The phosphatase activity of the plasma membrane Ca2+ pump. Activation by acidic lipids in the absence of Ca2+ increases the apparent affinity for Mg2+.

The purified PMCA supplemented with phosphatidylcholine was able to hydrolyze pNPP in a reaction media containing only Mg(2+) and K(+). Micromolar concentrations of Ca(2+) inhibited about 75% of the pNPPase activity while the inhibition of the remainder 25% required higher Ca(2+) concentrations. Acidic lipids increased 5-10 fold the pNPPase activity either in the presence or in the absence of Ca(2+). The activation by acidic lipids took place without a significant change in the apparent affinities for pNPP or K(+) but the apparent affinity of the enzyme for Mg(2+) increased about 10 fold. Thus, the stimulation of the pNPPase activity of the PMCA by acidic lipids was maximal at low concentrations of Mg(2+). Although with differing apparent affinities vanadate, phosphate, ATP and ADP were all inhibitors of the pNPPase activity and their effects were not significantly affected by acidic lipids. These results indicate that (a) the phosphatase function of the PMCA is optimal when the enzyme is in its activated Ca(2+) free conformation (E2) and (b) the PMCA can be activated by acidic lipids in the absence of Ca(2+) and the activation improves the interaction of the enzyme with Mg(2+).

A novel role of 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid as an activator of the phosphatase activity catalyzed by plasma membrane Ca2+-ATPase.

The hydrolysis of p-nitrophenyl phosphate catalyzed by the erythrocyte membrane Ca2+-ATPase is stimulated by low concentrations of the compound 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS), a classic inhibitor of anion transport. Enhancement of the phosphatase activity varies from 2- to 6-fold, depending on the Ca2+ and calmodulin concentrations used. Maximum stimulation of the pNPPase activity in ghosts is reached at 4-5 microM DIDS. Under the same conditions, but with ATP rather than pNPP as the substrate, the Ca2+-ATPase activity is strongly inhibited. Activation of pNPP hydrolysis by DIDS is equally effective for both ghosts and purified enzyme, and therefore is independent of its effect as an anion transport inhibitor. Binding of the activator does not change the Ca2+ dependence of the pNPPase activity. Stimulation is partially additive to the activation of the pNPPase activity elicited by calmodulin and appears to involve a strong affinity binding or covalent binding to sulfhydryl groups of the enzyme, since activation is reversed by addition of dithiothreitol but not by washing. The degree of activation of pNPP hydrolysis is greater at alkaline pH values. DIDS decreases the apparent affinity of the enzyme for pNPP whether in the presence of Ca2+ alone or Ca2+ and calmodulin or in the absence of Ca2+ (with 5 microM DIDS the observed Km shifts from 4.8 +/- 1.4 to 10.1 +/- 2.6, from 3.8 +/- 0.4 to 7.0 +/- 0.8, and from 9.3 +/- 0.7 to 15.5 +/- 1.1 mM, respectively). However, the pNPPase rate is always increased (as above, from 3.6 +/- 0.6 to 11.2 +/- 1.7, from 4.4 +/- 0.5 to 11.4 +/- 0.9, and from 2.6 +/- 0.6 to 18.6 +/- 3.9 nmol mg-1 min-1, in the presence of Ca2+ alone or Ca2+ and calmodulin or in the absence of Ca2+, respectively). ATP inhibits the pNPPase activity in the absence of Ca2+, both in the presence and in the absence of DIDS. Therefore, kinetic evidence indicates that DIDS does more than shift the enzyme to the E2 conformation. We propose that the transition from E2 to E1 is decreased and a new enzyme conformer, denoted E2*, is accumulated in the presence of DIDS.

[The activity and properties of K-pNPphosphatase in membrane preparations of erythrocytes with different polypeptide compositions in Wistar, Wistar-Kyoto and SHR strain rats]. / Aktivnost' i svoistva K-pNFfosfatazy v membrannykh preparatakh éritrotsitov s razlichnym polipeptidnym sostavom u krys linii Vistar, Vistar--Kioto i SHR.

A higher K-pNPPase activity was found in erythrocyte membrane preparations from the SHR as compared with the Wistar rats. Removal of peripheral proteins from the membrane skeleton depressed the K-phosphatase activity and eliminated the difference between the SHR and Wistar rats. No activating effect of the ATP and Ca on the enzyme was found. The data obtained suggest that the proteins of the erythrocyte membrane skeleton are connected with regulation of the enzyme transport in the erythrocyte membranes of the SHR via a modulating effect of intracellular ATP and Ca.

