Comparison of (Ca2+-Mg2+)-ATPase and Ca2+-ATPase in rat cardiac sarcolemma

The calcium dependency of the Ca2+-pump ATPase of rat cardiac sarcolemma was investigated in the presence and absence of EGTA and EDTA in combination with two free Mg2+-ion concentrations. The results showed: that Mg2+-ions are not essential for the turnover of the Ca2+-pump ATPase; that the Ca2+-affinity is regulated by the concentration of the calcium-chelator complex present in the medium; that (Ca2+-Mg2+)-ATPase and Ca2+-ATPase are probably expressions of the same Ca2+-pump ATPase in the plasma membrane of the cell.     

(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.     

Localization of (Ca2+ + Mg2+)-ATPase,Ca2+ pump and other ATPase activities in cardiac sarcolemma

N-Ethylmaleimide was employed as a surface label for sarcolemmal proteins after demonstrating that it does not penetrate to the intracellular space at concentrations below 1·10^?4 M. The sarcolemmal markers, ouabain-sensitive (Na⁺ + K⁺)-ATPase and Na⁺/Ca²⁺-exchange activities, were inhibited in N-ethylmaleimide perfused hearts. Intracellular activities such as creatine phosphokinase, glutamate-oxaloacetate transaminase and the internal phosphatase site of the Na⁺ pump (K⁺-p-nitrophosphatase) were not affected. Almost 20% of the (Ca²⁺ + Mg²⁺)-ATPase and Ca²⁺ pump were inhibited indicating the localization of a portion of this activity in the sarcolemma. Sarcolemma purified by a recent method (Morcos, N.C. and Drummond, G.I. (1980) Biochim. Biophys. Acta 598, 27–39) from N-ethylmaleimide-perfused hearts showed loss of approx. 85% of its (Ca²⁺ + Mg²⁺-ATPase and Ca²⁺ pump compared to control hearts. (Ca²⁺ + Mg²⁺)-ATPase and Ca²⁺ pump activities showed two classes of sensitivity to vanadate ion inhibition. The high vanadate affinity class ($\text{K}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}$ for inhibition approx. 1.5 μM) may be localized in the sarcolemma and represented approx. 20% of the total inhibitable activity in agreement with estimates from N-ethylmaleimide studies. Sucrose density fractionation indicated that only a small portion of Mg²⁺-ATPase and Ca²⁺-ATPase may be associated with the sarcolemma. The major portion of these activities seems to be associated with high density particles.     

Regulation of cardiac sarcoplasmic reticulum (Ca2+ + Mg2+)-ATPase

Summary The two high affinity calcium binding sites of the cardiac (Ca²⁺ + Mg²⁺)-ATPase have been identified with the use of Eu³⁺. Eu³⁺ competes for the two high affinity calcium sites on the enzyme. With the use of laser-pulsed fluorescent spectroscopy, the environment of the two sites appear to be heterogeneous and contain different numbers of H₂O molecules coordinated to the ion. The ion appears to be occluded even further in the presence of ATP. Using non-radiative energy transfer studies, we were able to estimate the distance between the two Ca²⁺ sites to be between 9.4 to 10.2 A in the presence of ATP. Finally, from the assumption that the calcium site must contain four carboxylic side chains to provide the 6–8 ligands needed to coordinate calcium, and based on our recently published data, we predict the peptidic backbone of the two sites.     

Cholesterol effect on thermostability of the (Ca2+, Mg2+)-ATPase from cardiac muscle sarcolemma

When the cholesterol concentration in the sarcolemmal system is raised, the (Ca2+,Mg2+)-ATPase activity acquires an important degree of thermostability; phenomena that is completely lost if the experiment is carried out with cholesterol depleted sarcolemma. In this system, a gradual depletion of sarcolemmal cholesterol, renders the ATPase remarkably sensitive to temperature. At different concentrations of ATP, it is found that cholesterol affects the Vmax of the (Ca2+,Mg2+)-ATPase but not its Km. These results support our earlier suggestion of a direct effect of cholesterol upon the enzyme, and opens a possible mode of action of cholesterol on the enzyme. It is suggested that the inverse relationship between catalysis and thermostability is due to differences in the flexibility of the enzyme directly related to hydrophobicity changes caused by cholesterol.     

