Plasma Membrane ATPase Activity following Reversible and Irreversible Freezing Injury

Plasma membrane ATPase has been proposed as a site of functional alteration during early stages of freezing injury. To test this, plasma membrane was purified from Solanum leaflets by a single step partitioning of microsomes in a dextran-polyethylene glycol two phase system. Addition of lysolecithin in the ATPase assay produced up to 10-fold increase in ATPase activity. ATPase activity was specific for ATP with a K_m around 0.4 millimolar. Presence of the ATPase enzyme was identified by immunoblotting with oat ATPase antibodies. Using the phase partitioning method, plasma membrane was isolated from Solanum commersonii leaflets which had four different degrees of freezing damage, namely, slight (reversible), partial (partially reversible), substantial and total (irreversible). With slight (reversible) damage the plasma membrane ATPase specific activity increased 1.5- to 2-fold and its K_m was decreased by about 3-fold, whereas the specific activity of cytochrome c reductase and cytochrome c oxidase in the microsomes were not different from the control. However, with substantial (lethal, irreversible) damage, there was a loss of membrane protein, decrease in plasma membrane ATPase specific activity and decrease in K_m, while cytochrome c oxidase and cytochrome c reductase were unaffected. These results support the hypothesis that plasma membrane ATPase is altered by slight freeze-thaw stress.     

A loss in the plasma membrane ATPase activity and its recovery coincides with incipient freeze-thaw injury and postthaw recovery in onion bulb scale tissue

Plasma membrane ATPase has been proposed to be functionally altered during early stages of injury caused by a freeze-thaw stress. Complete recovery from freezing injury in onion cells during the postthaw period provided evidence in support of this proposal. During recovery, a simultaneous decrease in ion leakage and disappearance of water soaking (symptoms of freeze-thaw injury) has been noted. Since reabsorption of ions during recovery must be an active process, recovery of plasma membrane ATPase (active transport system) functions has been implicated. In the present study, onion (Allium cepa L. cv Downing Yellow Globe) bulbs were subjected to a freeze-thaw stress which resulted in a reversible (recoverable) injury. Plasma membrane ATPase activity in the microsomes (isolated from the bulb scales) and ion leakage rate (efflux/hour) from the same scale tissue were measured immediately following thawing and after complete recovery. In injured tissue (30-40% water soaking), plasma membrane ATPase activity was reduced by about 30% and this was paralleled by about 25% higher ion leakage rate. As water soaking disappeared during recovery, the plasma membrane ATPase activity and the ion leakage rate returned to about the same level as the respective controls. Treatment of freeze-thaw injured tissue with vanadate, a specific inhibitor of plasma membrane ATPase, during postthaw prevented the recovery process. These results indicate that recovery of freeze-injured tissue depends on the functional activity of plasma membrane ATPase.     

Cl- -HCO3- dependent ATPase in gills of freshwater and seawater adapted eels (Anguilla anguilla L.)

The gills of both seawater and freshwater adapted eels have an ATPase activity which is stimulated by anions in the presence of Mg2+. Plasma membranes were distinguished from mitochondrial membranes with specific enzyme markers, the membrane fractions separated on a discontinuous sucrose gradient, and the ATPase activity of the plasma membranes studied. Activation by the anions of Cl- or HCO3- followed Michaelis-Menten kinetics and was competitively inhibited by SCN-. The Cl- and HCO3- activation characteristics were determined: no differences between the plasma membrane ATPase activities of freshwater and seawater-adapted fishes were observed. Maximal activity measurements after solubilization of the enzymes by Triton X 100 confirmed these findings. The function of a membrane anion-dependent ATPase in the brachial epithelium of euryhaline fish is discussed.     

Characterization of the plasma membrane ATPase of Candida tropicalis

1) Plasma membrane vesicles from Candida tropicalis were isolated from protoplasts by differential centrifugation and purified in a continuous sucrose gradient. 2) The plasma membrane bound ATPase was characterized. It is highly specific for ATP and requires Mg2+. It is stimulated by K+, Na+ and NH4+. Lineweaver-Burk plots for ATPase activity are linear with a Vmax of 4.2 mumoles of ATP hydrolyzed min-1.mg-1 protein and a Km for ATP of 0.76 mM. The ATPase activity is inhibited competitively by ADP with a Ki of 1.7 mM and non competitively by vanadate with a Ki of 3 microM. The activity is unaffected by oligomycin or azide but is sensitive to DCCD.     

