Calmodulin an activator of human erythrocyte (Ca2+ + Mg2+)-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 (Ca²⁺ + Mg²⁺)-ATPase could be a partial reflection of an increased rate of phosphorylation of the (Ca²⁺ + Mg²⁺)-ATPase of human erythrocyte membranes.     

A monoclonal antibody to the calmodulin-binding (Ca2+ + Mg2+)-dependent ATPase from pig stomach smooth muscle inhibits plasmalemmal (Ca2+ + Mg2+)-dependent ATPase activity.

A monoclonal antibody (2B3) directed against the calmodulin-binding (Ca2+ + Mg2+)-dependent ATPase from pig stomach smooth muscle was prepared. This antibody reacts with a 130,000-Mr protein that co-migrates on SDS/polyacrylamide-gel electrophoresis with the calmodulin-binding (Ca2+ + Mg2+)-ATPase purified from smooth muscle by calmodulin affinity chromatography. The antibody causes partial inhibition of the (Ca2+ + Mg2+)-ATPase activity in plasma membranes from pig stomach smooth muscle, in pig erythrocytes and human erythrocytes. It appears to be directed against a specific functionally important site of the plasmalemmal Ca2+-transport ATPase and acts as a competitive inhibitor of ATP binding. Binding of the antibody does not change the Km of the ATPase for Ca2+ and its inhibitory effect is not altered by the presence of calmodulin. No inhibition of (Ca2+ + Mg2+)-ATPase activity or of the oxalate-stimulated Ca2+ uptake was observed in a pig smooth-muscle vesicle preparation enriched in endoplasmic reticulum. These results confirm the existence in smooth muscle of two different types of Ca2+-transport ATPase: a calmodulin-binding (Ca2+ + Mg2+)-ATPase located in the plasma membrane and a second one confined to the endoplasmic reticulum.     

Inhibitory antibodies to plasmalemmal Ca2+-transporting ATPases. Their use in subcellular localization of (Ca2+ + Mg2+)-dependent ATPase activity in smooth muscle.

Antibodies directed against the purified calmodulin-binding (Ca2+ + Mg2+)-ATPase [(Ca2+ + Mg2+)-dependent ATPase] from pig erythrocytes and from smooth muscle of pig stomach (antral part) were raised in rabbits. Both the IgGs against the erythrocyte (Ca2+ + Mg2+)-ATPase and against the smooth-muscle (Ca2+ + Mg2+)-ATPase inhibited the activity of the purified calmodulin-binding (Ca2+ + Mg2+)-ATPase from smooth muscle. Up to 85% of the total (Ca2+ + Mg2+)-ATPase activity in a preparation of KCl-extracted smooth-muscle membranes was inhibited by these antibodies. The (Ca2+ + Mg2+)-ATPase activity and the Ca2+ uptake in a plasma-membrane-enriched fraction from this smooth muscle were inhibited to the same extent, whereas in an endoplasmic-reticulum-enriched membrane fraction the (Ca2+ + Mg2+)-ATPase activity was inhibited by only 25% and no effect was observed on the oxalate-stimulated Ca2+ uptake. This supports the hypothesis that, in pig stomach smooth muscle, two separate types of Ca2+-transport ATPase exist: a calmodulin-binding ATPase located in the plasma membrane and a calmodulin-independent one present in the endoplasmic reticulum. The antibodies did not affect the stimulation of the (Ca2+ + Mg2+)-ATPase activity by calmodulin.     

Enhancement of (Ca2+ + Mg2+)-ATPase activity of human erythrocyte membranes by hemolysis in isosmotic imidazole buffer. I. General properties of variously prepared membranes and the mechanism of the isosmotic imidazole effect.

1. Membranes prepared from human erythrocytes hemolyzed in isosmotic (310 imosM) imidazole buffer, pH 7.4, show enhanced and stabilized (Ca2+ + Mg2+)-ATPase activity compared with membranes prepared from erythrocytes hemolyzed in hypotonic (20 imosM) phosphate or imidazole buffer, pH 7.4. 2. Exposure of intact erythrocytes or well-washed erythrocyte membranes to isosmotic imidazole does not cause enhanced (Ca2+ + Mg2+)-ATPase activity. 3. Exposure of erythrocyte membranes, in the presence of isosmotic imidazole, to the supernatant of erythrocyte hemolysis or to a partially purified endogenous (Ca2+ + Mg2+)-ATPase activator, promotes enhanced (Ca2+ + Mg2+)-ATPase activity. Under appropriate conditions, NaCl can be shown to substitute for imidazole. The results demonstrate that imidazole does not act directly on the erythrocyte membrane but rather by promoting interaction between an endogenous (Ca2+ + Mg2+)-ATPase activator and the erythrocyte membrane.     

