrate-dependent and showed sensitivity to oligomycin. When Mg⁺⁺-ions were used instead of Ca⁺⁺-ions, a distinct reaction was also found on mitochondrial inner membranes.In contrast to the localization of the Ca⁺⁺ -ATPase activity, the K⁺-NPPase activity was demonstrated only on the plasmalemma of the inner segments, but not on the mitochondria, other cytoplasmic elements or the outer segment membranes. This reaction was almost completely abolished by ouabain or by elimination of K⁺ from the incubation medium.Fellow of the Alexander von Humboldt Foundation, Bonn, Federal Republic of Germany     

Immunocytochemical localization of Na+/K+-ATPase and V-H+-ATPase in the salivary glands of the cockroach,Periplaneta americana

The acinous salivary glands of the cockroach (Periplaneta americana) consist of four morphologically different cell types with different functions: the peripheral cells are thought to produce the fluid component of the primary saliva, the central cells secrete the proteinaceous components, the inner acinar duct cells stabilize the acini and secrete a cuticular, intima, whereas the distal duct cells modify the primary saliva via the transport of water and electrolytes. Because there is no direct information available on the distribution of ion transporting enzymes in the salivary glands, we have mapped the distribution of two key transport enzymes, the Na⁺/K⁺-ATPase (sodium pump) and a vacuolar-type H⁺-ATPase, by immunocytochemical techniques. In the peripheral cells, the Na⁺/K⁺-ATPase is localized to the highly infolded apical membrane surface. The distal duct cells show large numbers of sodium pumps localized to the basolateral part of their plasma membrane, whereas their highly folded apical membranes have a vacuolar-type H⁺-ATPase. Our immunocytochemical data are supported by conventional electron microscopy, which shows electrondense 10-nm particles (portasomes) on the cytoplasmic surface of the infoldings of the apical membranes of the distal duct cells. The apically localized Na⁺/K⁺-ATPase in the peripheral cells is probably directly involved in the formation of the Na⁺-rich primary saliva. The latter is modified by the distal duct cells by transport mechanisms energized by the proton motive force of the apically localized V-H⁺-ATPase.     

Evolutionary appearance of the plasma membrane H+-ATPase containing a penultimate threonine in the bryophyte

The plasma membrane H⁺-ATPase provides the driving force for solute transport via an electrochemical gradient of H⁺ across the plasma membrane, and regulates pH homeostasis and membrane potential in plant cells. However, the plasma membrane H⁺-ATPase in non-vascular plant bryophyte is largely unknown. Here, we show that the moss Physcomitrella patens, which is known as a model bryophyte, expresses both the penultimate Thr-containing H⁺-ATPase (pT H⁺-ATPase) and non-pT H⁺-ATPase as in the green algae, and that pT H⁺-ATPase is regulated by phosphorylation of its penultimate Thr. A search in the P. patens genome database revealed seven H⁺-ATPase genes, designated PpHA (Physcomitrella patens H⁺-ATPase). Six isoforms are the pT H⁺-ATPase; a remaining isoform is non-pT H⁺-ATPase. An apparent 95-kD protein was recognized by anti-H⁺-ATPase antibodies against an isoform of Arabidopsis thaliana and was phosphorylated on the penultimate Thr in response to a fungal toxin fusicoccin and light in protonemata, indicating that the 95-kD protein contains pT H⁺-ATPase. Furthermore, we could not detect the pT H⁺-ATPase in the charophyte alga Chara braunii, which is the closest relative of the land plants, by immunological methods. These results strongly suggest the pT H⁺-ATPase most likely appeared for the first time in bryophyte.     