Ca2+ - and Mg2+-dependent ATPase activities in the deoxycholate-treated rat heart sarcolemma

• 1.1. Isolated rat heart sarcolemma was treated with different concentrations of an ionic detergent, deoxycholate (DOC) and ATP hydrolysis in the presence of Ca²⁺ or Mg²⁺ was determined.
• 2.2. Both Ca²⁺-dependent ATPase and Mg²⁺-dependent ATPase activities were decreased in the DOC-treated membranes; however, the depression of Mg²⁺-dependent ATPase activity was greater than that of Ca²⁺-dependent ATPase.
• 3.3. The differential changes in Ca²⁺-dependent ATPase and Mg²⁺-dependent ATPase activities were apparent when incubations with DOC were carried out for different time intervals and at different temperatures.
• 4.4. In DOC-treated preparations, the K_m value for Ca²⁺-dependent ATPase was decreased whereas that for Mg²⁺-dependent ATPase was increased. The half maximal velocities of the Ca²⁺-dependent ATPase and Mg²⁺-dependent ATPase enzyme reactions in the treated preparations were obtained at a DOC: membrane protein ratio of 3.0 and 0.6, respectively.
• 5.5. In the DOC-treated membranes exhibiting the half maximal velocities of enzyme reactions, the K_i value for Ca²⁺-dependent ATPase was drastically reduced but remained unchanged for Mg²⁺-dependent ATPase.
• 6.6. The DOC treatment was associated with a loss of protein as well as phospholipids and resulted in changes in the ultrastructural integrity of the membrane.
• 7.7. Varying degrees of decreases in the activities of sarcolemmal adenylate cyclase. (Na⁻-K⁺)-ATPase. 5'-nucleotidase and calcium binding were seen upon DOC treatment.
• 8.8. The extent of reduction in Ca²⁺-dependent ATPase and Mg²⁺-dependent ATPase activities were also different when the membrane was treated with a non-ionic detergent, Lubrol PX.
• 9.9. These data suggest that Ca²⁺-dependent ATPase in heart sarcolemma is more resistant than Mg²⁺-dependent ATPase to detergent treatments and further indicate some differences in the properties of these enzymes.

     

Effect of nitric oxides and hydrogen peroxide on Ca2+, Mg(2+)-ATPase and Mg(2+)-ATPase activity in myometrium sarcolemma

The action of sodium nitroprusside, nitrite-anions and hydrogen peroxide on Ca2+, Mg(2+)-ATPase and Mg(2+)-ATPase (Ca(2+)-independent) enzymatic activity in myometrium sarcolemma fraction is investigated. It is established, that 0.1 mM sodium nitroprusside and 10(-8)-10(-5) M nitrite-anions essentially reduce Ca2+, Mg(2+)-ATPase activity whereas Mg(2+)-ATPase proved to be absolutely resistant to them. At rather high concentration of nitrite-anions (0.1 mM) appreciable stimulation of Ca2+, Mg(2+)-ATPase was observed. Hydrogen peroxide (10(-8)-10(-4)), depending on the concentration suppressed both enzymes activity. However, Ca2+, Mg(2+)-ATPase proved to be more sensitive to the action of H2O2 (seeming K(i) = 0.42 +/- 0.1 microM), than Mg(2+)-ATPase (seeming K(i) = 3.1 +/- 0.9 microM). At presence of 1 mM ditiothreitole (a reducer of SH groups of the membrane surface) action of investigated substances considerably decreased. Reagents on carboxic- (dicyclogexilcarbodiimid) and amino- groups of the membrane (trinitrobenzolsulfonic acid) inhibited both Ca2+, Mg(2+)-ATPase, and Mg(2+)-ATPase activity in membrane fractions. In the presence of noted reagents sodium nitroprusside and nitrite-anions action was not almost shown. Hence, nitrogen oxide, nitrite-anions and hydrogen peroxide suppress Ca2+, Mg(2+)-ATPase and Mg(2+)-ATPase (only hydrogen peroxide) activity in the plasmatic membrane of myometrium cells, and this action can be connected with direct updating of superficial chemical groups of the membrane.     

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.     