ATPase activity of partially purified P-glycoprotein from multidrug-resistant Chinese hamster ovary cells.

In vitro studies of multidrug-resistant cell lines have shown that a membrane protein, the P-glycoprotein, is responsible for resistance to a wide range of structurally and functionally dissimilar anti-cancer drugs. The amino-acid sequence of P-glycoprotein (Pgp) indicates two consensus sequences for ATP binding and the purified protein has been reported to possess a low level of ATPase activity. As part of our goal to further characterize the ATPase activity of P-glycoprotein, we have developed a procedure for rapid partial purification of the protein in a highly active form. Plasma membrane vesicles from multidrug-resistant CHRC5 Chinese hamster ovary cells were subjected to a two-step procedure involving selective extraction with different concentrations of the zwitterionic detergent CHAPS. The resulting extract was enriched in P-glycoprotein (around 30% pure) and displayed an ATPase activity (specific activity 543 nmol mg-1 min-1) that was not found in a similar preparation from drug-sensitive cells. The ATPase specific activity was over 10-fold higher than that previously reported for immunoprecipitated Pgp and 280-fold higher than that of immunoaffinity-purified Pgp. This ATPase activity could be distinguished from that of other ion-motive ATPases and membrane-associated phosphatases and is, thus, proposed to be directly attributable to P-glycoprotein. Optimal P-glycoprotein ATPase activity required Mg2+ at an ATP: Mg2+ molar ratio of 0.75:1 and the apparent Km for ATP was 0.88 mM. P-Glycoprotein ATPase could be completely inhibited by vanadate and by the sulfhydryl-modifying reagents N-ethylmaleimide, HgCl2 and p-chloromercuribenzenesulfonate. Certain drugs and chemosensitizers, including colchicine, progesterone, nifedipine, verapamil and trifluoperazine, produced up to 50% activation of P-glycoprotein ATPase activity.     

The isolation of plasma membrane and characterisation of the plasma membrane ATPase from the yeast Candida albicans

Plasma membrane ghosts were isolated from Candida albicans ATCC 10261 yeast cells following stabilisation of spheroplasts with concanavalin A, osmotic lysis and Percoll density gradient centrifugation. Removal of extrinsic proteins with NaCl and methyl alpha-mannoside gave increased ATPase and chitin synthase specific activities in the resultant plasma membrane fraction. Sonication of this fraction yielded unilamellar plasma membrane vesicles which exhibited ATPase and chitin synthase specific activities of 4.5-fold and 3.0-fold, respectively, over those of the plasma membrane ghosts. ATPase activity in the membrane ghosts was optimal at pH 6.4, showed high substrate specificity (for Mg X ATP) and was inhibited 80% by sodium vanadate but less than 4% by oligomycin and azide. The effects of a range of other inhibitors were also characterised. Temperature effects of ATPase activity were marked, with a maximum at 35 degrees C. Breaks in the Arrhenius plot, at 12.2 degrees C and 28.9 degrees C, coincided with endothermic heat flow peaks detected by differential scanning calorimetry. ATPase was solubilised from the plasma membranes with Zwittergent in the presence of glycerol and phenylmethylsulphonyl fluoride and partially purified by glycerol density gradient centrifugation. The solubilised enzyme hydrolysed Mg X ATP at Vmax = 20 mumol X min-1 X mg-1 in the presence of phospholipids, with optimal activity at pH 6.0--6.5.     

Subcellular localization of cytochrome b and ubiquinone in a tertiary granule of resting human neutrophils and evidence for a proton pump ATPase

