Similarity of lysosomal H+-ATPase to mitochondrial F0F1-ATPase in sensitivity to anions and drugs as revealed by solubilization and reconstitution

Lysosomal H+-translocating ATPase (H+-ATPase) was solubilized with lysophosphatidylcholine and reconstituted into liposomes (Moriyama, Y., Takano, T. and Ohkuma, S. (1984) J. Biochem. (Tokyo) 96, 927-930). In this study, the sensitivities of membrane-bound, solubilized and liposome-incorporated ATPase to various anions and drugs were measured in comparison with those of similar forms of mitochondrial H+-ATPase (mitochondrial F0F1-ATPase) with the following results. (1) Bicarbonate and sulfite activated solubilized lysosomal H+-ATPase, but not the membrane-bound ATPase or ATPase incorporated into liposomes. All three forms of mitochondrial F0F1-ATPase were activated by these anions. (2) All three forms of both lysosomal H+-ATPase and mitochondrial F0F1-ATPase were strongly inhibited by SCN-, NO3- and F-, but scarcely affected by Cl-, Br- and SO2-4. (3) The solubilized lysosomal H+-ATPase was strongly inhibited by azide, quercetin, 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole (NBD-Cl), 4-acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic acid (SITS), 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS) and oligomycin. Its sensitivity was almost the same as that of mitochondrial F0F1-ATPase. Neither membrane-bound ATPase nor ATPase incorporated into liposomes was affected appreciably by these drugs. These results indicate that the sensitivity to anions and drugs of lysosomal H+-ATPase depends on the form of the enzyme and that the sensitivity of the solubilized lysosomal H+-ATPase is very similar to that of mitochondrial F0F1-ATPase. On the other hand, the two ATPases differ in their sensitivity to N-ethylmaleimide and pyridoxal phosphate: only the mitochondrial ATPase is inhibited by pyridoxal phosphate whereas only the lysosomal ATPase is inhibited by N-ethylmaleimide.     

Electrogenic nature of lysosomal proton pump as revealed with a cyanine dye

Studies were carried out on the electrogenicity of the lysosomal proton pump using dipropylthiadicarbocyanine iodide (diS-C3-(5] as a membrane potential probe. Pure lysosome preparations (tritosomes) quenched the fluorescence of diS-C3-(5). The quenching correlated well with the potassium ion diffusion potential (inside negative) generated by K+ with or without valinomycin. The quenching caused by lysosomes was reversed by lipophilic cations, tetraphenylarsonium (TPA) or triphenylmethylphosphonium (TPMP). Mg-ATP also reversed the quenching, which was inhibited by a protonophore, 3,5-di-tert-butyl-4-hydroxybenzylidene-malononitrile (SF-6847). The properties of the ATP-induced recovery of the quenching were exactly the same as those of ATP-induced acidification, as measured with fluorescein isothiocyanate-dextran (FD) (Ohkuma, S., et al. (1982) Proc. Natl. Acad. Sci. U.S. 79, 2758-2762) and acridine orange (Moriyama, Y., et al. (1982) J. Biochem. 92, 1333-1336), except replacement of the anion by an impermeable one enhanced ATP-induced recovery of quenching, but reduced ATP-induced acidification. Amines which dissipate delta pH across the lysosomal membrane also enhanced the Mg-ATP-induced fluorescence recovery. These results suggest that isolated lysosomes exhibit an inside negative membrane potential, especially in low K+ medium, mostly due to the K+-diffusion potential, and that the Mg-ATP-driven proton pump causes membrane depolarization (in the direction of inside positive). These possibilities were supported by results on the uptake of the radioactive membrane-permeant ions [3H]TPMP and [14C]SCN. The present results provide evidence for the electrogenic nature of the lysosomal proton pump.     