Cytochemical localization of K(+)-dependent p-nitrophenyl phosphatase and adenylate cyclase by using one-step method in human washed platelets.

Ultrastructural cytochemical localization of ouabain-sensitive, potassium dependent p-nitrophenyl phosphatase (K(+)-NPPase) of the Na(+)-/K(+)-ATPase complex and adenylate cyclase (cAMPase) activities, in washed inactivated human platelets, are described. The one-step lead-citrate method, under similar incubation conditions, was used to determine both activities. K(+)-NPPase appeared in both plasma membrane and the surface-connected canalicular system (SCCS) of the platelets. These data suggest a uniform distribution of the enzyme throughout membrane systems which are in contact with the external medium. cAMPase activity was strictly localized in tubules of the dense tubular system (DTS) when incubation medium contained prostaglandin E1, prostaglandin D2 or forskolin, at concentrations known to stimulate the enzyme in platelets that are intact. This fact and the inhibition of cytochemical reaction by thrombin confirm that the one-step lead-citrate method is a useful procedure in determining adenylate cyclase, abolishing the unfavorable conditions of previously reported methods.

A p-nitrophenylphosphatase activity associated with the human erythrocyte membrane.

A p-nitrophenylphosphatase activity has been identified as a component of the human erythrocyte membrane. This activity is distinct from that associated with the cell's Na(+)+K(+)-dependent ATPase, Ca(2+)-dependent ATPase, or spectrin phosphatase. The activity described here is stimulated by Mn2+ but not by Ca2+ with or without calmodulin. A potential erythrocyte membrane substrate for this activity is a 95 kDa phosphoprotein that can be shown to undergo Mn(2+)-stimulated but not Mg(2+)-stimulated dephosphorylation.

The calmodulin-binding domain as an endogenous inhibitor of the p-nitrophenylphosphatase activity of the Ca2+ pump from human red cells.

Digestion of red cell membranes with chymotrypsin elicited p-nitrophenylphosphatase activity. During digestion, the p-nitrophenylphosphatase appeared in parallel with the activation of the Ca(2+)-ATPase (in the absence of calmodulin). The chymotrypsin-activated p-nitrophenylphosphatase was inhibited by C20W, a 20 amino acid peptide modelled after the sequence of the calmodulin-binding site of the red cell Ca2+ pump (Vorherr et al. (1990) Biochemistry 29, 355-365). On the contrary, the (ATP + Ca(2+)-dependent p-nitrophenylphosphatase activity of intact red cell membranes was not affected by C20W. Ca2+ inhibited the chymotrypsin-induced p-nitrophenylphosphatase (Ki for Ca2+ = 2 microM). In the absence of ATP, C20W and Ca2+ did not interact in apparent affinity as inhibitors of this activity. On the other hand, in the presence of 2 mM ATP, Ca2+ antagonized the inhibition produced by C20W. The results are consistent with the idea that the calmodulin-binding site is an 'autoinhibitory domain' of the Ca2+ pump, and that removal of this domain by proteolysis, or its modification by calmodulin binding is the reason for the activation of both the ATPase and the p-nitrophenylphosphatase activity of the pump. The results presented in this paper give new information about the mechanism of the two kinds of p-nitrophenylphosphatase and about the nature of the apparent competition between C20W and Ca2+.

[Characterization of changes in the activity of K+-n-nitrophenylphosphatase of erythrocyte ghosts under the influence of ouabain]. / Osobennosti izmeneniia aktivnosti K+-n-nitrofenilfosfatazy tenei éritrotsitov pod vliianiem uabaina.

Variation of K+-p-NPP-ase of human ghosts under the action of ouabain (10(-11)-10(-3) M) has been studied. In human ghosts the activity of ouabain-sensitive K+-p-NPP-ase has been found to make up 65% of the total enzyme activity. The activity of ouabain-sensitive K+-p-NPP-ase reaches a maximum level at pH 7.6-8.0. A decrease in the activity of this enzyme caused by ouabain is of two-phase character. In the range of ouabain concentration from 10(-10) M to 10(-6) M and from 10(-5) M to 10(-3) M the enzyme activity lowers significantly; in the range of 10(-7) M to 10(-5) M it reaches the plato. Two types of the enzyme are assumed to exist differing by 4-5 orders of magnitude in their sensitivity to ouabain, inhibitor affinity constants and Michaelis constants.

Allosteric transition of erythrocyte alkaline phosphatase from Duchenne muscular dystrophy (DMD) patients and Duchenne muscular dystrophy carriers (Homo sapiens).