The possible role of redox-associated protons in growth of plant cells

The protons excreted by plant cells may arise by two different mechanisms: (1) by the action of the plasma membrane H⁺-ATPase and (2) by plasma membrane redox reactions. The exact proportion from each source is not known, but the plasma membrane H⁺-ATPase is, by far, the major contributor to proton efflux. There is still some question of whether the redox-associated protons produced by NADH oxidation on the inner side of the plasma membrane traverse the membrane in a 1 : 1 relationship with electrons generated in the redox reactions. Membrane depolarization observed in the presence of ferricyanide reduction by plasma membranes of whole cells or tissues or the lag period between ferricyanide reduction and medium acidification argue that only scalar protons may be involved. The other major argument against tight coupling between protons and electrons involves the concept of strong charge compensation. When ferricyanide is reduced to ferrocyanide on the outside of cells or tissues, an extra negative charge arises, which is compensated for by the release of H⁺ or K⁺, so that the total ratio of increased H⁺ plus K⁺ equals the electrons transferred by transmembrane electron transport. These are strong arguments against a tight coupling between electrons and protons excreted by the plasma membrane. On the other hand, there is no question that inhibitor studies provide evidence for two mechanisms of proton generation by plasma membranes. When the H⁺-ATPase activity is totally inhibited, the addition of ferricyanide induces a burst of extra proton excretion, orvice versa, when plasma membrane redox reactions are inhibited, the H⁺-ATPase can function normally. Since plasma membrane redox reactions and associated H⁺ excretion are related to growth, it is possible that in plants the ATPase-generated protons have a different function from redox-associated protons. The H⁺-ATPase-generated protons have been considered for many years to be necessary for cell wall expansion, allowing elongation to take place. A special function of the redox-generated protons may be in initiating proliferative cell growth, based on the presence of a hormone-stimulated NADH oxidase in membranes of soybean hypocotyls and stimulation of root growth by low concentrations of oxidants. Here we propose that this NADH oxidase and the redox protons released by its action control growth. The mechanism for this may be the evolution of protons into a special membrane domain, from which a signal to initiate cell proliferation may originate, independent of the action of the H⁺-ATPase-generated protons. It is also possible that both expansion and proliferative growth are controlled by redox-generated protons.     

Reinvestigation of auxin and fusicoccin stimulation of the plasma-membrane H+-ATPase activity

In vivo treatment of maize (Zea mays L.) coleoptile segments with auxin (indole-3-acetic acid; IAA) and fusicoccin (FC) followed by plasma-membrane isolation was used to characterize the effects of these treatments on the plasma-membrane H⁺-ATPase. Both IAA and FC increased H⁺ extrusion and elongation rate of the coleoptile segments, FC more strongly than IAA. Plasma membranes isolated after in-vivo treatment with FC showed a twofold stimulation of ATP hydrolysis and a several-fold stimulation of H⁺ pumping, whereas no effect was observed after IAA treatment, irrespective of whether the plasma membranes were prepared by two-phase partitioning or sucrose-gradient centrifugation. A more detailed investigation of the kinetic properties and pH dependence of the enzyme showed that FC treatment led to a twofold increase in V _max, a decrease in K _m for ATP from 1.5 mM to 0.24 mM, and a change in pH dependence resulting in increased activity at physiological pH levels. Again, IAA treatment showed no effects. Quantitation of the H⁺-ATPase by immunostaining using four different antibodies revealed no difference between IAA-and FC-treated material, and controls. From these data we conclude that (i) neither IAA nor FC gives rise to an increase in the amount of H⁺ -ATPase molecules in the plasma membrane that can be detected after membrane isolation, and (ii) if the H⁺-ATPase is activated by IAA, this activation is, in contrast to FC activation, not detectable after membrane isolation.Abbreviations BTP 1,3-bis(tris[hydroxymethyl]methylamino)-propane - FC fusicoccin - lyso-PC lysophosphatidylcholine - Mes 2-(N-morpholino)ethanesulfonic acid This paper is dedicated to Prof. Dieter Klämbt on the occasion of his 65th birthdayWe thank Ann-Christine Holmström and Adine Karlsson for excellent technical assistance, Professor Ramón Serrano (Instituto de Biologia Molecular y Celular de Plantas, UPV-CSIC, Universidad Politecnica, Valencia, Spain) for a generous gift of antisera to the H⁺-ATPase and Professor Wolfgang Michalke (Institut für Biologie III, Albert-Ludwigs-Universität, Freiburg, Germany) for kindly providing the monoclonal antibody to the H⁺-ATPase. This work was supported by the Swedish Natural Science Research Council, the Deutsche Agentur für Raumfahrtangelegenheiten (DARA, Bonn) via AGRAVIS (Bonn) and by the Ministerium für Wissenschaft und Forschung (MWF, Düsseldorf). Thomas Jahn received scholarships from the Deutsche Graduiertenförderung des Landes Nordrhein-Westfalen and the Deutscher Akademischer Austauschdienst (DAAD, Bonn).     