Cyclic GMP-dependent protein kinase stimulates the plasmalemmal Ca2+ pump of smooth muscle via phosphorylation of phosphatidylinositol.

The effect of phosphorylation by cyclic GMP-dependent protein kinase (G-kinase) on the activity of the plasmalemmal Ca2+-transport ATPase was studied on isolated plasma membranes and on the ATPase purified from pig erythrocytes and from the smooth muscle of pig stomach and pig aorta. Incubation with G-kinase resulted, in both smooth-muscle preparations, but not in the erythrocyte ATPase, in a higher Ca2+ affinity and in an increase in the maximal rate of Ca2+ uptake. Cyclic AMP-dependent protein kinase (A-kinase) did not exert such an effect. The stimulation of the (Ca2+ + Mg2+)-dependent ATPase activity of the purified Ca2+ pump reconstituted in liposomes depended on the phospholipid used for reconstitution. The stimulation of the (Ca2+ + Mg2+)-ATPase activity by G-kinase was only observed in the presence of phosphatidylinositol (PI). G-kinase, but not A-kinase, stimulated the phosphorylation of PI to phosphatidylinositol phosphate (PIP) in a preparation of (Ca2+ + Mg2+)-ATPase obtained by calmodulin affinity chromatography from smooth muscle, but not in a similar preparation from erythrocytes. Adenosine inhibited both the phosphorylation of PI and the stimulation of the (Ca2+ + Mg2+)-ATPase by G-kinase. In the absence of G-kinase the (Ca2+ + Mg2+)-ATPase was stimulated by the addition of PIP, but not by PI. In contrast with previous results of Furukawa & Nakamura [(1987) J. Biochem (Tokyo) 101, 287-290], no convincing evidence for a phosphorylation of the (Ca2+ + Mg2+)-ATPase was found. Evidence is presented showing that the apparent phosphorylation occurs in a contaminant protein, possibly myosin light-chain kinase. It is proposed that G-kinase stimulates the plasmalemmal Ca2+ pump of smooth-muscle cells indirectly via the phosphorylation of an associated PI kinase.     

Decrease of apparent calmodulin affinity of erythrocyte (Ca2+ + Mg2+)-ATPase at low Ca2+ concentrations

The calmodulin activation of the (Ca2+ + Mg2+)-ATPase (ATP phosphohydrolase, EC 3.6.1.3) in human erythrocyte membranes was studied in the range of 1 nM to 40 microM of purified calmodulin. The apparent calmodulin-affinity of the ATPase was strongly dependent on Ca2+ and decreased approx. 1000-times when the Ca2+ concentration was reduced from 112 to 0.5 microM. The data of calmodulin (Z) activation were analyzed by the aid of a kinetic enzyme model which suggests that 1 molecule of calmodulin binds per ATPase unit and that the affinities of the calcium-calmodulin complexes (CaiZ) decreases in the order of Ca3Z greater than Ca4Z greater than Ca2Z greater than or equal to CaZ. Furthermore, calmodulin dissociates from the calmodulin-saturated Ca2+-ATPase in the range of 10(-7)-10(-6) M Ca2+, even at a calmodulin concentration of 5 microM. The apparent concentration of calmodulin in the erythrocyte cytosol was determined to be 3 to 5 microM, corresponding to 50-80-times the cellular concentration of Ca2+-ATPase, estimated to be approx. 10 nmol/h membrane protein. We therefore conclude that most of the calmodulin is dissociated from the Ca2+-transport ATPase in erythrocytes at the prevailing Ca2+ concentration (probably 10(-7)-10(-8) M) in vivo, and that the calmodulin-binding and subsequent activation of the Ca2+-ATPase requires that the Ca2+ concentration rises to 10(-6)-10(-5) M.     