1. The kinetic properties of the p-nitrophenylphosphatase (EC 3.1.3.1) from erythrocytes was investigated in DMD-patients and DMD-carriers. 2. A different allosteric behaviour in the p-nitrophenylphosphatase from DMD-patients and DMD-carriers compared to controls is supported by the following findings: (a) values of n altered in F- inhibition of (K+)-activated p-nitrophenylphosphatase with Hill coefficients -1.5, -2.2 and -3.1; (b) heterotropic effect of increased concentration of Mg2+ on F- inhibition which is reverted by K+ in DMD-carriers and in control, but not in DMD-patients. 3. Evidence is presented showing that in DMD-patients and in DMD-carriers the interaction membrane-enzyme is different from the corresponding controls.

Calmodulin and ATP dependence of the high and low calcium affinity p-nitrophenylphosphatase from human erythrocyte membranes.

In calmodulin depleted membranes from human erythrocytes, the Ca2+-dependent phosphatase showed different sensitivity to calmodulin and ATP with variable affinity towards free calcium concentrations: a calmodulin-dependent activity with high calcium affinity, K1/2 = 1.2 X 10(-7) mol/l calcium, that was fully activated at submicromolar calcium concentrations, higher concentrations being rather inhibitory; an ATP-dependent activity with lower calcium affinity, K1/2 = 10(-6) mol/l calcium, that was fully activated at 10(-5) mol/l calcium in the presence of 50-200 mumol/l ATP and was insensitive to calmodulin, and a calcium dependent phosphatase that was active at a wider ranger of free calcium, 10(-8)-10(-5) mol/l, and required the presence of both calmodulin and ATP.

Partial purification and characterization of phosphotyrosyl-protein phosphatase(s) from human erythrocyte cytosol.

Phosphotyrosyl-protein phosphatase activity of human erythrocyte cytosol can be resolved into two fractions by DEAE-cellulose chromatography followed by P-cellulose chromatography. Both 32P-Tyr-phosphatases are able to dephosphorylate 32P-Tyr of poly (Glu-Tyr) 4:1 but not angiotensin II and synthetic peptide Asp-Ala-Glu-Tyr-Ala-Ala-Arg-Arg-Gly, previously phosphorylated on tyrosine residues by rat spleen tyrosine-protein kinase. Both 32P-Tyr-phosphatase activities distinctly differ from either 32P-Ser-casein phosphatase activity or "acid" and "alkaline" p-nitrophenylphosphatase activities with regard to catalytic and physico-chemical properties such as substrate specificity, chromatographic behaviour, response to various effectors.

The p-nitrophenyl phosphatase activity of ubiquitin from bovine erythrocytes.

Ubiquitin, a unique protein with esterase and carbonic anhydrase activity, has been found to have also a p-nitrophenyl phosphatase activity. This phosphomonoesterase activity of ubiquitin has an acidic pH optimum; its true substrate appears to be the phosphomonoanion, with a Km of 1.8 X 10(-3) M. It is competitively inhibited by the typical acid phosphatase inhibitors, arsenate (Ki = 1.3 X 10(-3) M), molybdate (Ki = 1.2 X 10(-6) M), and phosphate (Ki = 1.4 X 10(-3) M). These inhibitors have no effect on the CO2 hydration and p-nitrophenyl acetate esterase activities of the ubiquitin. Acetazolamide slightly inhibited the p-nitrophenyl phosphatase activity.

Two Ca2+-requiring p-nitrophenylphosphatase activities of the highly purified Ca2+-pumping adenosinetriphosphatase of human erythrocyte membranes, one requiring calmodulin and the other ATP.

The highly purified Ca2+-pumping ATPase from human erythrocyte membranes displays two p-nitrophenylphosphatase (NPPase) activities: one of these requires calmodulin and low concentrations of Ca2+, while the other requires ATP and higher Ca2+ concentrations. The free Ca2+ concentrations required for the expression of the two NPPase activities differed very substantially. Both activities required high free Mg2+ concentrations and displayed simple hyperbolic kinetics toward p-nitrophenyl phosphate (NPP) with a Km in the range of 5-20 mM. Study of the dependence of the calmodulin-stimulated NPPase on Mg2+ and NPP indicated that the Mg-NPP complex is not the substrate of the enzyme. Under conditions optimal for ATP-requiring NPPase (1 mM free Ca2+), the Ca2+-ATPase displayed simple hyperbolic kinetics with a low Km for ATP. NPP competitively inhibited this activity, and the apparent Ki for NPP was less than 1 mM, much lower than the Km for NPP as a substrate. If NPP were inhibiting the ATPase by binding at the same site at which NPP is hydrolyzed, the apparent Ki for NPP as inhibitor would be the same as the Km for NPP as substrate. (Under these circumstances, the apparent Ki and the Km can be directly compared, since NPP was being hydrolyzed under both circumstances.) Since Ki was much lower than Km, NPP must have been inhibiting at another site; thus, these data show the existence of two types of NPP sites on the enzyme, one at which NPP is hydrolyzed and the other at which it inhibits ATP hydrolysis.(ABSTRACT TRUNCATED AT 250 WORDS)

Effect of benzyl alcohol on adenosine triphosphatase, p-nitrophenylphosphatase and acetylcholinesterase in rat erythrocyte membrane.