Proton ATPase in rat renal cortical endocytotic vesicles

To relate ATPase activity to the ATP-driven H+-pump in rat renal endocytotic vesicles we applied an in vitro coupled optical test and a Pi-liberation assay. Endocytotic vesicles contain an ouabain-, vanadate- and oligomycin-insensitive ATPase. The ionophores for K+ and H+, valinomycin and carbonylcyanide p-chloro-methoxyphenylhydrazone (CCCP), respectively, stimulated ATPase activity, indicating its relation to the electrogenic H+-pump. This conclusion is supported by a similar distribution on a Percoll gradient of ATP-driven H+ uptake into endosomes and ionophore-stimulated ATPase activity. Coupled optical and Pi-liberation assays were then used to characterize the H+-ATPase with respect to the requirement for pH, nucleotides, anions, and mono- and divalent cations. The H+-ATPase activity was decreased by widely used blockers: N-ethylmaleimide (NEM), dicyclohexylcarbodiimide (DCCD) and diethylstilbestrol (DES). Different sensitivities to these blockers proved that alkaline phosphatase and H+-ATPase are separate entities. To investigate whether the NEM-, DCCD- and DES-sensitive ATPase activity is confined to intact endocytotic vesicles, cellular membranes from rat kidney cortex were separated on a Percoll density gradient. Surprisingly, endocytotic vesicles contain only a small fraction of the total NEM-, DCCD- and DES-sensitive ATPase activity. The majority of the blocker-sensitive ATPases belongs to membranes of as yet undefined cellular origin.     

Spatial coordination of chloroplast and plasma membrane activities in <Emphasis Type="Italic">chara</Emphasis> cells and its disruption through inactivation of 14-3-3 proteins

In Chara corallina cells exposed to continuous light, external pH (pH(o)) and photosystem II (PSII) photochemical yield show correlated banding patterns. Photosynthetic activity is low in cell regions producing alkaline zones and high in the acid regions. We addressed the question whether (and how) photosynthetic activity and plasma membrane (PM) H+-pumping and H+-conductance are coupled in the different bands. First, PM H+-pump activity was stimulated with fusicoccin. This resulted in a more acidic pH in the acid bands without disturbing the correlation of photosynthetic electron transport and H+ fluxes across the PM. Next, H+-pump activity was reduced through microinjection of a phosphorylated peptide matching the canonical 14-3-3 binding motif RSTpSTP in the acid cell region. Microinjection induced a rapid (~5 min) rise in pH(o) by ca. 1.0 unit near the injection site, whereas the injection of the non-phosphorylated peptide had no effect. This pH rise confirms the supposed inhibition of the H+-pump upon the detachment of 14-3-3 proteins from the H+-ATPase. However, the PSII yield in the cell regions corresponding to the new alkaline peak remained high, which violated the normal inverse relations between the pH(o) and PSII photochemical yield. We conclude that the injection of the competitive inhibitor of the H+-ATPase disrupts the balanced operation of PM H+-transport and photosynthetic electron flow and promotes electron flow through alternative pathways.     

Acridine orange as a fluorescent probe for lysosomal proton pump

Acridine orange was found to accumulate in pure lysosomal particles (tritosomes) in vitro, and the quenching of its fluorescence correlated well with the delta pH (inside acid) across the lysosomal membrane. Use of this dye showed that Mg-ATP caused lysosomal acidification. This acidification was sensitive to N,N'-dicyclohexylcarbodiimide, N-ethylmaleimide, and azide, but not to oligomycin, ouabain or vanadate. These results supported the idea of the existence of a lysosomal H+-pump, suggested in a previous paper (Ohkuma et al. (1982) Proc. Natl. Acad. Sci. U.S. 79, 2758-2762).     

Calcium transport in human erythrocytes. Separation and reconstitution of high and low Ca affinity (Mg mca)-AT Pase activities in membranes prepared at low ionic strength.

Human erythrocyte membranes obtained by freeze-thawing of ghosts prepared in the absence or presence of EDTA, by washing with a 12 mosm medium at pH 7.7 or a 2 mosm medium at pH 6.5 contain both high and low Ca affinity (Mg + Ca)-ATPase activities. Incubation of ghosts in a less than 2 mosm medium at pH 7.5 or in 0.1 mm EDTA + 1 Him Tris-maleate (pH 8.0) results in removal of the high affinity (Mg + Ca)-ATPase activity from the membrane in a time dependent manner. Under similar conditions up to 25% of membrane proteins are removed. The soluble protein fraction extracted, although devoid of ATPase activity, reconstitutes with the remaining membrane residue with restoration of original (Mg + Ca)-ATPase activity. Addition of the soluble protein fraction to heat-treated membranes devoid of low affinity (Mg + Ca)-ATPase activity allows reconstitution of more than 33% of the original high affinity (Mg + Ca)-ATPase activity which has a Ca dissociation constant of approximately 1.6μm. Temperature and phospholipase A₂ studies indicate that low affinity (Mg + Ca)-ATPase activity is phospholipid dependent in contrast to high affinity (Mg + Ca)-ATPase activity. Ruthenium red and LaCl₃ inhibit both high and low affinity (Mg + Ca)-ATPase activities with similar potencies. The ease of removal of high affinity (Mg + Ca)-ATPase activity from the membrane by relatively mild conditions suggests that an activator protein or the high affinity (Mg + Ca)-ATPase itself is only loosely attached to the membrane. These studies show that low affinity (Mg + Ca)-ATPase activity is not an artifact and is distinct from high affinity (Mg + Ca)-ATPase activity. The low affinity (Mg + Ca)-ATPase activity is sensitive to Ca²⁺ in the concentration range from below 0.3 μm to 300 μm compatible with an association of this enzyme with Ca transport.     

