Characterization of a (Ca2+ + Mg2+)-ATPase activator bound to human erythrocyte membranes

Incubation of human erythrocyte ghosts with an equal volume of 0.2 mM EDTA in isotonic KCl decreased both the activity and Ca2+ sensitivity of the (Ca2+ + Mg2+)-ATPase remaining associated with the membrane. Readdition of the EDTA-extract activated the (Ca2+ + Mg2+)-ATPase activity. The activator activity was trypsin sensitive, heat stable and retained by a phenothiazine affinity column, consistent with properties expected of calmodulin. However, unlike calmodulin, the activity was not retained by DEAE Sephadex A-50 and it eluted from Sephacryl S-200 as heterogeneous peaks of activator activity of apparent molecular weight between 107,000 and 178,000. Nevertheless, the activator in the EDTA extract both before and after gel filtration contained calmodulin, as determined by radioimmunoassay and by its activation of calmodulin - deficient phosphodiesterase. SDS-gel electrophoresis of the activator isolated by gel filtration showed a protein of Mr 56,000 in addition to a low molecular weight protein corresponding to calmodulin. It is suggested that the red cell membrane contains a calmodulin binding protein which tightly binds calmodulin as a polymeric complex in a Ca2+-independent manner.     

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 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.     

Calmodulin-dependent Ca2+-pump ATPase of human smooth muscle sarcolemma

An enzymatically active Ca2+-stimulated ATPase has been isolated from the sarcolemmal sheets of human smooth muscle (myometrium). Ca2+-ATPase activity was quantitated in an assay medium which simulated the characteristic free ionic concentrations of the cytosol. New computer programs for calculating the composition of solutions containing metals (Ca, Mg, Na, K) and ligands (EGTA, ATP), based on the updated stability constants, were used. In detergent-soluble form the enzyme has a high Ca2+-affinity expressed by an apparent Km (Ca2+) of 0.25 +/- 0.04 microM. The maximum specific activity (about 20 nmol of Pi/mg protein/min) was found in the micromolar domain of free-Ca2+ concentrations, the same levels required for normal maximal contractions in smooth muscle. The variation of free-Ca2+ concentration in the assay medium over 4 orders of magnitude (pCa 9 to pCa 5) resulted in a sigmoidal dependence of enzymatic activity, with a Hill coefficient of 1.4, which suggested the regulation of Ca2+-ATPase by allosteric effectors. The presence and the activator role of endogenous calmodulin in smooth muscle sarcolemma was proved by calmodulin-depletion experiments and by using suitable anticalmodulinic concentrations of trifluoperazine. The addition of exogenous calmodulin restored the enzyme activity. Apparently, the concentration of calmodulin in isolated smooth muscle sarcolemma is about 0.1% of sarcolemmal proteins, as deduced from the comparison of calmodulin-depletion and calmodulin-readdition experiments. Calmodulin increased significantly the enzyme Ca2+-affinity and Vmax (by a factor of about 10). At variance with the sarcoplasmic reticulum Ca2+-ATPase, the sarcolemmal Ca2+-ATPase is extremely sensitive to orthovanadate, half-maximal inhibition being observed at 0.8 microM vanadate. In conclusion, the Ca2+-ATPase isolated from smooth muscle sarcolemma appears very similar to the well-known Ca2+-pump ATPases of erythrocyte membrane, heart sarcolemma or axolemma. We suggest that this high-affinity Ca2+-ATPase represents the calmodulin-regulated Ca2+-extrusion pump of the smooth muscle sarcolemma.     