The effect of calmodulin on the interaction of carbodiimides with the purified human erythrocyte (Ca2+ + Mg2+)-ATPase

The activity of the solubilized and purified (Ca2+ + Mg2+)-ATPase from human erythrocyte membranes was inhibited by N,N'-dicyclohexylcarbodiimide in a concentration-dependent manner. The carbodiimide prevented formation of the phosphorylated intermediate during the catalytic cycle of the enzyme. Treatment of the enzyme with N,N'-dicyclohexyl[14C]carbodiimide resulted in the formation of a 14C-labelled polypeptide corresponding to the enzyme monomer (molecular weight 136,000). The tryptic fragmentation of this 14C-labelled enzyme resulted in the formation of three major 14C-labelled fragments with molecular weights of 58,000, 36,500 and 23,000, the latter two probably representing transmembrane and calmodulin-binding domains of the enzyme, respectively. In the absence of calmodulin, 6.7 molecules of N,N'-dicyclohexyl[14C]carbodiimide covalently bound to each molecule of Ca2+-ATPase; in the presence of calmodulin, the number of molecules of carbodiimide bound was 13.1. The binding of N,N'-dicyclohexylcarbodiimide to the (Ca2+ + Mg2+)-ATPase greatly reduced its ability to bind to a calmodulin-agarose gel.     

Ca2+-stimulated, Mg2+-dependent ATPase in bovine thyroid plasma membranes

An isolated plasma membrane fraction from bovine thyroid glands contained a Ca2+-stimulated, Mg2+-dependent adenosine triphosphatase ((Ca2+ + Mg2+)-ATPase) activity which was purified in parallel to (Na+ + K+)-ATPase and adenylate cyclase. The (Ca2+ + Mg2+)-ATPase activity was maximally stimulated by approx. 200 microM added calcium in the presence of approx. 200 microM EGTA (69.7 +/- 5.2 nmol/mg protein per min). In EGTA-washed membranes, the enzyme was stimulated by calmodulin and inhibited by trifluoperazine.     

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.     

An endogenous inhibitor protein of synaptic plasma membrane (Ca2+ + Mg2+)-ATPase

An inhibitor protein of synaptic plasma membrane (Ca2+ + Mg2+)-ATPase was purified to apparent homogeneity from rat cerebrum by a molecular weight cut followed by chromatography of cytosol proteins with molecular weights between 10 000 and 3500 on DEAE-Sephadex at pH 5.2. The inhibitor could be partially inactivated by proteinases and dithiothreitol, but was heat-stable. Gel filtration gave a molecular weight of about 6000. Like the (Ca2+ + Mg2+)-ATPase inhibitor protein isolated from erythrocytes, the inhibitor from brain contains a characteristic high proportion of glutamic acid (36%) and glycine (37%) residues. Synaptic plasma membrane Mg2+-ATPase and microsomal membrane (Ca2+ + Mg2+)-ATPase did not respond to the inhibitor. Synaptic plasma membrane and erythrocyte membrane (Ca2+ + Mg2+)-ATPases, however, were affected. Inhibitory influence on synaptic membrane (Ca2+ + Mg2+)-ATPase was reversible, since inhibition could be relieved upon removal of inhibitor from saturable sites on the membrane. The inhibitor is not a calmodulin-binding protein, since the concentration of calmodulin for half-maximal activation of the ATPase was unaffected by its presence. Mode of inhibition of the (Ca2+ + Mg2+)-ATPase by the inhibitor was non-competitive.     

Isolation from haemolysate of a proteinaceous inhibition of the red cell Ca2+-pump ATPase. Its action on the kinetics of the enzyme

The purification to apparent homogeneity of a small protein from the cytosol of human red cells is described. The procedure consists of a combination of anion-exchange-chromatography, ultrafiltration, (NH4)2SO4- and heat-precipitation. The resulting protein is a potent inhibitor of (Ca2+ + Mg2+)-ATPase of erythrocyte membranes and of Ca2+-uptake into inside-out vesicles. Membrane (Na+ + K+)-ATPase is not affected by the inhibitor. The peptide migrates as a single band in SDS gels. Its apparent molecular weight is 19,000. It causes inhibition of the Ca2+-pump by decreasing Ca2+-affinity at all calmodulin concentrations.     