The irreversible effect of benzyl alcohol on ATPase, p-nitrophenylphosphatase and acetylcholinesterase in the erythrocyte membrane of rats was demonstrated. The ATPase activity in the membranes was stimulated with 10 -70 mM benzyl alcohol and inhibited by concentrations greater than 80 mM. p-Nitrophenylphosphatase was gradually inhibited by concentrations of benzyl alcohol greater than 30 mM. The acetylcholinesterase activity was not affected by concentrations below 100 mM and strongly inhibited by concentrations of benzyl alcohol greater than 150 mM. With the uptake studies of 14C-labeled benzyl alcohol by membranes, the highest uptake was obtained in the presence of 200 mM of benzyl alcohol. And SDS-polyacrylamide gel electrophoresis showed a binding of benzyl alcohol to the major protein bands of the erythrocyte membrane. Therefore, stimulation of the ATPase activity appeared to be the result of an increase in ion uptake due to an increase in the fluidity of the membrane lipid by benzyl alcohol, and the inhibition of the enzymes may be the result of benzyl alcohol-induced denaturation of the membrane components. The difference in the observed inhibition patterns among ATPase, p-nitrophenylphosphatase and acetylcholinesterase may be related to the sensitivity of benzyl alcohol on those enzymes.

Properties of p-nitrophenyl phosphatase activity from porcine neutrophils.

p-nitrophenyl phosphatase activity is high in porcine neutrophils and was found in plasma membrane and granule fractions isolated from sucrose density gradients after nitrogen cavitation to disrupt the cells. Very little activity was found in the cytosol. The enzyme has optimum activity at alkaline pHs with a pH optimum of 10.3. The pH profile was fairly broad with activity still remaining at physiological pH. Orthovanadate was shown to be a potent competitive inhibitor of the enzyme with a Ki of 14 microM. Phosphate also inhibited but at millimolar concentrations and the two inhibitors bind in a mutually exclusive fashion. Evidence from experiments using divalent ion chelators and zinc ions suggested that the phosphatase is a zinc metalloenzyme. Beryllium was found to be a very potent, non-competitive inhibitor of the neutrophil enzyme (Ki = 1.1 microM). Levamisole and theophylline were both shown to be uncompetitive inhibitors of the porcine phosphatase (Ki = 0.2 mM and 1.2 mM respectively). The neutrophil phosphatase was inhibited by L-homoarginine but unaffected by L-phenylalanine and L-glutamate.

4-nitrophenyl phosphatase activity of the red blood cell membrane in essential hypertension.

In this paper we report the levels of the 4-nitrophenyl phosphate hydrolysing activity of the red blood cell membrane in 46 hypertensive patients as compared to 41 normal controls and eight secondary hypertensives. This activity has at least two components; one of them is dependent on the presence of magnesium and potassium ions, and more sensitive to sodium, ATP, heat and -SH blockers than the cation-independent activity. This component appears increased in membranes from essential hypertension patients, correlating to the clinical seriousness of the condition, while remaining at control level in the secondary hypertension patients. The cation-independent component of this activity does not differ significantly in any of the groups studied.

Alterations of globin chain synthesis and of red cell membrane proteins in congenital dyserythropoietic anemia I and II.

Red cell membrane proteins were investigated in two unrelated children with congenital dyserythropoietic anemia (CDA) I and two siblings with CDA II. The CDA I patients displayed globin chain synthesis imbalance, with reduction of the non alpha/alpha ratio. One of the CDA II patients presented the reverse alteration. Whenever globin chain synthesis was unbalanced, the membrane p-nitrophenylphosphatase had an abnormally biphasic kinetics, consistent with substrate excess inhibition, as is observed in alpha- or beta-thalassemic syndromes. One CDA I patient displayed a decrease of electrophoretic band 4.1 along with an ectopic phosphorylated protein at the level of band 4.2. In CDA II and, to a lesser extent, in CDA I, the in vitro endogenous phosphorylation of band 2 + 2.1 was sharply reduced.