ATPase Of Erythrocyte Ghosts Research Articles

Long-term stabilization and crystallization of (Ca 2+ + Mg 2+)-ATPase of detergent-solubilized erythrocyte plasma membrane

Conditions which were optimal for the stabilization of Ca 2+-transporting ATPase in solubilized sarcoplasmic reticulum membranes (Pikula, S., Mullner, N., Dux, L. and Martonosi, A. (1988) J. Biol. Chem. 263, 5277–5286) were also found conductive for preservation of (Ca 2+ + Mg 2+)-ATPase activity in detergent-solubilized erythrocyte plasma membrane for up to 60 days. Of particular importance for the stabilization of calmodulin-stimulated Ca 2+-dependent activity of (Ca 2+ + Mg 2+)-ATPase of solubilized erythrocyte plasma membrane was the presence of Ca 2+ (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 (Ca 2+ + Mg 2+)-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 (Ca 2+ + Mg 2+)-ATPase differ from those of Ca 2+-ATPase of detergent-solubilized sarcopiasmic reticulum microsomes.

Preliminary findings on calmodulin-stimulated Ca2+-ATPase of erythrocyte ghosts in psychotic patients.

Kinetic properties of the calmodulin-stimulated erythrocyte ghost Ca2+-ATPase seem to be altered in some sub-groups of affective and schizophrenic psychoses. The sub-group of affective disorders concerned mostly unipolar manic and bipolar psychoses with predominantly manic episodes. In the corresponding cases from schizophrenics hyper- and parakinetic basic syndromes were predominantly diagnosed. An evaluation of our preliminary results was undertaken in connection with our biochemical hypothesis on possible alterations in the regulation of Ca2+ concentrations and Ca2+-calmodulin-mediated processes under certain psychotic conditions.

Comparison of the membrane-bound (Ca 2+ + Mg 2+)-ATPase in erythrocyte ghosts from some mammalian species

1. 1. The properties of the membrane-bound calcium-pumping protein, the (Ca 2+ + Mg 2+)-ATPase (ATP phosphohydrolase, EC 3.6.1.3) were compared in erythrocyte ghosts isolated from five mammalian species—human ( Homo sapiens), bovine ( Bos taurus), porcine ( Sus scrofa malitensis), ovine ( Ovis aries crassicandus) and caprine ( Capra hircus syriaca). 2. 2. The specific activity of the enzyme in porcine erythrocytes is one order of magnitude higher than in the other species. It was also stimulated to various extents by the regulator protein, calmodulin, and by phosphatidylinositol in all the species. 3. 3. Analysis of membrane proteins revealed a number of differences which seem to suggest that the molecular architecture of the red cell membrane influences the activity of the enzyme.

Ca 2+-stimulated, Mg 2+-independent ATP hydrolysis and the high affinity Ca 2+-pumping ATPase. Two different activities in rat kidney basolateral membranes

The Mg 2+-dependency of Ca 2+-induced ATP hydrolysis is studied in basolateral plasma membrane vesicles from rat kidney cortex in the presence of CDTA and EGTA as Mg 2+- and Ca 2+-buffering ligands. ATP hydrolysis is strongly stimulated by Mg 2+ with a K m of 13 μ M in the absence or presence of 1 μ M free Ca 2+. At free Mg 2+ concentrations of 1 μ M and lower, ATP hydrolysis is Mg 2+ -independent, but is strongly stimulated by submicromolar Ca 2+ concentrations K m  0.25 μM, V max  24 μmol P i/h per mg protein). The Ca 2+-stimulated ATP hydrolysis strongly decreases at higher Mg 2+ concentrations. The Ca 2+-stimulated Mg 2+-independent ATP hydrolysis is not affected by calmodulin or trifluoperazine and shows no specificity for ATP over ADP, ITP and GTP. In contrast, at high Mg 2+ concentrations calmodulin and trifluoperazine affect the high affinity Ca 2+-ATPase activity significantly and ATP is the preferred substrate. Control studies on ATP-dependent Ca 2+-pumping in renal basolaterals and on Ca 2+-ATPase in erythrocyte ghosts suggest that the Ca 2+-pumping enzyme requires Mg 2+. In contrast, a role of the Ca 2+-stimulated Mg 2+-independent ATP hydrolysis in active Ca 2+ transport across basolateral membranes is rather unlikely.

Palytoxin binds to and inhibits kidney and erythrocyte Na+, K+-ATPase.

Hog kidney Na+, K+-ATPase, purified to the microsomal stage and activated with detergent, binds palytoxin, as shown by the nearly complete competition of the toxin with 3H-ouabain. The Ki-values of palytoxin, but not of ouabain, depend on the protein concentration; this indicates additional binding sites for the toxin on kidney membranes. - Palytoxin inhibits the enzymatic activity of the detergent-activated preparation nearly completely (IC50 8 X 10(-7) mol/l). Inhibition of ATPase activity and of ouabain binding are promoted by borate, a known activator of palytoxin. - Palytoxin also inhibits the Na+,K+-ATPase of erythrocyte ghosts in the same dose range. The data are discussed in context with the hypothesis (Chhatwal et al. 1983) that palytoxin raises the cellular permeability by altering the state of Na+,K+-ATPase or its environment.