Two Ca2+-dependent ATPases in rat liver plasma membrane. The previously purified (Ca2+-Mg2+)-ATPase is not a Ca2+-pump but an ecto-ATPase

We have shown that the rat liver plasma membrane has at least two (Ca2+-Mg2+)-ATPases. One of them has the properties of a plasma membrane Ca2+-pump (Lin, S.-H. (1985) J. Biol. Chem. 260, 7850-7856); the other one, which we have purified (Lin, S.-H., and Fain, J.N. (1984) J. Biol. Chem. 259, 3016-3020) and characterized (Lin, S.-H. (1985) J. Biol. Chem. 260, 10976-10980) has no established function. In this study we present evidence that the purified (Ca2+-Mg2+)-ATPase is a plasma membrane ecto-ATPase. In hepatocytes in primary culture, we can detect Ca2+-ATPase and Mg2+-ATPase activities by addition of ATP to the intact cells. The external localization of the active site of the ATPase was confirmed by the observation that the Ca2+-ATPase and Mg2+-ATPase activities were the same for intact cells, saponin-treated cells, and cell homogenates. Less than 14% of total intracellular lactate dehydrogenase, a cytosolic enzyme, was released during a 30-min incubation of the hepatocytes with 2 mM ATP. This indicates that the hepatocytes maintained cytoplasmic membrane integrity during the 30-min incubation with ATP, and the Ca2+-ATPase and Mg2+-ATPase activity measured in the intact cell preparation was due to cell surface ATPase activity. The possibility that the ecto-Ca2+-ATPase and Mg2+-ATPase may be the same protein as the previously purified (Ca2+-Mg2+)-ATPase was tested by comparing the properties of the ecto-ATPase with those of (Ca2+-Mg2+)-ATPase. Both the ecto-ATPase and the (Ca2+-Mg2+)-ATPase have broad nucleotide-hydrolyzing activity, i.e. they both hydrolyze ATP, GTP, UTP, CTP, ADP, and GDP to a similar extent. The effect of Ca2+ and Mg2+ on the ecto-ATPase activity is not additive indicating that both Ca2+- and Mg2+-ATPase activities are part of the same enzyme. The ecto-ATPase activity, like the (Ca2+-Mg2+)-ATPase, is not sensitive to oligomycin, vanadate, N-ethylmaleimide and p-chloromercuribenzoate; and both the ecto-ATPase and purified (Ca2+-Mg2+)-ATPase activities are insensitive to protease treatments. These properties indicate that the previously purified (Ca2+-Mg2+)-ATPase is an ecto-ATPase and may function in regulating the effect of ATP and ADP on hepatocyte Ca2+ mobilization (Charest, R., Blackmore, P.F., and Exton, J.H. (1985) J. Biol. Chem. 260, 15789-15794).     

Leishmania plasma membrane Mg2+-ATPase is a H+/K+-antiporter involved in glucose symport. Studies with sealed ghosts and vesicles of opposite polarity

