Partial purification and characterization of the (Ca2+ + Mg2+)-ATPase from squid optic nerve plasma membrane

A membrane fraction enriched in axolemma was obtained from optic nerves of the squid (Sepiotheutis sepioidea) by differential centrifugation and density gradient fractionation. The preparation showed an oligomycin- and NaN₃-insensitive (Ca²⁺ + Mg²⁺)-ATPase activity. The dependence of the ATPase activity on calcium concentration revealed the presence of two saturable components. One had a high affinity for calcium ($\text{K}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}{}^1\text{= 0.12 μ}\text{M}$) and the second had a comparatively low affinity ($\text{K}{}^2{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}\text{= 49.5 μ}\text{M}$). Only the high-affinity component was specifically inhibited by vanadate (K₁ = 35 μM). Calmodulin (12.5 μ/ml) stimulated the (Ca²⁺ + Mg²⁺)-ATPase by approx. 50%, and this stimulation was abolished by trifluoperazine (10 μM). Further treatment of the membrane fraction with 1% Nonidet P-40 resulted in a partial purification of the ATPase about 15-fold compared to the initial homogenate. This (Ca²⁺ + Mg²⁺)-ATPase from squid optic nerve displays some properties similar to those of the uncoupled Ca²⁺-pump described in internally dialyzed squid axons, suggesting that it could be its enzymatic basis.     

A (Ca2+, Mg2+)-ATPase activity in plasma membrane fragments isolated from squid nerves

A (Ca2+, Mg2+)-ATPase activity and a (Ca2+, Mg2+)-dependent phosphorylation from ATP have been found in plasma membrane fragments from squid optical nerves under conditions where contamination by intracellular organelles is unlikely. The properties of this (Ca2+, Mg2+)-ATPase activity are almost identical to those of the ATP-dependent uncoupled Ca2+ efflux observed in dialyzed squid giant axons. This gives further support to the notion that the mechanism responsible for maintaining the low levels of ionized Ca concentration in nerves at rest is not a Na+-Ca2+ exchange system but an ATP-driven uncoupled Ca2+ pump.     

Partial purification and reconstitution of the (Ca2+ + Mg2+)-ATPase of erythrocyte membranes.

Benzenemethane Sulfonylfluoride (329-98-6) is an irreversible inactivator of many esterases including mammalian acetylcholinesterases. However, previous reports indicated that acetylcholinesterase from the electric eel, Electrophorus electricus (EC 3.1.1.7) failed to react with benzenemethane sulfonylfluoride at measurable rates. We report here that eel acetylcholinesterase reacts with this inactivator at a low rate. Hydrolysis of the sulfonylating agent is so much faster than enzyme inactivation that, under most conditions, there will be only slight inactivation. Like the reaction of other active site acylating agents with this enzyme, inactivation can be accelerated in the presence of certain organic cations. We introduce a rate equation for enzyme sulfonylation which incorporates both the hydrolysis of the inactivator and the complication that fluoride resulting from hydrolysis of the inactivator is a potent competitive inhibitor of this enzyme. This rate equation accurately describes the time course of enzyme inactivation.     

Partial purification and characterization of (Na++K+)-ATPase from garfish olfactory nerve axon plasma membrane

Summary The (Na⁺+K⁺)-ATPase of garfish olfactory nerve axon plasma membrane was purified about sixfold by treatment of the membrane with sodium dodecyl sulfate followed by sucrose density gradient centrifugation. The estimated molecular weights of the two major polypeptide components of the enzyme preparation on sodium dodecyl sulfate gels were 110,000 and 42,000 daltons, which were different from those of the corresponding peptides of rabbit kidney (Na⁺+K⁺)-ATPase. No carbohydrate was detected in the 42,000-dalton component either by the periodic acid-Schiff reagent or by the more sensitive concanavalin A-peroxidase staining procedure. The molecular properties of the garfish (Na⁺+K⁺)-ATPase, such as theK _m for ATP, pH optimum, energies of activation, Na and K ion dependence and vanadium inhibition, were, however, similar to those of the kidney enzyme.The partially purified garfish (Na⁺+K⁺)-ATPase was reconstituted into phospholipid vesicles by a freeze-thaw-sonication procedure. The reconstituted enzyme was found to catalyze a time and ATP dependent²²Na⁺ transport. The ratio of²²Na⁺ pumped to ATP hydrolyzed was about 1; under the same reconstitution and assay conditions, eel electroplax (Na⁺+K⁺)-ATPase, however, gave a²²Na⁺ pumped to ATP hydrolyzed ratio of nearly 3.     

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.     