Localization and properties of a high-affinity (Ca2+ + Mg2+)-ATPase in isolated kidney cortex plasma membranes

Surface membrane fractions from Paramecium tetraurelia cells contain a calmodulin-stimulated Ca²⁺-ATPase responding to low levels of free Ca²⁺ and with features characteristic of a membrane-bound ATPase responding to low levels of free Ca²⁺ and with features characteristics of a membrane-bound ATPase. Among the different strains analyzed this enzyme was practically absent selectively from the ‘non-discharge” mutant nd9—28°C (from J. Beisson); if cultured at a permissive temperature (18°C), this strain showed identical values of calmodulin-stimulated Ca²⁺-ATPase activity as wild-type cells (7S) or strains with mutations which do not affect exocytosis performance. We conclude that this calmodulin-stimulated Ca²⁺-activated ATPase might be a prerequisite for membrane fusion in the course of exocytosis performance.     

Ca2+-Dependent activities of (Na+ + K+)-ATPase

Experiments on the effects of varying concentrations of Ca²⁺ on the Mg²⁺ + Na⁺-dependent ATPase activity of a highly purified preparation of dog kidney (Na⁺ + K⁺)-ATPase showed that Ca²⁺ was a partial inhibitor of this activity. When Ca²⁺ was added to the reaction mixture instead of Mg²⁺, there was a ouabain-sensitive Ca²⁺ + Na⁺-dependent ATPase activity the maximal velocity of which was 30 to 50% of that of Mg²⁺ + Na⁺-dependent activity. The apparent affinities of the enzyme for Ca²⁺ and CaATP seemed to be higher than those for Mg²⁺ and MgATP. Addition of K⁺, along with Ca²⁺ and Na⁺, increased the maximal velocity and the concentration of ATP required to obtain half-maximal velocity. The maximal velocity of the ouabain-sensitive Ca²⁺ + Na⁺ + K⁺-dependent ATPase was about two orders of magnitude smaller than that of Mg²⁺ + Na⁺ + K⁺-dependent activity. In agreement with previous observations, it was shown that in the presence of Ca²⁺, Na⁺, and ATP, an acid-stable phosphoenzyme was formed that was sensitive to either ADP or K⁺. The enzyme also exhibited a Ca²⁺ + Na⁺-dependent ADP-ATP exchange activity. Neither the inhibitory effects of Ca²⁺ on Mg²⁺-dependent activities, nor the Ca²⁺-dependent activities were influenced by the addition of calmodulin. Because of the presence of small quantities of endogenous Mg²⁺ in all reaction mixtures, it could not be determined whether the apparent Ca²⁺-dependent activities involved enzyme-substrate complexes containing Ca²⁺ as the divalent cation or both Ca²⁺ and Mg²⁺.     

Decreased Ca2+-binding and Ca2+-ATPase activities in heart sarcolemma upon phospholipid methylation

Heart sarcolemma has been shown to possess three catalytic sites (I, II and III) for methyl transferase activity (Panagia V, Ganguly PK and Dhalla NS. Biochim Biophys Acta 792: 245–253, 1984). In this study we examined the effect of phosphatidylethanolamine N-methylation on ATP-independent Ca²⁺ binding and ATPase activities in isolated rat heart sarcolemma. Both low affinity (1.25 mM Ca²⁺) and high affinity (50 µM Ca²⁺) Ca²⁺ binding activities were decreased following incubation of sarcolemmal membranes with AdoMet under optimal conditions for site II and III. Similarly, Ca²⁺ ATPase activities measured at 1.25 mM and 4 mM Ca²⁺ were depressed by phospholipid N-methylation. S-adenosyl homocysteine, a specific inhibitor of phospholipid N-methylation, prevented the depression of low affinity Ca²⁺ binding and Ca²⁺ ATPase activities, whereas the methylation-induced effect on the high affinity Ca²⁺ binding was not influenced by this agent. Pretreatment of sarcolemma with methyl acetimidate hydrochloride, an amino group blocking agent, also prevented the methylation-induced inhibition of both Ca²⁺ binding and Ca²⁺ ATPase. A further decrease in Ca²⁺ binding and Ca²⁺ ATPase activities together with a marked increase in the intramembranal level of PC was seen when membranes were methylated under the site III conditions in the presence of phosphatidyldimethylethanolamine as exogenous substrate. There was no effect of phospholipid methylation on sarcolemmal Na⁺-K⁺ ATPase and Mg²⁺ ATPase activities. These results indicate a role of phospholipid N-methylation in the regulation of sarcolemmal Ca²⁺ ATPase and low affinity ATP-independent Ca²⁺ binding.     