Experiments from other laboratories conducted with Leishmania donovani promastigote cells had earlier indicated that the plasma membrane Mg2+-ATPase of the parasite is an extrusion pump for H+. Taking advantage of the pellicular microtubular structure of the plasma membrane of the organism, we report procedures for obtaining sealed ghost and sealed everted vesicle of defined polarity. Rapid influx of H+ into everted vesicles was found to be dependent on the simultaneous presence of ATP (1 mm) and Mg2+ (1 mm). Excellent correspondence between rate of H+ entry and the enzyme activity clearly demonstrated the Mg2+-ATPase to be a true H+ pump. H+ entry into everted vesicle was strongly inhibited by SCH28080 (IC50 = approximately 40 microm) and by omeprazole (IC50 = approximately 50 microm), both of which are characteristic inhibitors of mammalian gastric H+,K+-ATPase. H+ influx was completely insensitive to ouabain (250 microm), the typical inhibitor of Na+,K+-ATPase. Mg2+-ATPase activity could be partially stimulated with K+ (20 mm) that was inhibitable (>85%) with SCH28080 (50 microm). ATP-dependent rapid efflux of 86Rb+ from preloaded vesicles was completely inhibited by preincubation with omeprazole (150 microm) and by 5,5'-dithiobis-(2-nitrobenzoic acid) (1 mm), an inhibitor of the enzyme. Assuming Rb+ to be a true surrogate for K+, an ATP-dependent, electroneutral stoichiometric exchange of H+ and K+(1:1) was established. Rapid and 10-fold active accumulation of [U-(14)C]2-deoxyglucose in sealed ghosts could be observed when an artificial pH gradient (interior alkaline) was imposed. Rapid efflux of [U-(14)C]d-glucose from preloaded everted vesicles could also be initiated by activating the enzyme, with ATP. Taken together, the plasma membrane Mg2+-ATPase has been identified as an electroneutral H+/K+ antiporter with some properties reminiscent of the gastric H+,K+-ATPase. This enzyme is possibly involved in active accumulation of glucose via a H+-glucose symport system and in K+ accumulation.     

Investigation of adenosinetriphosphatase activity of rat liver and thymus cell nuclei]

The activity of ATPase was studied in highly purified rat liver and thymus cell nuclei, HCO3-, CO3(2-) and SO3(2-) stimulated nuclear ATPase in 1.5--2 times. HSO3- did not affect the enzyme activity, and NO3-, J-, ClO4-,F- and SCN- inhibited it. Bicarbonate increased V and decreased Ka for ATP. SCN- inhibited HCO3--ATPase activity non-competitively with respect to HCO3-. Mg2+-ATPase activity did not depend on pH, and HCO3-component of the activity was decreased under alkaline pH. Mg2+, Mn2+ and Co2+ increased the initial ATPase activity and helped its stimulation with HCO3-. Ba2+, Ni2+ and Zn2+ inhibited the ATPase activity, and Ca2+ did not affect it, Nuclear ATPase is sensitive to 2,4-dinitrophenol and DNAase. It is suggested that cell nuclei have their own H+-ATPase differing for some characteristics from mitochondrial H+-ATPase.     

Purification and properties of H+-translocating, Mg2+-adenosine triphosphatase from vacuolar membranes of Saccharomyces cerevisiae

H+-translocating, Mg2+-ATPase was solubilized from vacuolar membranes of Saccharomyces cerevisiae with the zwitterionic detergent N-tetradecyl-N,N-dimethyl-3-ammonio-1-propanesulfonate and purified by glycerol density gradient centrifugation. Partially purified vacuolar membrane H+-ATPase, which had a specific activity of 18 units/mg of protein, was separated almost completely from acid phosphatase and alkaline phosphatase. The purified enzyme required phospholipids for maximal activity and hydrolyzed ATP, GTP, UTP, and CTP, with this order of preference. Its Km value for Mg2+-ATP was determined to be 0.21 mM and its optimal pH was 6.9. ADP inhibited the enzyme activity competitively, with a Ki value of 0.31 mM. The activity of purified ATPase was strongly inhibited by N,N'-dicyclohexylcarbodiimide, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide, tributyltin, 7-chloro-4-nitrobenzoxazole, diethylstilbestrol, and quercetin, but was not affected by oligomycin, sodium azide, sodium vanadate, or miconazole. It was not inhibited at all by antiserum against mitochondrial F1-ATPase or mitochondrial F1-ATPase inhibitor protein. These results indicated that vacuolar membrane H+-ATPase is different from either yeast plasma membrane H+-ATPase or mitochondrial F1-ATPase. The vacuolar membrane H+-ATPase was found to be composed of two major polypeptides a and b of Mr = 89,000 and 64,000, respectively, and a N,N'-dicyclohexylcarbodiimide binding polypeptide c of Mr = 19,500, whose polypeptide composition was also different from those of either plasma membrane H+-ATPase or mitochondrial F1-ATPase of S. cerevisiae.     