Further characterization of the membrane-bound (Ca2+ + Mg2+)-ATPase from porcine erythrocytes

1. The kinetic and physicochemical properties of the calcium-pumping protein, (Ca2+ + Mg2+)-ATPase (ATP phosphohydrolase, EC 3.6.1.3) were studied in ghost membranes isolated from porcine erythrocytes. 2. The membrane-bound enzyme in situ has a specific activity of 3.12 +/- 0.08 micron/mg protein/hr and a Vmax of 3.47 +/- 0.21 mumol/mg protein/hr in the absence of calmodulin. 3. Its activity was stimulated by calmodulin about 5-fold. The enzyme is also highly sensitive to inhibition by vanadate (Ki = 1.6 +/- 0.2 microM). 4. Calmodulin also affects the pH- and Ca2+-sensitivity of the enzyme. The optimum pH, in the presence of calmodulin, is 7.5 and the optimum temperature is 38 degrees C with an activation energy of 11.9 kcal/mol.     

Fast reversal of the initial reaction steps of the plasma membrane (Ca2+ + Mg2+)-ATPase

(1) Calmodulin-depleted red cell membranes catalyse a Ca²⁺, Mg²⁺-dependent ATP-[³H]ADP exchange at 37° C. The Ca²⁺, Mg²⁺-dependent exchange, measured at 20 μM CaCl₂, 1.5 mM MgCl₂, 1.5 mM ADP and 1.5 mM ATP, is comparable to the (Ca²⁺ + Mg²⁺)-ATPase activity, between 0.3 and 0.8 mmol/litre original cells per h. (2) EDTA-washed membranes present a Ca²⁺-dependent ATP-ADP exchange whose rate is not more than 7% of that found in a Mg²⁺-containing medium, while their Ca²⁺-dependent ATPase is essentially zero. Addition of 1.5 mM MgCl₂ to the medium restores both activities to the levels found with membranes not treated with EDTA. (3) Calmodulin (16 μg/ml) produces an eight-fold stimulation of the Ca²⁺-dependent ATP-ADP exchange, slightly less than it stimulates the Ca²⁺-dependent ATP hydrolysis. The effect of 1.5 mM MgCl₂ on the exchange is greater in the presence than in the absence of calmodulin. (4) It is proposed that the reversal of the initial phosphorylation of the Ca²⁺ pump, occurring at a fast rate at 37° C, involves a conformational change in the phosphoenzyme. Thus, it would be an ADP-liganded phosphoenzyme of the form EP(ADP) that would experience the fast conformational transition at 37° C. The great difficulty in producing an overall reversal of the Ca²⁺ pump should then be due to one or more reaction steps later than and including Ca²⁺ release and dephosphorylation.     

Inhibition of membrane erythrocyte (Ca2+ + Mg2+)-ATPase by hemin

Red blood cell lysis is a common symptom following severe or prolonged oxidative stress. Oxidative processes occur commonly in sickle cells, probably mediated through denatured hemoglobin and the accumulation of ferric hemes in the membranes. Calmodulin-stimulated (Ca2+ + Mg2+)-ATPase from sickle red cell membranes is partially inactivated (Leclerc et al. (1987) Biochim. Biophys. Acta 897, 33-40). In this study (Ca2+ + Mg2+)-ATPase activity from normal adult erythrocyte membranes was measured in the presence of hemin. We report a time- and concentration-dependent inhibition of the activity of the enzyme by hemin due to a decrease in the maximum velocity. Only a mild inhibitory effect was observed in the presence of iron-free protoporphyrin IX, indicating the catalytic influence of the iron. Experiments carried out with hemin (ferric iron) liganded with imidazole or with reduced protoheme (ferrous iron) liganded with carbon monoxide, demonstrated that the inhibition requires that hemin be capable of binding additional ligands. The inhibition was not influenced by the absence of oxygen but was prevented by addition of bovine serum albumin. Addition of butylated hydroxytoluene, a protective agent of lipid peroxidation, failed to prevent the inhibition of calmodulin-stimulated (Ca2+ + Mg2+)-ATPase. As dithiothreitol partially restores the enzyme activity, we postulated that hemin interacts with the thiol groups of the enzyme.     

Partial purification and characterization of rabbit-kidney brush-border (Ca2+ or Mg2+)-dependent adenosine triphosphatase