Ca2+- and calmodulin-dependent stimulation of smooth muscle actomyosin Mg2+-ATPase by fodrin

Fodrin, a spectrin-like actin and calmodulin binding protein, was purified to electrophoretic homogeneity from a membrane fraction of bovine brain. The effect of fodrin on smooth muscle actomyosin Mg2+-ATPase activity was examined by using a system reconstituted from skeletal muscle actin and smooth muscle myosin and regulatory proteins. The simulation of actomyosin Mg2+-ATPase by fodrin showed a biphasic dependence on fodrin concentration and on the time of actin and myosin preincubation at 30 degrees C. Maximal stimulation (50-70%) was obtained at 3 nM fodrin following 10 min of preincubation of actin and myosin. This stimulation was also dependent on the presence of tropomyosin. In the absence of myosin light chain kinase, the fodrin stimulation of Mg2+-ATPase could not be demonstrated with normal actomyosin but could be demonstrated with acto-thiophosphorylated myosin, suggesting that fodrin stimulation depends on the phosphorylation of myosin. Fodrin stimulation was shown to require the presence of both Ca2+ and calmodulin when acto-thiophosphorylated myosin was used. These observations suggest a possible functional role of fodrin in the regulation of smooth muscle contraction and demonstrate an effect on Ca2+ and calmodulin on fodrin function.     

The stoichiometry of the Ca2+ pump in human erythrocyte vesicles: modulation by Ca2+, Mg2+ and calmodulin

Active Ca2+ uptake and the associated (Ca2+ + Mg2+)-ATPase activity were studied under the same conditions in an inside-out vesicle preparation of human red blood cells made essentially by the procedure of Quist and Roufogalis (Journal of Supramolecular Structure 6, 375-381, 1977). Some preparations were treated with 1 mM EDTA at 30 degrees to further deplete them of endogenous levels of calmodulin. As the Ca2+ taken up by the EDTA-treated inside-out vesicles, as well as the non-EDTA treated vesicles, was maintained after addition of 4.1 mM EGTA, the vesicles were shown to be impermeable to the passive leak of Ca2+ over the time course of the experiments. In the absence of added calmodulin, both active Ca2+ uptake and (Ca2+ + Mg2+)-ATPase were sensitive to free Ca2+ over a four log unit concentration range (0.7 microM to 300 microM Ca2+) at 6.4 mM MgCl2. Below 24 microM Ca2+ the stoichiometry of calcium transported per phosphate liberated was close to 2:1, both in EDTA and non-EDTA treated vesicles. Above 50 microM Ca2+ the stoichiometry approached 1:1. When MgCl2 was reduced from 6.4 mM to 1.0 mM, the stoichiometry remained close to 2:1 over the whole range of Ca2+ concentrations examined. In contrast to the results at 6.4 mM MgCl2, the Ca2+ pump was maximally activated at about 2 microM free Ca2+ and significantly inhibited above this concentration at 1 mM MgCl2. Calmodulin (0.5-2.0 microgram/ml) had little effect on the stoichiometry in any of the conditions examined. The possible significance of a variable stoichiometry of the Ca2+ pump in the red blood cell is discussed.     

Hydrolysis by horse muscle acylphosphatase of (Ca2+ + Mg2+)-ATPase phosphorylated intermediate

Horse muscle acylphosphatase (EC 3.6.1.7) was found to hydrolyze the labeled phosphorylated intermediate of (Ca²⁺ + Mg²⁺)-ATPase from rabbit muscle. In addition, the phosphorylated peptides obtained by pepsin digestion of the labeled phosphorylated microsomes were completely hydrolyzed by acylphosphatase. These findings suggest a possible regulatory role of this enzyme in vivo on the calcium transport process by sarcoplasmic reticulum.     

Relation between Ca2+-ATPase and endogenous calmodulin of human erythrocyte membranes

Short incubation of erythrocyte membranes with oleic acid releases Ca2+-independently bound endogenous calmodulin together with a minor fraction of membrane-associated proteins without destruction of the membranes. The released endogenous calmodulin is similar if not identical to cytosolic calmodulin reversibly bound to ghosts in a Ca2+-dependent manner. The release of endogenous calmodulin proceeds without affecting the activity of Ca2+-ATPase when ghosts are incubated with oleic acid in the presence of Ca2+ plus ATP and thereafter freed from oleic acid by washings with serum albumin. Kinetic parameters of Ca2+-ATPase of ghosts with and without endogenous calmodulin are identical as are amounts of exogenous calmodulin bound to these ghosts. Thus, endogenous calmodulin does not function as an essential part of Ca2+-ATPase.     

