ATPase Of Human Erythrocyte Membranes Research Articles

2,4-Dinitrophenol and Carbonylcyanide p-Trifluoromethoxyphenylhydrazone Activate the Glutathione S-Conjugate Transport ATPase of Human Erythrocyte Membranes

2,4-Dinitrophenol (DNPOH) and carbonylcyanide p-trifluoromethoxyphenylhydrazone (FCCP), two classical uncouplers of mitochondrial oxidative phosphorylation, were found to stimulate human erythrocyte membrane vesicle ATPase activity. Both compounds competed with S-(2,4-dinitrophenyl) glutathione (DNPSG) for activation of the glutathione S-conjugate transport ATPase. Stimulation of the ATPase by DNPOH or FCCP occurred with V max values 4-6 times greater than that with DNPSG. The K 0.5 for DNPOH (195 μM) was similar to that of DNPSG (196 μM), while that for FCCP (4.3 μM) was 40 times lower. Vanadate inhibits both the DNPOH- and FCCP-stimulated ATPase activities, as previously reported for the glutathione S-conjugate ATPase. The stimulation of erythrocyte vesicle ATPase activities by these classical uncoupling agents does not result from increased proton conductance across the vesicle membrane: monensin, gramicidin and nystatin, all of which increase proton conductance, but by different mechanisms, do not stimulate erythrocyte vesicle ATPase activity. Verapamil, a known P-glycoprotein ATPase activator also does not stimulate human erythrocyte membrane ATPase activity. These results show that relatively small, monoanionic lipophilic compounds such as DNPOH and FCCP can activate the glutathione S-conjugate transport ATPase. The higher V max values for activation by these agents than by DNPSG make possible a more sensitive assay of this transport ATPase activity. The results raise the question of whether these substances and other small anionic, lipophilic compounds are also transported by this system.

Stimulation of a human erythrocyte membrane ATPase by glutathione conjugates

An ATP-dependent transport process for S-(2,4-dinitrophenyl) glutathione (Dnp-SG) mediated by a novel ATPase designated as Dnp-SG ATPase has been demonstrated in human erythrocytes (LaBelle et al., FEBS Lett. 228, 51–53, 1988). In order to investigate whether the Dnp-SG ATPase system represents a generalized mechanism for the transport of xenobiotic conjugates of glutathione (GSH), stimulation of this ATPase by different GSH conjugates was studied in membrane vesicles prepared from human erythrocytes. Kinetic parameters for several GSH conjugates including S-(methyl)glutathione, S-( n-propyl)glutathione, S-( n-pentyl)glutathione, S-( n-decyl)glutathione, S-( p-chlorophenacyl)glutathione, S-( p-nitrobenzyl)glutathione, and the GSH conjugate of 9,10-epoxystearic acid were determined in order to evaluate their affinity for Dnp-SG ATPase. These studies reveal that all these conjugates stimulated Dnp-SG ATPase of human erythrocyte membrane. The apparent K m values of Dnp-SG ATPase for different conjugates were found to be in the range of 0.26–0.66 m m with V max values ranging from 0.55 to 4.44 nmol/min/mg protein. The results of these studies indicate that erythrocyte membrane Dnp-SG ATPase represents a generalized mechanism for the transport of GSH conjugates formed with xenobiotics as well as with the endogenously generated electrophilic compounds such as epoxystearic acid. It is suggested that Dnp-SG ATPase in conjunction with GSH and GSH S-transferase may play an important role in the protection of erythrocytes from exogenous as well as endogenous electrophilic toxicants.