Characterization of Syrian hamster gastric mucosal H+,K+-ATPase

Employing a simple one-step sucrose gradient fractionation method, gastric mucosal membrane of Syrian hamster was prepared and demonstrated to be specifically enriched in H⁺,K⁺-ATPase activity. The preparation is practically devoid of other ATP hydrolyzing activity and contains high K⁺-stimulated ATPase, activity of at least 4–5 fold compared to basal ATPase activity. The H⁺,K⁺-ATPase showed hydroxylamine-sensitive phosphorylation and K⁺-dependent dephosphorylation of the phospho-enzyme, characteristic inhibition by vanadate, omeprazole and SCH 28080, and nigericin-reversible K⁺-dependent H⁺-transport — properties characteristic of gastric proton pump One notable difference with H⁺,K⁺-ATPase of other species has been the observation of valinomycin-independent H⁺ transport in such membrane vesicles. It is proposed that such H⁺,K⁺-ATPase-rich hamster gastric mucosal membrane preparation might provide a unique model to study physiological aspects of H⁺,K⁺-ATPase-function in relation to HCl secretion.     

Lack of immunological cross reactivity between the transport enzymes (Na+ + K+)-ATPase and (K+ + H+)-ATPase

Goat antisera against (Na⁺ + K⁺)-ATPase and its isolated subunits and against (K⁺ + H⁺)-ATPase have been prepared in order to test for immune cross-reactivity between the two enzymes, whose catalytic subunits show great chemical similarity. None of the (Na⁺ + K⁺)-ATPase antisera cross-reacted with (K⁺ + H⁺)-ATPase or inhibited its enzyme activity. The same was true for the (K⁺ + H⁺)-ATPase antiserum with regard to (Na⁺ + K⁺)-ATPase and its subunits and its enzyme activity. So not withstanding the chemical similarity of their subunits, there is no immunological cross-reactivity between these two plasma membrane ATPases.Number LIII in the series Studies on (Na⁺ + K⁺)-Activated ATPase.     

Identification and biochemical characterization of the plasma-membrane H+-ATPase in guard cells of Vicia faba L.

The plasma-membrane H⁺-pump in guard cells generates the driving force for the rapid ion fluxes required for stomatal opening. Since our electrophysio-logical studies revealed a two fold higher pump-current density in guard cells than in mesophyll cells of Vicia faba L. we elucidated the biochemical properties of this proton-translocating ATPase in plasma-membrane vesicles isolated from both cell types. The capability of the H⁺ —ATPase to create an H⁺ gradient is maintained in plasma-membrane vesicles derived from purified guard cells via blender maceration, high-pressure homogenization and polymer separation. The H⁺-pumping activity of these vesicles coincides with the presence of two polypeptides of approx. 100 and 92 kDa which are recognized by a monoclonal antibody raised against the plasma-membrane H⁺-ATPase from Zea mays L. coleoptiles. Comparison of H⁺-pumping activities of isolated membranes revealed an approximately two fold higher activity in guard cells than in mesophyll cells with respect to the total membrane protein content. Furthermore, we demonstrated by western blotting that the difference in pump activities resulted from a higher abundance of the electroenzyme per unit membrane protein in guard-cell plasma membranes. We suggest that the high H⁺-pump capacity is necessary to enable guard cells to respond to sudden changes in the environment by a change in stomatal aperture.     