The role of a (Ca2+ + Mg2+)-ATPase of the rough endoplasmic reticulum in regulating intracellular Ca2+ during cholinergic stimulation of rat pancreatic acini

Rough endoplasmic reticulum membranes, purified from isolated rat pancreatic acini stimulated by carbachol, had a decreased Ca2+ content and increased (Ca2+ + Mg2+)-ATPase activity. Ca2+ was regained and ATPase activity reduced to control levels only after blockade by atropine. The (Ca2+ + Mg2+)-ATPase was activated by free Ca2+ (half-maximal at 0.17 microM; maximal at 0.7 microM) over the concentration range which occurs in the cell cytoplasm. Pretreatment with EGTA, at a high concentration (5 mM), inhibited ATPase activity which, our results suggest, was due to removal of a bound activator such as calmodulin. The rate of (Ca2+ + Mg2+)-ATPase actively declined during the 10-min period over which maximal active accumulation of Ca2+ by membrane vesicles occurs. In the presence of ionophore A23187, which released actively accumulated Ca2+ and stimulated the (Ca2+ + Mg2+)-ATPase, this time-dependent decline in activity was not observed. Our data provide evidence that the activity of the Ca2+-transporting ATPase of the rough endoplasmic reticulum is regulated by both extra and intravesicular Ca2+ and is consistent with a direct role of this enzyme in the release and uptake of Ca2+ during cholinergic stimulation of pancreatic acinar cells.     

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+.     

The calmodulin-stimulated (Ca2+ + Mg2+)-ATPase in hemoglobin S erythrocyte membranes: effects of sickling and oxidative agents

A decrease in the reactivity of erythrocyte membrane (Ca2+ + Mg2+)-ATPase to calmodulin stimulation has been observed in aging red cells and in various types of hemolytic anemias, particularly in sickle red cell membranes. Unlike the aging process, the defect in the (Ca2+ + Mg2+)-ATPase from SS red blood cells is not secondary to a decrease in calmodulin activity and is already present in the least dense SS red blood cells separated on a discontinuous density gradient. Deoxygenated AS red cells were forced to sickle by lowering the pH, raising the osmolarity of the buffer (sickling pulse). Under these conditions an inhibition of the calmodulin-stimulated enzyme was observed only if several cycles of oxygenation/deoxygenation were applied. No alteration of the enzyme could be detected after submitting AS red blood cells to other conditions or in AA red blood cells submitted to the same treatments. This suggests that oxidative processes are involved in the alterations of the (Ca2+ + Mg2+)-ATPase activity. Treatment of membranes from AA erythrocytes by thiol group reagents and malondialdehyde, a by-product of auto-oxidation of membrane unsaturated lipids and a cross-linking agent of cytoskeletal proteins, led to a partial inhibition of the calmodulin-stimulated (Ca2+ + Mg2+)-ATPase. We postulate that the hyperproduction of free radicals described in the SS red blood cells and involved in the destabilization of the membrane may be also responsible for the (Ca2+ + Mg2+)-ATPase failure.     

Characterization of the phosphorylated intermediate of the isolated high-affinity (Ca2+ + Mg2+)-ATPase of human erythrocyte membranes

Phosphorylation of solubilized and purified high-affinity (Ca2+ + Mg2+)-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 microM. Ca2+-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 (Ca2+ + Mg2+)-ATPase of human erythrocyte membranes.     

Protein kinase C phosphorylates the carboxyl terminus of the plasma membrane Ca(2+)-ATPase from human erythrocytes.

Purified Ca(2+)-stimulated, Mg(2+)-dependent ATPase (Ca(2+)-ATPase) from human erythrocytes was phosphorylated with a stoichiometry of about 1 mol of phosphate/mol of ATPase at both threonine and serine residues by purified rat brain type III protein kinase C. In the presence of calmodulin, the phosphorylation was markedly reduced. Labeled phosphate from [gamma-32P]ATP was retained on an 86-kDa calmodulin-binding tryptic fragment of Ca(2+)-ATPase but not on 82- and 77-kDa non-calmodulin-binding fragments. Similarly, fragmentation of the phosphorylated Ca(2+)-ATPase by calpain I revealed that calmodulin-binding fragments (127 and 125 kDa) retained phosphate label whereas a non-calmodulin-binding fragment (124 kDa) did not. The calmodulin-binding domain, located about 12 kDa from the carboxyl terminus of the Ca(2+)-ATPase, was thus located as a site of protein kinase C phosphorylation. A synthetic peptide corresponding to a segment of the calmodulin-binding domain (H2 N-R-G-L-N-R-I-Q-T-Q-I-K-V-V-N-COOH) was indeed phosphorylated at the single threonine residue within this sequence. The additional serine phosphorylation site was carboxyl terminal to the calmodulin domain. Phosphorylation by purified type III protein kinase C (canine heart) antagonized the calmodulin activation of the Ca(2+)-ATPase, particularly at lower Ca2+ concentrations (0.2-1.0 microM). By contrast, a purified but unresolved protein kinase C isoenzyme mixture from rat brain stimulated the activity of Ca(2+)-ATPase prepared in asolectin, but not glycerol, by more than 2-fold in the presence of the ionophore A23187, without increasing its Ca2+ sensitivity. The results clearly indicate that human erythrocyte Ca(2+)-ATPase is a substrate of protein kinase C, but the effect of phosphorylation on the activity of the enzyme depends on the isoenzyme form of protein kinase C used and on the lipid associated with the Ca(2+)-ATPase.     