Ca 2+-mediated activation of human erythrocyte membrane Ca 2+-ATPase

Ca 2+-ATPase of human erythrocyte membranes, after being washed to remove Ca 2+ after incubation with the ion, was found to be activated. Stimulation of the ATPase was related neither to fluidity change nor to cytoskeletal degradation of the membranes mediated by Ca 2+. Activation of the transport enzyme was also unaffected by detergent treatment of the membrane, but was suppressed when leupeptin was included during incubation of the membranes with Ca 2+. Stimulation of the ATPase by a membrane-associated Ca 2+-dependent proteinase was thus suggested. Much less 138 kDa Ca 2+-ATPase protein could be harvested from a Triton extract of membranes incubated with Ca 2+ than without Ca 2+. Activity of the activated enzyme could not be further elevated by exogenous calpain, even after treatment of the membranes with glycodeoxycholate. There was also an overlap in the effect of calmodulin and the Ca 2+-mediated] stimulation of membrane Ca 2+-ATPase. While K m(ATP) of the stimulated ATPase remained unchanged, a significant drop in the free-Ca 2+ concentration for half-maximal activation of the enzyme was observed.

Activation of the Ca 2+-ATPase of human erythrocyte membrane by an endogenous Ca 2+-dependent neutral protease

Limited proteolysis of the plasma membrane calcium transport ATPase (Ca 2+-ATPase) from human erythrocytes by trypsin produces a calmodulin-like activation of its ATP hydrolytic activity and abolishes its calmodulin sensitivity. We now demonstrate a similar kind of activation of the human erythrocyte membrane Ca 2+-ATPase by calpain (calcium-dependent neutral protease) isolated from the human red cell cytosol. Upon incubation of red blood cell membranes with purified calpain in the presence of Ca 2+ the membrane-bound Ca 2+-ATPase activity was increased and its sensitivity to calmodulin was lost. In contrast to the action of other proteases tested, proteolysis by calpain favors activation over inactivation of the Ca 2+-ATPase activity, except at calpain concentrations more than 2 orders of magnitude higher. Exogenous calmodulin protects the Ca 2+-ATPase against calpain-mediated activation at concentrations which also activate the Ca 2+-ATPase activity. Calcium-dependent proteolytic modification of the Ca 2+-ATPase could provide a mechanism for the irreversible activation of the membrane-bound enzyme.

Inhibition of Ca ++-ATPase of rat erythrocyte membranes by K-opioid agonists

The effect of various opioid agonists on Ca ++, Mg ++-ATPase activity of rat erythrocyte membranes was studied. The Ca ++-stimulated component of this enzyme (Ca ++-ATPase) showed properties similar to those of the Ca ++-pumping ATPase of human erythrocyte membranes, that is, high affinity for Ca ++, potentiation by calmodulin, and insensitiveness to Na +. Ethylketocyclazocine (EKC) dose-dependently inhibited this Ca ++-ATPase activity at a concentration less than one nM without changing basal Mg ++-ATPase activity and this action was reversed by the antagonist Win 44,441. Other opioid agonists mimicked this EKC effect and the rank order of potency was dynorphin (1-13)=EKC>levorphanol=morphine=B-endorphin=dihydromorphine>leu-enkphalin>(D-Ala) 2-(D-leu) 5-enkephalin (DADL)=morphiceptin. It is concluded that rat erythrocyte membranes possess k-type opioid receptors through which the Ca ++-pump is inhibited.

Stimulation of (Ca 2+ + Mg 2+)-ATPase activity in human erythrocyte membranes by synthetic lysophosphatidic acids and lysophosphatidylcholines. Effects of chain length and degree of unsaturation of the fatty acid groups

Synthetic lysophosphatidic acids and lysophosphatidylcholines were examined for their effects on the (Ca 2+ + Mg 2+)-ATPase of human erythrocyte membranes. Addition of these compounds to erythrocyte ghosts caused significant changes in ATPase activity. The degree of unsaturation and the length of the sn-1 long chain hydrocarbon moiety were both contributing factors. All lysophosphatidic acids tested stimulated (Ca 2+ + Mg 2+)-ATPase activity. Of the species having a saturated acyl group, the most active was the myristoyl derivative. Linoleoyllysophosphatidic acid was the most potent of the unsaturated species. Saturated lysophosphatidylcholines with a short chain fatty acyl group (C 10 to C 14) exhibited only a moderate stimulatory activity, whereas the longer chain homologues, i.e., C 16 and C 18 were inhibitory at high concentrations. On the other hand, unsaturated lysophosphatidylcholines had stimulatory activities comparable to the unsaturated lysophosphatidic acids. These results suggest that the acidic moiety of lysophosphatidic acid is not an important structural determinant for expressing ATPase stimulatory activity in ghosts. Rather the nature of the hydrocarbon chain as well as the lyso structure of these compounds appear most critical under these conditions. The stimulatory effects of lysophosphatidic acids or lysophosphatidylcholines were additive to that induced with calmodulin, suggesting that these lysophospholipids affect the (Ca 2+ + Mg 2+)-ATPase by a mechanism which is different from that seen with calmodulin.