Long-term stabilization and crystallization of (Ca2+ + Mg2+)-ATPase of detergent-solubilized erythrocyte plasma membrane.

Conditions which were optimal for the stabilization of Ca2(+)-transporting ATPase in solubilized sarcoplasmic reticulum membranes (Piku?la, S., Mullner, N., Dux, L. and Martonosi, A. (1988) J. Biol. Chem. 263, 5277-5286) were also found conducive for preservation of (Ca2+ + Mg2+)-ATPase activity in detergent-solubilized erythrocyte plasma membrane for up to 60 days. Of particular importance for the stabilization of calmodulin-stimulated Ca2(+)-dependent activity of (Ca2+ + Mg2+)-ATPase of solubilized erythrocyte plasma membrane was the presence of Ca2+ (10-20 mM), glycerol, anti-oxidants, proteinase inhibitors and appropriate detergents. Among eight detergents tested octaethylene glycol dodecyl ether, polyoxyethylene glycol(10) lauryl alcohol and polydocanol were found to be promotive in long-term preservation of the enzyme activity. Under these conditions (Ca2+ + Mg2+)-ATPase of erythrocyte ghosts became highly stable and developed microcrystalline arrays after storage for 35 days. Electron micrographs of the negatively stained and thin sectioned material indicated that crystals of purified, detergent-solubilized, lipid-stabilized erythrocyte (Ca2+ + Mg2+)-ATPase differ from those of Ca2(+)-ATPase of detergent-solubilized sarcoplasmic reticulum microsomes.     

The lipid requirement of the (Ca2+ + Mg2+)-ATPase in the human erythrocyte membrane, as studied by various highly purified phospholipases.

1. When complete hydrolysis of glycerophosphlipids and sphingomyelin in the outer membrane leaflet is brought about by treatment of intact red blood cells with phospholipase A2 and sphingomyelinase C, the (Ca2+ + Mg2+)-ATPase activity is not affected. 2. Complete hydrolysis of sphingomyelin, by treatment of leaky ghosts with spingomyelinase C, does not lead to an inactivation of the (Ca2+ + Mg2+)-ATPase. 3. Treatment of ghosts with phospholipase A2 (from either procine pancreas of Naja naja venom), under conditions causing an essentially complete hydrolysis of the total glycerophospholipid fraction of the membrane, results in inactivation of the (Ca2+ + Mg2+)-ATPase by some 80--85%. The residual activity is lost when the produced lyso-compounds (and fatty acids) are removed by subsequent treatment of the ghosts with bovine serum albumin. 4. The degree of inactivation of the (Ca2+ + Mg2+)-ATPase, caused by treatment of ghosts with phospholipase C, is directly proportional to the percentage by which the glycerophospholipid fraction in the inner membrane layer is degraded. 5. After essentially complete inactivation of the (Ca2+ + Mg2+)-ATPase by treatment of ghosts with phospholipase C from Bacillus cereus, the enzyme is reactivated by the addition of any of the glycerophospholipids, phosphatidylserine, phosphatidylcholine, phosphatidylethanolamine or lysophosphatidylcholine, but not by addition of sphingomyeline, free fatty acids or the detergent Triton X-100. 6. It is concluded that only the glycerophospholipids in the human erythrocyte membrane are involved in the maintenance of the (Ca2+ + Mg2+)-ATPase activity, and in particular that fraction of these phospholipids located in the inner half of the membrane.     

ATP-dependent H+ -pump activity in inverted vesicles of Methanosarcina mazei Gö1 and characterization of membrane ATPase.

ATP-dependent H+ -pump activity was found in inverted vesicles of Methanosarcina mazei Gö1 by using acridine orange as a fluorescent probe. The H+ -pump activity specifically required both Mg and sulfite ions, but azide, an inhibitor of F0F1-ATPase, did not inhibit the activity. The membranes prepared from M. mazei also had an Mg-ATPase activity, and at least the presence of vacuolar-type ATPase was detected.     