The subcellular distribution of cytochrome b and ubiquinone in resting human neutrophils was investigated by rate zonal sedimentation of postnuclear supernatants on continuous sucrose gradients. Both cytochrome b and ubiquinone were mainly localized in small organelles, tertiary granules, that were resolved from the specific and azurophilic granules as well as from the cell membrane fraction. This cytochrome b- and ubiquinone-rich granule was shown to contain dicyclohexylcarbodiimide (DCCD)-sensitive, Mg2+-dependent ATPase as well as low amounts, less than a third, of the acid hydrolases beta-glucuronidase and N-acetyl-beta-glucosaminidase. Cytochrome b was also found in smaller proportions in plasma membranes and specific granules. A significant proportion of the ubiquinone was located in the region of the gradients where specific granules and mitochondria sedimented. However, quantitative measurements of oligomycin-sensitive ATPase indicated that this second localization of ubiquinone could not be entirely attributed to mitochondrial contamination. Plasma membrane contained small amounts of ubiquinone. In addition, the existence and location of a putative proton pump ATPase were also investigated. The ATPase was mainly located in the plasma membrane and in the upper half of the gradients (tertiary and specific granules), with the highest specific activity occurring in the tertiary granules. This activity was inhibited by 100 microM DCCD. Furthermore, ATP-dependent uptake of [14C]methylamine by tertiary and specific granules was observed. These results suggest that the DCCD-sensitive ATPase may function as a proton pump. DCCD inhibited the release of enzymes from specific granules that occurred when human neutrophils were activated by phorbol myristate acetate. However, higher concentrations of DCCD were required to achieve the same degree of inhibition of O2 uptake (I50 of 0.4 mM for secretion versus 1 mM for O2 uptake). These results suggest that specific granules do not play a crucial role in oxygen metabolism.     

The involvement of a vanadate-sensitive ATPase in plasma membranes of a salt tolerant cyanobacterium

Plasma membranes of the marine cyanobacterium Spirulina subsalsa were tested for ATPase activity, and for involvement in salt stress. Transition of cells from saline to hypersaline medium enhances the respiratory activity associated with extrusion of Na⁺ and Cl⁻, and persisting salt stress induces synthesis of respiratory enzymes in the plasma membranes. The membranes possess an ATPase, specific for ATP and Mg²⁺ and sensitive to orthovanadate and dicyclohexylcarbodiimide. Immunoblot analysis of plasma membrane polypeptides from Spirulina subsalsa with anti- Arabidopsis H⁺-ATPase serum identified a single polypeptide of 100 kDa, which cross-reacted with the antibodies. An unusual feature of this ATPase is a specific stimulation by Na⁺ ions. Prolonged adaptation of S. subsals cells to hypersaline conditions induced an increase in ATPase activity in subsequent plasma membrane preparations, as well as a higher content of the 100 kDa polypeptide. It is suggested that the ATPase investigated is an H⁺-pump, which is involved in extrusion of Na⁺ and in conferring resistance to salt stress.     

Isolation of plasma membranes from corn roots by sucrose density gradient centrifugation: an anomalous effect of ficoll

An investigation was conducted into the isolation of plasma membrane vesicles from primary roots of corn (Zea mays L., WF9 × M14) by sucrose density gradient centrifugation. Identification of plasma membranes in cell fractions was by specific staining with the periodic-chromic-phosphotungstic acid procedure. Plasma membrane vesicles were rich in K⁺-stimulated ATPase activity at pH 6.5, and equilibrated in linear gradients of sucrose at a peak density of about 1.165 g/cc. It was necessary to remove mitochondria (equilibrium density of 1.18 g/cc) from the homogenate before density gradient centrifugation to minimize mitochondrial contamination of the plasma membrane fraction. Endoplasmic reticulum (NADH-cytochrome c reductase) and Golgi apparatus (latent IDPase) had equilibrium densities in sucrose of about 1.10 g/cc and 1.12 to 1.15 g/cc, respectively. A correlation (r = 0.975) was observed between K⁺-stimulated ATPase activity at pH 6.5 and the content of plasma membranes in various cell fractions. ATPase activity at pH 9 and cytochrome c oxidase activity were also correlated.     

Characterization and Effect of Light on the Plasma Membrane H+-ATPase of Bean Leaves

Proton excretion from bean (Phaseolus vulgaris L.) leaf cells is increased by bright white light. To test whether this could be due, at least in part, to an increase in plasma membrane (PM) ATPase activity, PM vesicles were isolated from primary leaves by phase partitioning and used to characterize PM ATPase activity and changes in response to light. ATPase activity was characterized as magnesium ion dependent, vanadate sensitive, and slightly stimulated by potassium chloride. The pH optimum was 6.5, the K_m was approximately 0.30 millimolar ATP, and the activity was about 60% latent. PM vesicles were prepared from leaves of plants grown for 11 days in dim red light (growing slowly) or grown for 10 days in dim red light and then transferred to bright white-light for 1 day (growing rapidly). For both light treatments, ATPase specific activity was approximately 600 to 700 nanomoles per milligram protein per minute, and the latency, K_m, and sensitivity to potassium chloride were also similar. PM vesicles from plants grown in complete darkness, however, exhibited a twofold greater specific activity. We conclude that the promotion of leaf growth and proton excretion by bright white light is not due to an increase in ATPase specific activity. Light does influence ATPase activity, however; both dim red light and bright white light decreased the ATPase specific activity by nearly 50% as compared with dark-grown leaves.     