Effects of fatty acids on activity and calmodulin binding of Ca 2+-ATPase of human erythrocyte membranes

Activation and inhibition of Ca 2+-ATPase of calmodulin-depleted human erythrocyte membranes by oleic acid and a variety of other fatty acids have been measured. Low concentrations of oleic acid stimulate the enzyme activity, both in the presence and in the absence of calmodulin. Concomitantly, the affinity of the membrane bound enzyme to calmodulin progressively decreases due to competitive interactions of calmodulin and oleic acid with the enzyme. Removal of oleic acid from the membrane by serum albumin extinguishes the activating effect of oleic acid and restores the ability of the enzyme to bind calmodulin with high affinity. High concentrations of oleic acid induce an almost complete and irreversible loss of enzyme activity which cannot be abolished by removal of oleic acid. Despite a complete loss of enzyme activity, binding of calmodulin to membranes is approximately normal after removal of oleic acid. Activities of ( Na + + K +)- ATPase , Mg 2+-ATPase and acetylcholine esterase, as well as the total protein content, show no gross changes upon treatment of membranes with increasing amounts of oleic acid, which seems to exclude that membrane solubilisation by oleic acid causes an inactivation of the enzyme.

Inhibition of calmodulin-activated Ca 2+-ATPase by propranolol and nadolol

Propranolol, at concentrations ranging from 0.05 to 0.5 mM, inhibits the calmodulin-activated Ca 2+-ATPase of human erythrocyte membranes. In the same concentration range it is without effect on the basal Ca 2+-ATPase. The inhibition is competitive and appears to be due to membrane binding, rather than to combination with cytoplasmic calmodulin as is the case for phenothiazines. This effect of propranolol may explain its ability to open the calcium-gated potassium channel, and could also be related to its action as a β-adrenergic blocker. Nadolol, another β-adrenergic blocker, is also an inhibitor of calmodulin-activated Ca 2+-ATPase.

Acidic phospholipids, unsaturated fatty acids, and limited proteolysis mimic the effect of calmodulin on the purified erythrocyte Ca2+ - ATPase.

The purified Ca2+-pumping ATPase of human erythrocyte membranes (Niggli, V., Adunyah, E. S., Penniston, J. T., and Carafoli, E. (1981) J. Biol. Chem. 256, 395-401) can be stimulated, in the absence of calmodulin, by other treatments. 1. A variety of acidic phospholipids (phosphatidylserine, cardiolipin, phosphatidylinositol, and phosphatidic acid) stimulate the Vmax and decrease the Km (Ca2+) of the isolated enzyme to the same extent as calmodulin. Unsaturated fatty acids (oleic and linoleic acid) have the same effect as phospholipids but at lower concentrations. Neutral phospholipids (phosphatidylcholine, sphingomyelin, and phosphatidylethanolamine) have no effect on the enzyme. The minimal proportion of acidic phospholipids in the environment of the enzyme necessary for full stimulation is about 40%. 2. The isolated enzyme, after reconstitution in phosphatidylcholine liposomes in the absence of calmodulin, can be activated by limited proteolysis. The trypsinized enzyme has the same high Vmax and high affinity for Ca2+ of the enzyme in the presence of calmodulin.