Interaction of Cd2+ with the calmodulin-activated (Ca2+ + Mg2+)-ATPase activity of human erythrocyte ghosts

Treatment of erythrocyte ghosts with micromolar concentrations of Cd2+ results in a noncompetitive inhibition of the calmodulin-dependent (Ca2+ + Mg2+)-ATPase activity. Higher concentrations of Cd2+ are required for inhibition of the (Ca2+ + Mg2+)-ATPase activity of calmodulin-depleted ghosts. The interaction of Cd2+ is time-dependent with an apparent rate constant around 0.12/min. The inhibition is relieved by addition of EGTA with a rate constant around 0.15/min. If Cd2+ is allowed to interact with calmodulin prior to the association of the protein with the ghosts, the inhibition is mainly competitive. The results suggest that the inhibitory effect caused by Cd2+ is due to an interaction with calmodulin. The slow interaction of Cd2+ suggests that calmodulin bound to the (Ca2+ + Mg2+)-ATPase is inaccessible to Cd2+.     

Effects of glycyrrhizin and glycyrrhetinic acid on (Na+ + K+)-ATPase of renal basolateral membranes in vitro

Studies were made on the direct effects of glycyrrhizin and its aglycone, glycyrrhetinic acid on the activities of (Na+ + K+)-ATPase and (Ca2+ + Mg2+)-ATPase, a membrane bound Na+ and Ca2+-extrusion pump enzyme of the basolateral membranes (BLM) of canine kidney. Glycyrrhetinic acid inhibited the activity of the Na+-pump enzyme dose-dependently (IC50 = 1.5 x 10(-4) M), but had no effect on that of the Ca2+-pump enzyme of kidney BLM and homogenates. Glycyrrhizin also inhibited the Na+-pump enzyme activity but had less effect (IC50 = 2 x 10(-3) M). The effects of these compounds were due to competitive inhibition with ATP binding to the enzyme (Ki = 12 microM) and so were different from that of ouabain, which inhibits the Na+-pump by binding to its extracellular K+-binding site. The direct effect of glycyrrhetinic acid on the membrane may be important role in the multiple actions of licorice.     

The assignment of the Ca2+-ATPase activity of chromaffin granules to the proton translocating ATPase

CaATP is shown to function as a substrate for the proton translocating ATPase of chromaffin granule ghosts at concentrations which are comparable to that of MgATP. Using the initial rate of the proton pump activity as the measure (delta pH/delta t), an apparent Km-value of 139 +/- 8 microM was estimated for CaATP and 59 +/- 3 microM for MgATP. The maximal rate was markedly higher with MgATP than with CaATP, partly due to an inhibition of the hydrolytic activity at the higher concentrations of CaATP. The proton pump activity with CaATP was inhibited by N-ethylmaleimide and N,N'-dicyclohexylcarbodiimide at concentrations similar to that found for MgATP. No inhibition was observed with sodium vanadate in the concentration range 0-15 microM. Calmodulin and trifluoperazine had no effect on the overall ATPase activity with CaATP. These findings establish this activity as an intrinsic property of the chromaffin granules, i.e., linked to the H+-ATPase. No evidence was obtained for the presence of a Ca2+-translocating ATPase [Ca2+ + Mg2+)-ATPase) in the chromaffin granules.     

Modulation of human erythrocyte Ca2+-ATPase activity by glycerol: The role of calmodulin

The effect of an intracellular cryoprotectant glycerol on human erythrocyte Ca2+-ATPase activity and possible involvement of calmodulin in the regulation of Ca2+-pump under these conditions were investigated. The experiments were carried out using saponin-permeabilized cells and isolated erythrocyte membrane fractions (white ghosts). Addition of rather low concentrations of glycerol to the medium increased Ca2+-ATPase activity in the saponin-permeabilized cells; the maximal effect was observed at 10% glycerol. Subsequent increase in glycerol concentrations above 20% was accompanied by inhibition of Ca2+-ATPase activity. Lack of stimulating effect of glycerol on white ghost Ca2+-ATPase may be attributed to removal of endogenous compounds regulating activity of this ion transport system. Inhibitory analysis using R24571 revealed that activation of Ca2+-ATPase by 10% glycerol was observed only in the case of inhibitor administration after modification of cells with glycerol; in the case of inhibitor addition before erythrocyte contact with glycerol, this phenomenon disappeared. These data suggest the possibility of regulation of human erythrocyte Ca2+-ATPase by glycerol; this regulatory effect may be attributed to both glycerol-induced structural changes in the membrane and also involvement of calmodulin in modulation of catalytic activity of the Ca2+-pump.     