Sodium—Potassium Pump Activity in Mineralocorticoid- and Glucocorticoid-Induced Hypertension

1. The activity of ouabain-sensitive Na+,K+-dependent ATPase and ouabain-sensitive ATPase was measured in the microsomal fractions of kidney, liver and heart tissue of Sprague-Dawley rats treated with DOCA (10 mg day−1 kg−1 for 6 days, systolic blood pressure 143 ± 13 mmHg, n = 9) or 6 α-methylprednisolone (100 mg day−1 kg−1 for 6 days, systolic blood pressure 140 ± 19 mmHg, n = 9) and of a control group (systolic blood pressure 124 ± 12 mmHg, n = 9). 2. In the kidney, the ouabain-sensitive Na+,K+-ATPase activity (μmol of phosphate h−1 mg−1 of protein) was increased in the DOCA-treated (33.0 ± 6.9) and in the prednisolone-treated groups (30.8 ± 6.9) compared with that in the control group (26.4 ± 3.4) (P < 0.05). In the liver, the ouabain-sensitive Na+,K+-ATPase activity was elevated in the prednisolone-treated animals only (4.8 ± 1.3 vs 3.1 ± 0.8 in the controls, P < 0.025). The ouabain-sensitive Na+,K+-ATPase activity in heart tissue was similar in all three groups. The ouabain-insensitive ATPase activity was not altered by DOCA or prednisolone in the tissues studied. 3. In a separate study, the activity of the ouabain-sensitive Na+,K+-ATPase in erythrocyte ghosts was found to be elevated in 10 patients with Cushing's syndrome (0.91 ± 0.35 μmol of phosphate h−1 mg−1 of protein) compared with five patients with primary aldosteronism and compared with 12 normotensive control subjects (0.38 ± 0.08) (P < 0.005). 4. These data demonstrate an increased Na-K pump activity in the kidney in mineralocorticoid-induced hypertension. Glucocorticoids in addition activate the Na-K pump in a variety of other tissues. This could partially explain the redistribution of volume from the intracellular to the extracellular space.

Effects of calmodulin on the (Ca 2+ + Mg 2+)ATPase partially purified from erythrocyte membranes

The stimulation of the (Ca 2+ + Mg 2+)ATPase of erythrocyte ghosts by calmodulin was observed not only in intact ghosts, but also in the solubilized (Triton X-100) and partially purified, reconstituted (phosphatidylserine liposomes) forms. Since the solubilized form of the enzyme migrated on Sepharose 6B at a position corresponding to a molecular weight of about 150,000, these results show that calmodulin stimulates by direct interaction with the ATPase complex. Additionally, the effects of calmodulin on erythrocyte ghosts prepared by the Dodge-EDTA method (hypotonic ghosts) and by the method of Ronner et al. (involving lysis followed by an isotonic wash repeated several times) were compared (P. Ronner, P. Gazzotti, and E. Carafoli, 1977, Arch. Biochem. Biophys. 179, 578–583) . The (Ca 2+ + Mg 2+)ATPase of the hypotonic ghosts was low and was stimulated by added calmodulin while that of the isotonic ghosts was high and changed only slightly upon calmodulin addition; this difference in response to calmodulin persisted in the solubilized and reconstituted forms. Hypotonic ghosts bound 125I-labeled calmodulin, while isotonic ghosts did not. This comparison of two types of ghosts showed that isotonic ghosts possess an intact calmodulin-(Ca 2+ + Mg 2+)ATPase complex, and that the calmodulin remained with the ATPase during solubilization and reconstitution. The isotonic preparation is a particularly useful method of preparing ghosts with an intact calmodulin-ATPase complex, since it requires no special equipment and produces an enzyme activity which is stable to freezing.

Erythrocyte ghosts (Na+ + K+) ATPase activity in Duchenne's dystrophy and myotonia.

In Duchenne muscular dystrophy the activity of (Na+ + K+)ATPase in erythrocyte ghosts is reduced and its reaction to ouabain is paradoxical both in low sodium and high sodium systems. No such changes were seen in a case of Becker dystrophy, in limb-girdle dystrophy, and in neurogenic atrophy of muscles. In myotonic dystrophy and congenital myotonia the activity of ATPase and its inhibition by ouabain were depressed.

Ouabain and erythrocyte-ghost adenosine triphosphatase. Effects in human muscular dystrophies.

An adenosine triphosphatase (ATPase) of erythrocyte ghosts from all six patients with Duchenne dystrophy and three of six patients with limb-girdle dystrophy was stimulated by 10-4 molar ouabain but inhibited by the same concentration of ouabain in 11 normal subjects, one patient with Becker dystrophy, and eight other neurological or muscular disorders. The inhibited response was also observed in two possible carriers of Duchenne dystrophy. The abnormality of erythrocyte ghost ATPase in patients with Duchenne dystrophy was not related to age, disability, or serum creatine phosphokinase. The mechanism of the abnormality of ATPase in erythrocyte ghosts from all patients with Duchenne dystrophy and some patients, but not all, with limb-girdle dystrophy is not known (24:187-200, 1971). Key Words.— Ouabain, erythrocyte-ghost ATPase, membrane ATPase, sodium-potassium-ATPase, human muscular dystrophies, Duchenne dystrophy, limb-girdle dystrophy.