Enhanced activation of human erythrocyte Ca 2+-ATPase by calmodulin after storage or brief exposure to disulfite

Ca 2+-ATPase of human erythrocyte membranes which are prepared from freshly drawn human blood can be activated by the calmodulin present in the hemolysate to 1.5-times the basal level. However, when the membranes are prepared from blood stored for 5–14 days the activation by calmodulin reaches 2.5-times the basal level. An enhanced reactivity to calmodulin of similar magnitude was produced by brief exposure of fresh erythrocytes to 25 mM Na 2S 2O 5 prior to isolation of the membranes. Reincubation of the activated cells in a disulfite-free medium restored the membrane-bound Ca 2+-ATPase to a state of normal reactivity to calmodulin. It is hypothesized that these results are related to the level of cytoplasmic Ca 2+ which is partly controlled by complex formation with 2,3-diphosphoglycerate, the concentration of which is diminished when its specific phosphatase is activated by Na 2S 2O 5.

Calmodulin an activator of human erythrocyte (Ca 2+ + Mg 2+)-ATPase phosphorylation

The effect of purified calmodulin on the calcium-dependent phosphorylation of human erythrocyte membranes was studied. Under the conditions employed, only one major peak of phosphorylation was observed when solubilized membrane proteins were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The molecular weight of this phosphorylated protein band was estimated to be 130 000 and in the presence of purified red blood cell calmodulin, the rate of phosphorylation of this band was increased. These data suggest that calmodulin activation of (Ca 2+ + Mg 2+)-ATPase could be a partial reflection of an increased rate of phosphorylation of the (Ca 2+ + Mg 2+)-ATPase of human erythrocyte membranes.

Calmodulin affinity chromatography yields a functional purified erythrocyte (Ca+ + Mg2+)-dependent adenosine triphosphatase.

The (Ca2+ + Mg2+)-dependent ATPase of human erythrocyte membranes was solubilized with deoxycholate and purified by calmodulin affinity chromatography to yield a functional enzyme. The method gave an enzyme purified 207-fold as compared with that of the erythrocyte membranes. The molecular weight of the ATPase was in the range 135 000-150 000, as revealed by a single major band after electrophoresis on dodecyl sulphate/polyacrylamide gels. The isolated enzyme was highly sensitive to calmodulin, since the activity was increased about 9-fold. At 37 degrees C and in the presence of calmodulin the purified ATPase had a specific activity of 10.1 mumol/min per mg of protein. Triton X-100 or deoxycholate stimulated the calmodulin-deficient enzyme in a concentration-dependent fashion whereby the calmodulin-sensitivity was lost. The purification method is suitable for studying the lipid-sensitivity of the ATPase, since the lipids can easily be exchanged without a significant loss of activity. A purification procedure described by Niggli, Penniston & Carafoli [(1979) J. Biol. Chem. 254, 9955-9958] resulted in an enzyme that indeed was pure but was lacking a predominant feature, namely the modulation by calmodulin.

Characterization of the phosphorylated intermediate of the isolated high-affinity (Ca 2+ + Mg 2+)-ATPase of human erythrocyte membranes

Phosphorylation of solubilized and purified high-affinity (Ca 2+ + Mg 2+)-ATPase (ATP phosphohydrolase, EC 3.6.1.3) of human erythrocyte membranes shows no dependence on cyclic AMP concentration in the range 0.1–1000 μM. Ca 2+-dependent phosphoprotein is sensitive to hydroxylamine and molybdate treatment. The phosphate linkage shows maximum stability at low pH values, which is progressively lost as the pH rises, with a shoulder around pH 6. SDS gel electrophoresis of the phosphorylated protein yields a peak which shows relative mobility corresponding to a molecular weight of 145 000 and sensitivity to MgATP-chase and hydroxylamine treatment. This indicates that the phosphoprotein represents the phosphorylated intermediate of the high-affinity (Ca 2+ + Mg 2+)-ATPase of human erythrocyte membranes.

Evidence of voltage-induced channel opening in Na/K ATPase of human erythrocyte membrane.