Characterisation of the H+-ATPase in plasma membranes isolated from the green alga Chlamydomonas reinhardtii

Plasma membranes from the green alga Chlamydomonas reinhardtii were purified by differential centrifugation and two-phase partitioning in an aqueous polymer system. The isolated plasma membranes were virtually free from contaminating chloroplasts, mitochondria, endoplasmic reticulum and Golgi membranes as shown by marker enzyme and pigment analysis. The isolated plasma membranes exhibited vanadate sensitive ATPase activity, indicating the presence of a P-type ATPase. This was verified by using antibodies against P-type ATPase from Arabidopsis , which crossreacted with a protein of 109 kDa. The ATPase activity was inhibited to more than 90% by vanadate (K_i= 0.9 μ M ) but not affected by inhibitors specific for F- or V-type ATPases. demonstrating the purity of the plasma membranes. Mg-ATP was the substrate, and the rate of ATP-hydrolysis followed simple Michaelis-Menten kinetics giving a K_m= 0.46 m M . Free Mg²⁺ stimulated the activity, K_1/2= 0.68 m M . Maximal activity was obtained at pH 8. The ATPase activity was latent but stimulated 10 to 20-fold in the presence of detergents. This indicates that the isolated plasma membrane vesicles were tightly sealed and mostly right-side-out, making the ATPase inaccessible to the hydrophilic substrate ATP. In the presence of the Brij 58, the isolated plasma membranes performed ATP dependent H⁺-pumping as shown by the optical pH probe acridine orange. H⁺-pumping was dependent on the presence of valinomycin and K⁺ ions and completely abolished by vanadate. Addition of Brij 58 has been shown to produce 100% sealed inside-out vesicles of plant plasma membranes (Johansson et al. 1995, Plant J. 7: 165–173) and this was also the case for plasma membranes from the green alga Chlamydomonas reinhardtii.     

Response of Tonoplast and Plasma Membrane ATPases in Chilling-Sensitive and -Insensitive Rice (Oryza sativa L.) Culture Cells to Low Temperature

Tonoplast and plasma membrane vesicles were prepared from chilling-sensitive(CS) and chilling-insensitive (CI) cultured cells of rice (Oryzasativa L.) to examine how they would respond to low temperature.With CS cells, the specific activity of ATPase in tonoplastvesicles was relatively higher than that of plasma membraneATPase. Tonoplast ATPase activity was decreased by low temperaturetreatment, and a slight decrease in plasma membrane ATPase activitywas also observed. The decrease in the specific activity ofthe tonoplast ATPase by low temperature may reflect a decreasein V_max. However, no change was noted in K_m. The break pointof the Arrhenius plots of the tonoplast ATPase was ca. 32?C,this value being ca. 9?C higher than that of the plasma membraneATPase. With CI cells, the specific activity of tonoplast ATPasewas somewhat less than that of the plasma membrane ATPase. TonoplastATPase activity was decreased by low temperature at 5?C, whereasan increase in plasma membrane ATPase activity was observed.The break point of the tonoplast ATPase activity was ca. 22?C,which was 3?C higher than that of the plasma membrane ATPase.Using ATPase solubilized from the plasma membrane or tonoplast,the Arrhenius plots of log ATPase activity against the reciprocalof absolute temperature gave a straight line fit from 5?C to45?C with no obvious break point. The break point appeared onadding a phospholipid mixture (asolectin) to a reaction mixturecontaining solubilized enzyme. The slope of the curve of theArrhenius plot was very different between the CS and CI cells.The plasma membrane and tonoplast ATPases from the CS cellshad a higher E_a above 20?C, whereas that from the CI cells hada lower one. These findings indicate that the tonoplast ATPase in a riceplant is more sensitive to low temperature than the plasma membraneATPase, with this response possibly being due to interactionsbetween the proteins and phospholipids. (Received January 6, 1988; Accepted July 5, 1988)     