Investigation of (Ca2+ + Mg2+)-ATPase phosphoprotein formation in erythrocyte membranes of patients with cystic fibrosis

The (Ca2+ + Mg2+)-ATPase present per mg of protein in erythrocyte membranes of controls and patients with cystic fibrosis (CF) was determined by estimation of the levels of its phosphoprotein. In the presence of 10 mM free Ca2+, which inhibits phosphoprotein decomposition, significantly less phosphoprotein intermediate, ECaP, was found in erythrocyte membranes from CF patients than in age- and sex-matched controls; this correlated with a significant decrease in (Ca2+ + Mg2+)-ATPase activity. These observations indicate a decrease in the number of functional (Ca2+ + Mg2+)-ATPase molecules in erythrocyte membranes from CF patients or an alteration in either the structure of the pump protein or the composition of its environment.     

Relation between phosphorylation and adenosine triphosphate-dependent Ca2+ binding of swine and bovine erythrocyte membranes

The correlation between the ATP-dependent Ca2+ binding and the phosphorylation of the membranes from swine and bovine erythrocytes was studied. The Ca2+ binding was measured by using 45CaCl2, and the phosphorylation by [gamma-32P]ATP was studied with the technique of SDS polyacrylamide gel electrophoresis. 200 mM NaCl and KCl markedly repressed the Ca2+ binding of swine erythrocyte membranes. The radioactivity of 32P-labelled membranes was revealed mainly in 250,000 dalton protein and a lipid fraction. NaCl and KCl also repressed the phosphorylation of the lipid which was identified as triphosphoinositide by paper chromatography. The membranes prepared from trypsin-digested erythrocytes completely retained the Ca2+-binding activity, and lost 30% of (Ca2+ + Mg2+)-ATPase activity. The Ca2+-binding and ATPase activity of isolated membranes decreased to 55% and to 0%, respectively, by tryptic digestion. Neither the Ca2+ binding nor the phosphorylation of polyphosphoinositides were detected in bovine erythrocyte membranes. These results suggest that the formation of triphosphoinositide rather than the (C2+ + Mg2+)-ATPase of membranes is linked to the ATP-dependent Ca2+ binding of erythrocyte membranes.     

Ca2+-ATPase activity in pancreatic acinar plasma membranes. Regulation by calmodulin and acidic phospholipids

A high degree of ATP hydrolytic activity present in purified rat pancreatic acinar cells was localized to plasma membranes. This activity was stimulated almost equally by Mg2+ or Ca2+. Kinetic analysis revealed that the enzyme had a higher affinity for Ca2+ (Kd = 1.73 microM) than Mg2+ (Kd = 2.98 microM) but a similar maximal rate of activity. A comparison of substrate requirements revealed very similar profiles for the Mg2+- and Ca2+-stimulated activities. Combinations of saturating concentrations of Mg2+ or Ca2+ produced the same degree of maximal activity. Investigation of the partial reactions of the ATPase activity revealed two phosphoprotein intermediates (Mr = 115,000 and 130,000) in the presence of Ca2+ and Mg2+. A significant stimulation of the Ca2+-ATPase activity by calmodulin was observed (Kd = 0.7 microM). Calmodulin increased the Ca2+-sensitivity of this enzyme system; Mg2+ appeared to be required for this effect. The Ca2+-ATPase activity was also stimulated by acidic phospholipids. Using an 125I-labeled calmodulin gel overlay technique, calmodulin was shown to bind in a Ca2+-dependent fashion to 133,000- and 230,000-dalton proteins present in the plasma membrane-enriched fraction. Under conditions that favor Ca2+-dependent kinase activity, calmodulin enhanced the phosphorylation of a 30,000- and 19,000-dalton protein. The major ATP hydrolytic activity in pancreatic acinar plasma membranes was present as an ectoenzyme.     

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.