Previous studies have shown that human erythrocytes when subjected to a high voltage pulsation, in the microsecond time range, lysed in an isotonic medium. The hemolysis was the result of the colloid osmotic swelling, which, in turn, was caused by the voltage perforation of the red cell membranes. In this work we demonstrate that in a low ionic medium at least 35% of the pores was related to the opening of Na+/K4 ATPase channels. The membrane conductance generated by the externally applied electric field could be partially blocked by a specific inhibitor, ouabain, or by a specific cross-linkin g reagent, Cu++-phenanthroline, of the ATPase. The effect of ouabain was saturable and had a mid-point of saturation at 0.15 microM. This value agrees with the physiological inhibition constant of the drug. K+ ion in the external medium suppressed the effect of ouabain, as has also been demonstrated n physiological studies. Experiment presented in this communicaton also suggests that the Na+/K+ ATPase was not perforable in a high ionic medium, and that a large fraction of the voltage-induced pores occurred at as yet unidentified sites.

Inhibition of human erythrocyte Ca ++-transport ATPase by phenothiazines and butyrophenones

Antipsychotic agents such as the phenothiazines-trifluoperazine, fluphenazine and chlorpromazine- and the butyrophenones-penfluridol, fluspirilene and pimozide — which were shown to interact with calmodulin in a Ca ++-dependent fashion, competitively inhibit Ca ++-transport ATPase of human erythrocyte membranes. Activation of the ATPase by calmodulin is half-maximally antagonized by these agents in the concentration range of 2.6–22 μM whereas inhibition of the basal ATPase (in the absence of calmodulin) is achieved at about 10-fold higher concentrations.

Purification of the Ca2+-stimulated ATPase activator from human erythrocytes. Its membership in the class of Ca2+-binding modulator proteins

The activator of the Ca2+-stimulated ATPase of erythrocyte membranes was purified 13,000-fold to homogeneity from human erythrocytes. The protein gave a single band upon electrophoresis both with and without detergent, and upon isoelectric focusing. This protein was compared with Ca2+-binding modulator proteins from bovine brain and rat testis. All three proteins were homogeneous and co-migrated on electrophoresis both in the presence of detergent and without detergent at pH values on both sides of the isoelectric point of the protein. The amino acid compositions of the three proteins were nearly indistinguishable, and all three proteins contained 1 residue of the unusual amino acid, trimethyllysine. All three were also indistinguishable as measured by their ability to further stimulate the Ca2+-stimulated ATPase of human erythrocyte membranes. Thus, we conclude that they represent functionally the same protein. Upon storage of all three proteins, a second band was detectable by detergent gel electrophoresis; the biochemical activity and the behavior on nondetergent gels were not changed. The presence of this second band is probably responsible for previous reports of differences between the rat testis and bovine brain modulator protein. The possibility is discussed that this protein is a general intracellular Ca2+ receptor, which mediates the activities of Ca2+ as an intracellular messenger.

Calcium ion-dependent phosphorylation of human erythrocyte membranes

Calcium ions promote the rapid transfer of the terminal phosphate of ATP to a protein of human erythrocyte membranes. The concentration of Ca2+ for half-maximal effect is 7 muM. At nonlimiting ATP concentrations the level of 32P incorporated by the membranes is independent of the presence or absence of Mg2+. The number of phosphorylating sites in a single erythrocyte membrane is about 700. The influence of pH on the rate of hydrolysis of the bound phosphate and its rapid release on exposure to hydroxylamine are both consistent with an acylphosphate bond. The phosphate in the protein undergoes rapid turnover. Enzymatic splitting of the phosphate is stimulated by Mg2+ but not by Ca2+. It is proposed that Mg2+ accelerates the splitting of the phosphate by favoring the conversion of the phosphoprotein from a state of low reactivity to a state of high reactivity towards water. The reactions described probably are intermediate steps in the hydrolysis of ATP catalyzed by the Ca2+-dependent ATPase of human erythrocyte membranes.