Phosphorylated intermediates of (Ca2+ + Mg2+)-ATPase and alkaline phosphatase in renal plasma membranes

Renal basal-lateral and brush border membrane preparations were phosphorylated in the presence of [gamma-32P]ATP. The 32P-labeled membrane proteins were analysed on SDS-polyacrylamide gels. The phosphorylated intermediates formed in different conditions are compared with the intermediates formed in well defined membrane preparations such as erythrocyte plasma membranes and sarcoplasmic reticulum from skeletal muscle, and with the intermediates of purified renal enzymes such as (Na+ + K+)-ATPase and alkaline phosphatase. Two Ca2+-induced, hydroxylamine-sensitive phosphoproteins are formed in the basal-lateral membrane preparations. They migrate with a molecular radius Mr of about 130 000 and 100 000. The phosphorylation of the 130 kDa protein was stimulated by La3+-ions (20 microM) in a similar way as the (Ca2+ + Mg2+)-ATPase from erythrocytes. The 130 kDa phosphoprotein also comigrated with the erythrocyte (Ca2+ + Mg2+)-ATPase. In addition in the same preparation, another hydroxylamine-sensitive 100 kDa phosphoprotein was formed in the presence of Na+. This phosphoprotein comigrates with a preparation of renal (Na+ + K+)-ATPase. In brush border membrane preparations the Ca2+-induced and the Na+-induced phosphorylation bands are absent. This is consistent with the basal-lateral localization of the renal Ca2+-pump and Na+-pump. The predominant phosphoprotein in brush border membrane preparations is a 85 kDa protein that could be identified as the phosphorylated intermediate of renal alkaline phosphatase. This phosphoprotein is also present in basal-lateral membrane preparations, but it can be accounted for by contamination of those membranes with brush border membranes.     

A proton-translocating ATPase regulates pH of the bacterial cytoplasm

Regulatory mechanisms of cytoplasmic pH in Streptococcus faecalis with no respiratory chain were investigated. In a mutant defective in cytoplasmic alkalization conducted by a proton-translocating ATPase (H+-ATPase), the cytoplasmic pH is approximately 0.4 to 0.5 pH units lower than the medium pH, at pH 5.5 to 9.0. The cytoplasmic pH of the wild-type strain was always higher than that of the mutant at a pH below 8 and was the same as that of the mutant at an alkaline pH over 8. Thus, the cytoplasmic pH is regulated only by the cytoplasmic alkalization, and there is no regulation at alkaline pH in S. faecalis. A generation of the protonmotive force conducted by the H+-ATPase depended on the cytoplasmic pH rather than the medium pH, and the generation decreased rapidly when the cytoplasmic pH was increased over 7.7. The decrease at alkaline pH was not caused by increases in the rate of proton influx. These results suggest that cytoplasmic alkalization is diminished when alkaline pH of the cytoplasm is over 7.7, because of a low activity of proton extrusion by the H+-ATPase, and consequently, the cytoplasmic pH is regulated at about 7.7. The cytoplasmic pH was regulated at a high level in cells that had a high level of H+-ATPase. I conclude that in S. faecalis, the cytoplasmic pH is regulated by H+-ATPase.     

Mechanism of action of "ruthenium red" compounds on Ca2+ ionophore from sarcoplasmic reticulum (Ca2+ + Mg2+)- adenosine triphosphatase and lipid bilayer.

Sarcoplasmic reticulum (Ca2+ + Mg2+)-ATPase was previously shown to have Ca2+-dependent and -selective ionophoric activity when tested in oxidized cholesterol lipid bilayer membranes (Shamoo, A. E., and MacLennan, D. H. (1974) Proc. Natl. Acad. Sci. U. S. A. 71, 3522). ruthenium red, a known inhibitor of (Ca2+ + Mg2+)-ATPase, is found to inhibit the Ca2+-ionophoric activity associated with (Ca2+ + Mg2+)-ATPase. Furthermore, ruthenium red alone acts as an anion-selective ionophore in lipid bilayers with the the following selectivity sequence for anions: l- greater than Cl-, Br- greater than F- greater than NO3-. The PCl-/PNa+ ratio was approximately 4/l. The presence of ruthenium red in excess of Ca2+ ionophore in lipid bilayer experiments converts the cation selectivity of the bilayer due to Ca2+ ionophore into anion selectivity.