Partial purification and characterization of (Na++K+)-ATPase from garfish olfactory nerve axon plasma membrane

Summary The (Na⁺+K⁺)-ATPase of garfish olfactory nerve axon plasma membrane was purified about sixfold by treatment of the membrane with sodium dodecyl sulfate followed by sucrose density gradient centrifugation. The estimated molecular weights of the two major polypeptide components of the enzyme preparation on sodium dodecyl sulfate gels were 110,000 and 42,000 daltons, which were different from those of the corresponding peptides of rabbit kidney (Na⁺+K⁺)-ATPase. No carbohydrate was detected in the 42,000-dalton component either by the periodic acid-Schiff reagent or by the more sensitive concanavalin A-peroxidase staining procedure. The molecular properties of the garfish (Na⁺+K⁺)-ATPase, such as theK _m for ATP, pH optimum, energies of activation, Na and K ion dependence and vanadium inhibition, were, however, similar to those of the kidney enzyme.The partially purified garfish (Na⁺+K⁺)-ATPase was reconstituted into phospholipid vesicles by a freeze-thaw-sonication procedure. The reconstituted enzyme was found to catalyze a time and ATP dependent²²Na⁺ transport. The ratio of²²Na⁺ pumped to ATP hydrolyzed was about 1; under the same reconstitution and assay conditions, eel electroplax (Na⁺+K⁺)-ATPase, however, gave a²²Na⁺ pumped to ATP hydrolyzed ratio of nearly 3.     

Drought stress stimulates p-nitrophenyl phosphate hydrolysis rate of the plasma membrane H+-ATPase from wheat leaves

The influence of drought stress on the ATP and p-nitrophenyl phosphate (PNPP) hydrolysis activity by plasma membrane H⁺-ATPase was investigated using purified plasma membrane vesicles from wheat leaves by two-phase partitioning. Drought stress increased the ATPase activity, and the optimal pH was shifted from 6.5 to about 7.0. Drought stress also stimulated the PNPP hydrolysis rate. The K_m for PNPP hydrolysis was moved from 4.49 ± 0.33 mM to 3.64 ± 0.12 mM. In addition, the PNPP hydrolysis was more sensitive to vanadate under drought compared to the control. However, the inhibitory effect of hydroxylamine on the ATPase was not changed by the present drought stress. In addtion, drought stress also decreased the trypsin activation of PNPP hydrolysis by PM H⁺-ATPase. These results suggested that drought stress altered the catalytic mechanism of the plasma membrane H⁺-ATPase, and the stimulation of its activity by drought stress was mainly due to increase of the catalytic activity of its phosphatase domain. It is also suggested that drought stress might alter the structure or property of the C-terminal end of PM H⁺-ATPase, therefore increasing the catalytic activity of the phosphatase domain.     

Characterization of a plasma membrane H+-ATPase from the extremely acidophilic algaDunaliella acidophila

Summary Dunaliella acidophila is an unicellular green alga which grows optimally at pH 0–1 while maintaining neutral internal pH. A plasma membrane preparation of this algae has been purified on sucrose density gradients. The preparation exhibits vanadatesensitive ATPase activity of 2 mol P_i/mg protein/min, an activity 15 to 30-fold higher than that in the related neutrophilic speciesD. salina. The following properties suggest that the ATPase is an electrogenic plasma membrane H⁺ pump. (i) ATP induces proton uptake and generates a positive-inside membrane potential as demonstrated with optical probes. (ii) ATP hydrolysis and proton uptake are inhibited by vanadate, diethylstilbestrol, dicyclohexylcarbodiimide and erythrosine but not by molybdate, azide or nitrate. (iii) ATP hydrolysis and proton uptake are stimulated by fussicoccin in a pH-dependent manner as found for plants plasma membrane H⁺-ATPase. Unusual properties of this enzyme are: (i) theK _m for ATP is around 60 M, considerably lower than in other plasma membrane H⁺-ATPases, and (ii) the ATPase activity and proton uptake are stimulated three to fourfold by K⁺ and to a smaller extent by other monovalent cations. These results suggest thatD. acidophila possesses a vanadate-sensitive H⁺-ATPase with unusual features enabling it to maintain the large transmembrane pH gradient.