Kinetics of (Na +,K +)-ATPase of human erythrocyte membranes : I. Activation by Na + and K +

The results of kinetic investigations on the ouabain-sensitive (Na +,K +)-ATPase (ATP phosphohydrolase, EC 3.6.1.3) of human erythrocyte membranes suggest the following sequence of reactions in the simultaneous presence of Na + and K +: 1. 1. The initial step is the binding of 1 K + per active centre. 2. 2. The formation of an enzyme-K + complex is a prerequisite for the binding of 2 Na + and MgATP 2- in a random reaction. Thus, the intermediates Na 2 +EK +, EK +- MgATP 2− and Na 2 +EK +- MgATP 2− can be formed. 3. 3. Only the Na 2 +EK +-- MgATP 2− intermediate decomposes into the phosphorylated intermediate Na 2 +EK +- P 1 and MgADP −. 4. 4. The final step of the rection is the splitting of Na 2 +EK +- P i into Na 2 +EK ++ P i .

Kinetics of (Na +,K +)-ATPase of human erythrocyte membranes : II. Inhibition by ouabain

Kinetic studies on the inhibition of (Na +,K +)-ATPase (ATP phosphohydrolase, EC 3+ .6.1.3) of human erythrocyte membranes by ouabain have led to the following conclusions: 1. 1. (Na +,K +)-ATPase is uncompetitively inhibited by ouabain at concentrations of Na + and K +, which are normally required for optimal activation. 2. 2. Inhibition experiments with ouabain refer to the existence of two types of phosphorylated intermediates, i.e. E K + - P i and Na 2 +E K +- P i . 3. 3. Ouabain reacts with both types of phosphorylated intermediates of the enzyme. The affinities between ouabain and E K + - P i , and Na 2 +E K+- P i , respectively, do not differ significantly. 4. 4. The binding of ouabain causes an elimination of K + from the phosphorylated intermediates. The relative degree of this ouabain inhibition is closely related to the K + concentration: The degree of inhibition is decreased by increasing K + concentrations and vice versa. In detail, the antagonism between K + and ouabain is comparable with a partially competitive type of inhibition. 5. 5. Since E K + - P i is not able to split into the end products E K + + P i , not only K +, but also Na + is essential for the release of inorganic phosphate from the active phosphorylated intermediate Na 2 +E K − - P i . 6. 6. The complete reaction mechanism, as derived from experiments with varied concentrations of Na +, K +, MgATP 2−, and ouabain includes the following enzyme intermediates: E K + , E K + - MgATP 2− , Na 2 +E K + , Na 2 +E K + - MgATP 2− , Na 2 +EI K +-P i , Na 2 +EI- P i , E K + - P i , EI K + - P i , and EI- P i .

Interaction of [formula omitted] and Ca 2+ with human erythrocyte membrane ATPase

Human erythrocyte membranes were treated with N- ethylmaleimide and then assayed for cation-sensitive components of ATPase activity. Inhibition of Na +-activated ATPase and (Na +-K +)-activated ATPase (ATP phosphohydrolase, EC 3.6.1.3), observed following N- ethylmaleimide treatment, was abolished when ethylene glycol bis-(β-aminoethyl)-N,N- tetraacetate (EGTA) was included in the assay system. N- Ethylmaleimide pretreatment reduced Mg 2+-activated ATPase activity both in the absence and presence of EGTA. The Mg 2+-dependent components of [ 14C]ADP-ATP exchange and membrane phosphorylation using [ γ- 32 P] ATP were reduced following N- ethylmaleimide pretreatment whereas the Na +-stimulated components of these activities were unaffected. At very low ATP concentration, chelation of endogenous Ca 2+ increases the sensitivity of the ATPase system to stimulation by low concentrations of Na +. In contrast a Ca 2+-requirement at a Na +-dependent step of (Na +-K +) activated ATPase reaction sequence was observed at higher ATP concentration. These results suggest both inhibitory and activating effects of Ca 2+ on alkali cation-sensitive ATPase.