Isolation and sequence of tryptic peptides from the proton-pumping ATPase of the oat plasma membrane

In crude extracts of plant tissue, the M_r = 100,000 proton-pumping ATPase constitutes less than 0.01% of the total cell protein. A large-scale purification procedure is described that has been used to obtain extensive protein sequence information from this enzyme. Plasma membrane vesicles enriched in ATPase activity were obtained from extracts of oat roots by routine differential and density gradient centrifugation. Following a detergent wash, the ATPase was resolved from other integral membrane proteins by size fractionation at 4°C in the presence of lithium dodecyl sulfate. After carboxymethylation of cysteine residues and removal of detergent, the ATPase was digested with trypsin and resultant peptide fragments separated by reverse phase high performance liquid chromatography. Peptides were recovered with high yield and were readily sequenced by automated Edman degradation on a gas-phase sequencer. Of the eight peptides sequenced, six showed strong homology with known amino acid sequences of the fungal proton-pumping and other cation-transporting ATPases.     

Partial purification of plant plasma membrane K+, Mg2+-ATPase by ion exchange chromatography

A purification procedure is presented which differs in three respects from other procedures for the purification of plant plasma membrane H⁺-pumping ATPase (EC 3.6.1.35) from various plants. Soybean ( Glycine max L. cv. Williams) hypocotyls were homogenized in the presence of physiological ionic strength and plasma membrane vesicles were purified by aqueous polymer two-phase partitioning. Plasma membrane vesicles were then solubilized in one step by using non-ionic detergent (either Triton X-100 or C₁₂E₈). The Mg-ATPase was separated by ion exchange chromatography from other solubilized membrane proteins. ATPase molecules bound to phosphocellulose fibers were eluted by a 0–1 M gradient of NaCl. The NaCl-eluted fractions contained a Mg-ATPase which showed the characteristics of Mg-ATPase present in the plasma membranes. The specific activity of the partially purified enzyme was 2–5 μmol mg⁻¹ min⁻¹ when it was reconstituted into proteoliposomes. This value is in good agreement with data obtained by other purification methods in the literature.     

The plasma membrane of Streptococcus cremoris: isolation and partial characterization

Plasma membrane was isolated from Streptococcus cremoris using mutanolysin from a streptomycete as the cell wall-degrading enzyme and phenylmethylsulfonyl fluoride as protease inhibitor. The specific activity of membrane-bound enzyme, adenosine triphosphatase (ATPase), was 4 μmol/mg protein per min, which was 5–10 times higher than the activity found in other fractions obtained during the isolation procedure. The number of polypeptides in the plasma membrane was approximately 50 with molecular weights 13 500–100 000, minor changes in the polypeptide pattern were observed when the plasma membrane was isolated without a protease inhibitor. The chemical composition of the membrane preparation was 49.7% protein, 21.9% lipid, 5.1% aminosugars, 17.3% RNA and 0.03% DNA. Electron microscopic examination confirmed the membrane to be practically devoid of cell wall components. Our results indicate that the membrane integrity is well retained and therefore the membrane preparation is suitable for detailed studies on vectorial metabolism and its enzymes, e.g. ATPase.     

Isolation and enzymatic characterization of the plasmalemma from bovine spermatozoa

An improved method for the isolation of pure plasma and acrosomal membranes from bull spermatozoa is presented. Plasma membranes were isolated from the spermatozoa of bulls of different breeds, and some enzymatic activity, such as (Na+-K+) ATPase, Ca++ ATPase, Mg++ ATPase, alkaline and acidic phosphatases were assayed. Such enzymatic activity levels differ noticeably from those published by other authors, whose preparations were probably contaminated by other cellular components. Highly statistically significant differences of these activities have been found among the several breeds.     