The ATPase activity of rabbit-kidney brush border can be activated almost equally well by Ca²⁺ and Mg²⁺ and, therefore, should be called (Ca²⁺ or Mg²⁺)-ATPase. This enzyme was solubilized and enriched 14-fold by the following steps: pretreatment with papain removed 69% of alkaline phosphatase without attacking a significant portion of the ATPase activity. Addition of 1% cholate removed 65% of the protein but no ATPase activity. The combination of cholate (0.5%) and deoxycholate (0.4%) solubilized most of the ATPase activity and most of the remaining protein. A column chromatography of the extract on Sepharose CL-2B resulted in an 6.5-fold increase of specific ATPase activity. A precipitation by ammonium sulfate (40% saturation) produced an additional 1.9-fold increase. The yield of this partial purification was 16%. Towards the nucleotides UTP and GTP the enzyme showed an activity slightly higher, and towards ITP and CTP an activity slightly lower than that with ATP. ADP was split about half as fast as ATP. AMP was not accepted by the enzyme. Replacing MgCl₂ by CaCl₂ resulted in an ATPase activity of 92% of that with MgCl₂. Using calcium- and magnesium-ATP as substrates, apparent $\text{K}{}_{\mathrm{<mtext>m</mtext>}}$ values of 0.22 and 0.33 mM, respectively, were obtained. The gel electrophoresis revealed the enrichment of a protein with an apparent $\text{M}{}_{\mathrm{<mtext>r</mtext>}}$ of 95 000 and also that of microvillus actin.     

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.     

Activation of an islet cell plasma membrane (Ca2+ + Mg2+)-ATPase by calmodulin and Ca-EGTA

Islet cell plasma membranes contain a calcium-stimulated and magnesium-dependent ATPase (Ca2+ + Mg2+)-ATPase) which requires calmodulin for maximum enzyme activity (Kotagal, N., Patke, C., Landt, M., McDonald, J., Colca, J., Lacy, P., and McDaniel, M. (1982) FEBS Lett. 137, 249-252). Investigations indicated that exogenously added calmodulin increases the velocity and decreases the Km for Ca2+ of the high affinity (Ca2+ + Mg2+)-ATPase. These studies routinely employed the chelator ethylene glycol bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA) to maintain Ca2+ concentrations in the submicromolar range. During the course of these investigations, it was found unexpectedly that increasing the concentrations of EGTA (0.1-4 mM) and total calcium in the media, while maintaining constant free Ca2+ levels, increased the velocity of the high affinity (Ca2+ + Mg2+)-ATPase. The free calcium concentrations under these conditions were verified by a calcium-sensitive electrode. The (Ca2+ + Mg2+)-ATPase maximally activated by 2-4 mM EGTA was not further stimulated by calmodulin, whereas camodulin stimulation increased as the concentration of EGTA in the media was decreased. A similar enhancement by Ca-EGTA was observed on active calcium transport by the plasma membrane-enriched fraction. Moreover, Ca-EGTA had a negligible effect on both active calcium transport as well as Ca2+-stimulated ATPase activity by the islet cell endoplasmic reticulum, processes which are not stimulated by calmodulin. The results indicate that stimulation by Ca-EGTA may be used to differentiate calcium transport systems by these subcellular organelles. Furthermore, the concentration of EGTA routinely employed to maintain free Ca2+ levels may itself obscure effects of calmodulin and other physiological agents on calcium-dependent activities.     

A protein modulator of erythrocyte membrane (Ca2+ + Mg2+)-ATPase inhibitor protein

A protein modulator of erythrocyte membrane (Ca²⁺ + Mg²⁺)-ATPase inhibitor protein was purified to apparent homogeneity from pig membrane-free hemolysate by a combination of carboxymethyl-Sephadex chromatography, gel filtration, chromatofocusing (pH 7-4) and subsequent removal of trace inhibitor protein by salt treatment. Gel filtration gave a molecular weight of 57 500 for the purified protein modulator, while SDS-polyacrylamide gel electrophoresis of dithiothreitol-treated modulator revealed one single band with a molecular weight of 29 000. Isoelectric focusing of the dithiothreitol-treated protein revealed one band (isoelectric pH 4.85), while untreated modulator gave an extra band (isoelectric pH 4.96). It contains no methionine and has an acidic content 73% higher than that of its basic residues. Freshly prepared or dithiothreitol-treated modulator suppressed both pig and human erythrocyte (Ca²⁺ + Mg²⁺)-ATPase inhibitor protein activity, but did not affect ATPase and calmodulin activities. Modulator-coupled Affi-Gel 15 could be employed for purification of the protein inhibitor.     

(Ca2+ + Mg2+)-ATPase in enriched sarcolemma from dog heart

An enriched fraction of plasma membranes was prepared from canine ventricle by a process which involved thorough disruption of membranes by vigorous homogenization in dilute suspension, sedimentation of contractile proteins and mitochondria at 3000 X g followed by sedimentation of a microsomal fraction at 200 000 X g. The microsomal suspension was then fractionated on a discontinuous sucrose gradient. Particles migrating in the density range 1.0591--1.1083 were characterized by (Na+ + K+)-ATPase activity and [3H]ouabain binding as being enriched in sarcolemma and were comprised of nonaggregated vesicles of diameter approx. 0.1 micron. These fractions contained (Ca2+ + Mg2+)-ATPase which appreared endogenous to the sarcolemma. The enzyme was solubilized using Triton X-100 and 1 M KCl and partially purified. Optimal Ca2+ concentration for enzyme activity was 5--10 microM. Both Na+ and K+ stimulated enzyme activity. It is suggested that the enzyme may be involved in the outward pumping of Ca2+ from the cardiac cell.     