Some properties of the purified (Ca2+ + Mg2+)-ATPase from human red cell membranes

The (Ca2+ + Mg2+)-ATPase from red cell membranes, purified by means of a calmodulin-containing affinity column according to the method of Gietzen et al. (Gietzen, K., Tejcka, M. and Wolf, H.U. (1980) Biochem. J. 189, 81-88) with either phosphatidylcholine or phosphatidylserine as phospholipid is characterized. The phosphatidylcholine preparation can be activated by calmodulin, while the phosphatidylserine preparation is fully activated without calmodulin. The enzyme shows a biphasic ATP dependence with two Km values of 3.5 and 120 microM. The enzyme is phosphorylated by ATP in the presence of Ca2+ only.     

Comparative changes of levels of nitrendipine Ca2+ channels, of tetrodotoxin-sensitive Na+ channels and of ouabain-sensitive (Na+ + K+)-ATPase following denervation of rat and chick skeletal muscle

Three major ion transport systems, the nitrendipine-sensitive Ca2+ channels, the tetrodotoxin-sensitive Na+ channel and the ouabain-sensitive (Na+ + K+)-ATPase, have been studied in skeletal muscle from rat and chick after chronic denervation. It is shown that the situation found for the Ca2+ channel differs dramatically from that found for the Na+ channel and the (Na+ + K+)-ATPase and that regulation of the nitrendipine-sensitive Ca2+ channel in denervated muscle also differs widely from that of the tetrodotoxin-sensitive Na+ channel and the ouabain-sensitive (Na+ + K+)-ATPase which show a quite similar evolution.     

Uptake, retention, and efflux of Ca2+ by mitochondrial preparations from skeletal muscle

Functionally intact mitochondria, substantially free of contamination, were isolated from rabbit gastrocnemius muscle after protease digestion and their Ca2+-handling properties examined. When judged by their capacity to retain large Ca2+ loads and the magnitude of basal and Na+-stimulated Ca2+ effluxes, the most suitable isolation method was digestion of finely minced muscle in buffered isoosmotic KCl with low levels (0.4 mg/g) of trypsin or the bacterial protease nagarse, followed by differential centrifugation. Polytron disruption of skeletal muscle in both sucrose- and KCl-based media released mitochondria deficient in cytochrome c. Use of the divalent ion chelator EDTA rather than EGTA in the isolation medium sharply reduced Ca2+-dependent respiratory control and tolerance of the mitochondria to Ca2+ loads, probably by removing Mg2+ essential to membrane integrity. ADP-dependent respiratory control was not altered in mitochondria prepared in an EDTA-containing isolation medium. Purification of mitochondria on a Percoll density gradient did not improve their Ca2+-handling ability despite removal of minor contaminants. Mitochondria prepared by the protease method could accumulate micromole loads of Ca2+/mg while maintaining a low basal Ca2+ efflux. Addition of BSA to the assay medium slightly improved Ca2+ retention but was not essential either during isolation or assay. Ca2+-dependent state 3 respiration was maximal at pH 6.5-7.0 while respiratory control and Ca2+/O were optimal at pH 7.0-7.5. Neither Pi nor oxaloacetate induced Ca2+ release from loaded mitochondria when monitored for 30 min after ruthenium red addition. Na+-stimulated Ca2+ efflux had sigmoidal kinetics with a Hill coefficient of 3. Since skeletal muscle mitochondria can be isolated and assayed in simple media, functional deficiencies of mitochondria from diseased muscle are unlikely to be masked.     

Mechanism of stimulation of Ca2+ plus Mg2+ -dependent phospodiesterase from rat cerebral cortex by the modulator protein and Ca2+

The activity of phosphodiesterase (“Ca²⁺ plus Mg²⁺-dependent” phosphodiesterase) of a preparation from brain was found to depend on the presence of both Ca²⁺ and a protein factor called modulator. It was shown by gel filtration that the active enzyme-modulator complex (MW, about 200,000) was formed from the modulator (MW, 28,000) and an inactive enzyme (MW, about 150,000) in the presence of Ca²⁺. When EGTA was added, this active enzyme-modulator complex dissociated into inactive enzyme and modulator. These results, together with the finding of Teo and Wang that Ca²⁺ binds to the modulator, could explain the stimulatory effect of Ca²⁺ on this enzyme as follows: The “Ca²⁺ plus Mg²⁺-dependent” phosphodiesterase may exist as the inactive free form in equilibrium with the active enzymemodulator (Ca²⁺) complex, and Ca²⁺, through binding to the modulator, may shift the equilibrium towards formation of the active enzyme-modulator (Ca²⁺) complex, thereby increasing the activity of the mixture. On decreasing the concentration of Ca²⁺, the process is reversible.     