Seasonal Changes in Plasma Membranes and Mitochondria Isolated From Jerusalem Artichoke Tubers. Possible Relationship to Cold Hardiness

Plasma membranes and mitochondria were isolated from Jerusalemartichoke tubers during cold acclimation from September to December.The protein and lipid contents of the membranes were analyzedwith reference to physiological properties of the tubers, especiallycold hardiness. As cold hardiness increased from autumn to winter,the content of phospholipids and sterols on a mg protein basisincreased by 20–30% in plasma membranes, but little changewas observed in mitochondria. Minor changes were observed inthe fatty acid composition of phospholipids either in plasmamembranes or mitochondria. Membrane fluidity, assessed by fluorescentpolarization of 1,6-diphenyl-1,3,5-hexatriene, was found tobe relatively constant in both membranes during the season.One dimensional SDS-polyacrylamide gel electrophoresis revealedseasonal changes in proteins and glycoproteins in plasma membranes,but not in mitochondrial membranes. Plasma membrane ATPase increasedin specific activity from September to December, which was morenoticeable at higher assay temperatures. However, irrespectiveof the season, the plasma membrane ATPase had an inflectionon the slope of the Arrhenius plot around 15C. These resultssuggest that plasma membranes, in contrast to mitochondria,undergo several molecular changes from autumn to winter, whichmay be related to cold acclimation of the tubers. ¹ Contribution No. 2668 from the Institute of Low TemperatureScience. ² Present address: Crop Development Centre, University of Saskatchewan,Saskatoon, Canada S7N 0W0.     

Presence of a Na+-stimulated P-type ATPase in the plasma membrane of the alkaliphilic halotolerant cyanobacterium Aphanothece halophytica

Aphanothece cells could take up Na(+) and this uptake was strongly inhibited by the protonophore, carbonyl cyanide m-chlorophenylhydrazone (CCCP). Cells preloaded with Na(+) exhibited Na(+) extrusion ability upon energizing with glucose. Na(+) was also taken up by the plasma membranes supplied with ATP and the uptake was abolished by gramicidin D, monensin or Na(+)-ionophore. Orthovanadate and CCCP strongly inhibited Na(+) uptake, whereas N, N'-dicyclohexylcarbodiimide (DCCD) slightly inhibited the uptake. Plasma membranes could hydrolyse ATP in the presence of Na(+) but not with K(+), Ca(2+) and Li(+). The K(m) values for ATP and Na(+) were 1.66+/-0.12 and 25.0+/-1.8 mM, respectively, whereas the V(max) value was 0.66+/-0.05 mumol min(-1) mg(-1). Mg(2+) was required for ATPase activity whose optimal pH was 7.5. The ATPase was insensitive to N-ethylmaleimide, nitrate, thiocyanate, azide and ouabain, but was substantially inhibited by orthovanadate and DCCD. Amiloride, a Na(+)/H(+) antiporter inhibitor, and CCCP showed little or no effect. Gramicidin D and monensin stimulated ATPase activity. All these results suggest the existence of a P-type Na(+)-stimulated ATPase in Aphanothece halophytica. Plasma membranes from cells grown under salt stress condition showed higher ATPase activity than those from cells grown under nonstress condition.     

Partial purification of a tonoplast ATPase from corn coleoptiles

The tonoplast ATPase from corn coleoptile membranes was solubilized using a two-step procedure consisting of a pretreatment with 0.15% (w/v) deoxycholate to remove 60% of the protein, and 40 millimolar octyl-glucoside to solubilize the ATPase. During ultracentrifugation, the solublized ATPase entered a linear sucrose gradient faster than the majority of the protein, resulting in an 11-fold purification over the initial specific activity. The partially purified ATPase was almost completely inhibited by KNO₃ with an estimated K_i of 10 millimolar. The specific activity of the KNO₃-sensitive ATPase was increased 29-fold during purification. N,N′-Dicyclohexylcarbodiimide also completely inhibited the ATPase with half-maximal effects at a concentration of 4 micromolar. Neither vanadate nor azide inhibited enzyme activity. The purified ATPase was stimulated by Cl⁻ and preferred Mg-ATP as substrate. Analysis of frations from the sucrose gradient by sodium dodecyl sulfate-polyacrylamide gel electrophoresis led to the identification of two major polypeptides at 72,000 and 62,000 daltons which were best correlated with ATPase activity. Several minor bands also appeared to copurify with enzyme activity, but were less consistent. Radiation inactivation experiments with intact membranes indicated that the functional molecular size of the tonoplast ATPase was nearly 400,000 daltons. This suggests that the ATPase is composed of several polypeptides, possibly including the 72,000- and 62,000-dalton proteins.