Cross-linking of the (Ca2+ + Mg2+)-ATPase protein.

The addition of cupric-1,10,-phenanthroline, a cross-linking catalyst, to sarcoplasmic reticulum membranes caused protein sulfhydryl groups to form disulfide bridges. Following a short exposure to the catalyst (15 s, 22 degrees C) most of the protein was in a dimeric form (Mr = 248 000). Longer exposure times resulted in the formation of trimers, tetramers and other oligomers too large to enter the gel. At low temperatures (4 degrees C) dimer formation predominates even for exposure times as long as 5 min. Cross-linking in the presence of 7.5 mM Triton X-100 (a concentration that resulted in clearing of the membrane suspension and thus solubilization of the membrane components) showed the appearance of a considerable dimer fraction, however, most of the (Ca2+ + Mg2+)-ATPase protein appeared as a monomer. Following 1 min of cross-linking at 22 degrees C, freeze-etched membranes showed no alteration in the number or appearance of 80 A intramembranous particles. Thus extensive cross-linking of the (Ca2+ + Mg2+)-ATPase protein can occur without disruption of the normal position of the intramembrane portion of the molecule.     

Isolation and purification of Ca2+,Mg2+-ATPase from plasma membranes of the myometrium

The preparation of the purified Ca2+, Mg2(+)-ATPase has been isolated from triton X-100 solubilizate of plasma membranes of the pig myometrium using the method of affinity chromatography on calmodulin-Sepharose 4B. The specific activity of the enzyme shows its 52-fold purification. The enzymic preparation practically has no Mg2(+)-ATPase activity. By the data of DS-Na-electrophoresis in PAAG the Ca2+, Mg2+ ATPase preparation consists of two polypeptides with Mm 130 and 205 kDa. Autoradiography shows their Ca2(+)-dependent phosphorylation. The purified enzyme is highly sensitive to the inhibitory effect of orthovanadate.     

Novel effects of calmodulin and calmodulin antagonists on the plasma membrane (Ca2+ + Mg2+)-ATPase from rabbit kidney proximal tubules.

In this work we report an unusual pattern of activation by calmodulin on the (Ca2+ + Mg2+)-ATPase from basolateral membranes of kidney proximal tubule cells. The activity of the ATPase depleted of calmodulin is characterized by a high Ca2+ affinity (Km = 2.2-3.4 microM) and a biphasic dependence on ATP concentration. The preparation responded to the addition of calmodulin by giving rise to a new Ca2+ site of very high affinity (Km less than 0.05 microM). Calmodulin antagonists had diverse effects on ATPase activity. Compound 48/80 inhibited calmodulin-stimulated activity by 70%, whereas calmidazolium did not modify this component. In the absence of calmodulin, 48/80 still acted as an antagonist, increasing the Km for Ca2+ to 5.7 microM and reducing enzyme turnover by competing with ATP at the low affinity regulatory site. Calmidazolium did not affect Ca2+ affinity, but it did displace ATP from the regulatory site. At fixed Ca2+ (30 microM) and ATP (5 mM) concentrations, Pi protected against 48/80 and potentiated inhibition by calmidazolium. At 25 microM ATP, Pi protected against calmidazolium inhibition. We propose that the effects of ATP and Pi arise because binding of the drugs to the ATPase occurs mainly on the E2 forms.     

Reconstitution of the purified calmodulin-dependent (Ca2+ + Mg2+)-ATPase from smooth muscle

The purified calmodulin dependent (Ca2+ + Mg2+)-ATPase (CaMg ATPase) from porcine antral smooth muscle transports Ca2+ after reconstitution in lipid vesicles indicating that this enzyme is indeed a Ca2+-transport ATPase. For CaMg ATPase reconstituted in asolectin vesicles a good correlation was found between the time course of Ca2+ accumulation and the corresponding changes in CaMg ATPase activity. The ATPase activity was stimulated 8-fold by A23187, which further indicates a tight coupling between ATP hydrolysis and Ca2+ transport. Asolectin vesicles with incorporated enzyme accumulated Ca2+ with a ratio approaching one Ca2+ ion transported for each ATP hydrolyzed. For CaMg ATPase reconstituted in phosphatidylcholine vesicles on the other hand, Ca2+ transport and CaMg ATPase were poorly coupled as is shown by the approximately 3.5 fold stimulation by A23187. The activity of the CaMg ATPase when reconstituted in asolectin vesicles was stimulated 1.25 fold by calmodulin while in phosphatidylcholine a value of 4.25 was obtained. The CaMg ATPase activity of the enzyme reconstituted either in asolectin or phosphatidylcholine was, after its stimulation by A23187, still further stimulated by detergent by a factor of 5.