The phospholipid requirement of the (Ca2+ + Mg2+)-ATPase from human platelets

The phospholipid requirement of the ($\text{Ca}{}^{\mathrm{2+}}\text{+}\text{Mg}{}^{\mathrm{2+}}\text{)-}\text{ATPase}$ present in a membrane fraction from human platelets was studied using various purified phospholipases. Only those phospholipases, which hydrolyse the negatively charged phospholipids, inhibited the ($\text{Ca}{}^{\mathrm{2+}}\text{+}\text{Mg}{}^{\mathrm{2+}}\text{)-}\text{ATPase activity}$. The ATPase activity could be restored by adding mixed micelles of Triton X-100 and phosphatidylserine or phosphatidylinositol. Micelles with phosphatidic acid, phosphatidylcholine, phosphatidylethanolamine or sphingomyelin could not be used for reconstitution and inhibited the activity of the native enzyme.     

Cholesterol effect on enzyme activity of the sarcolemmal (Ca2+ + Mg2+)-ATPase from cardiac muscle

The effect of cholesterol incorporation and depletion of the cardiac sarcolemmal sacs on (Ca2+ + Mg2+)-ATPase activity was examined. Cholesterol incorporation to the sarcolemmal sacs was achieved utilizing an in vivo and an in vitro procedure. Cholesterol depleted membranes were obtained in vitro after incubation of the sarcolemmal sacs with inactivated plasma. Arrhenius plots of the (Ca2+ + Mg2+)-ATPase activity showed a triphasic curve when the assays were carried out using a temperature range between 0 and 40 degrees C. The sarcolemmal (Ca2+ + Mg2+)-ATPase activity was shown to be inversely proportional to the cholesterol concentration of the membranes, showing a low ATPase activity with a high cholesterol content and a high ATPase activity when the cholesterol concentration was low. Although the (Ca2+ + Mg2+)-ATPase activity was found to be inhibited in the cholesterol incorporated sarcolemmal sacs, the withdrawal of small amounts of cholesterol from the membranes produced an important stimulatory effect. Changes in (Ca2+ + Mg2+)-ATPase activity due to variation in the membrane cholesterol concentration were shown to be reversible. Our results indicate the possibility of a slow exchange of cholesterol between the tightly bound lipid surrounding the (Ca2+ + Mg2+)-ATPase and the bulk lipid of the sarcolemma.     

Inhibition by quercetin of thyroid hormone stimulation in vitro of human red blood cell Ca2+-ATPase activity

Human red blood cell membrane Ca2+-ATPase activity is stimulated in vitro by physiological concentrations of thyroid hormone. Quercetin, a flavonoid that inhibits several membrane-linked ATPases, suppressed thyroid hormone action on red cell Ca2+-ATPase activity and also interfered with binding of the hormone by red cell membranes. These effects of quercetin were dose-dependent over a range of concentrations (1-50 microM). In contrast, in the absence of thyroid hormone, quercetin at low concentrations stimulated Ca2+-ATPase activity and at 50 microM inhibited the enzyme. The effects of quercetin at low concentrations (1-10 microM), namely, stimulation of Ca2+-ATPase and inhibition of membrane-binding of thyroid hormone, mimic those of thyroid hormone and are consistent with the thyronine-like structure of quercetin. At high concentrations, quercetin is generally inhibitory of Ca2+-ATPase activity. Chalcone, fisetin, hesperetin and tangeretin are other flavonoids shown to reduce susceptibility of membrane Ca2+-ATPase to hormonal stimulation.     

(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.