Inhibition of Ca2+-dependent protein kinase and Ca2+/Mg2+-ATPase in cardiac sarcolemma by the anti-calmodulin drug calmidazolium

Sarcolemma (SL) vesicles, isolated from pig heart, contain both a Ca2+-calmodulin-dependent protein kinase (CaM-PK) and a Ca2+-dependent Mg2+-ATPase (Ca2+/Mg2+)-ATPase). Some of their properties have been compared: their affinity for Ca2+ ions, dependence on exogenous calmodulin (CaM) and sensitivity to the anti-CaM drug calmidazolium (R24571). The properties of Ca2+-CaM-dependent brain phosphodiesterase (PDE) have also been examined. R24571 appeared to be the most potent inhibitor from brain PDE. For the three enzymes studied, exogenously added CaM was able to antagonize the R24571 inhibition, although the efficiency to counteract was rather low in the case of the SL Ca2+/Mg2+-ATPase. R24571 decreased the affinity of the Ca2+/Mg2+-ATPase for Ca2+ ions (KCa 0.35 versus 0.75 microM) and exerted an inhibition non-competitive with Ca2+ ions on the other CaM-dependent enzymes. Membrane-bound CaM, which is involved in controlling the Ca2+/Mg2+-ATPase, appeared to be present in a stoichiometry varying from 1:1 to 1:4 compared to the 32P-intermediate of the ATPase. R24571 treatment of SL vesicles selectively solubilized a number of proteins in the molecular range of 13-20 kD, which may include CaM. The results suggest that different mechanisms are involved in the CaM control of the two SL enzymes studied.     

Characterization of Mg2+- and (Ca2+ + Mg2+)-ATPase activity in adipocyte endoplasmic reticulum

The presence of an energy-dependent calcium uptake system in adipocyte endoplasmic reticulum (D. E. Bruns, J. M. McDonald, and L. Jarett, 1976, J. Biol. Chem.251, 7191–7197) suggested that this organelle might possess a calcium-stimulated transport ATPase. This report describes two types of ATPase activity in isolated microsomal vesicles: a nonspecific, divalent cation-stimulated ATPase (Mg²⁺-ATPase) of high specific activity, and a specific, calcium-dependent ATPase (Ca²⁺ + Mg²⁺-ATPase) of relatively low activity. Mg²⁺-ATPase activity was present in preparations of mitochondria and plasma membranes as well as microsomes, whereas the (Ca²⁺ + Mg²⁺)-ATPase activity appeared to be localized in the endoplasmic reticulum component of the microsomal fraction. Characterization of microsomal Mg²⁺-ATPase activity revealed apparent K_m values of 115 μm for ATP, 333 μm for magnesium, and 200 μm for calcium. Maximum Mg²⁺-ATPase activity was obtained with no added calcium and 1 mm magnesium. Potassium was found to inhibit Mg²⁺-ATPase activity at concentrations greater than 100 mm. The energy of activation was calculated from Arrhenius plots to be 8.6 kcal/mol. Maximum activity of microsomal (Ca²⁺ + Mg²⁺)-ATPase was 13.7 nmol ³²P/mg/min, which represented only 7% of the total ATPase activity. The enzyme was partially purified by treatment of the microsomes with 0.09% deoxycholic acid in 0.15 m KCl which increased the specific activity to 37.7 nmol ³²P/mg/min. Characterization of (Ca²⁺ + Mg²⁺)-ATPase activity in this preparation revealed a biphasic dependence on ATP with a Hill coefficient of 0.80. The apparent K_m^s for magnesium and calcium were 125 and 0.6–1.2 μm, respectively. (Ca²⁺ + Mg²⁺)-ATPase activity was stimulated by potassium with an apparent K_m of 10 mm and maximum activity reached at 100 mm potassium. The energy of activation was 21.5 kcal/mol. The kinetics and ionic requirements of (Ca²⁺ + Mg²⁺)-ATPase are similar to those of the (Ca²⁺ + Mg²⁺)-ATPase in sarcoplasmic reticulum. These results suggest that the (Ca²⁺ + Mg²⁺)-ATPase of adipocyte endoplasmic reticulum functions as a calcium transport enzyme.     

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.