Phospholamban expressed in slow-twitch and chronically stimulated fast-twitch muscles minimally affects calcium affinity of sarcoplasmic reticulum Ca(2+)-ATPase.

Chronic excitation, at 2 Hz for 6-7 weeks, of the predominantly fast-twitch canine latissimus dorsi muscle promoted the expression of phospholamban, a protein found in sarcoplasmic reticulum (SR) from slow-twitch and cardiac muscle but not in fast-twitch muscle. At the same time that phospholamban was expressed, there was a switch from the fast-twitch (SERCA1) to the slow-twitch (SERCA2a) Ca(2+)-ATPase isoform. Antibodies against Ca(2+)-ATPase (SERCA2a) and phospholamban were used to assess the relative amounts of the slow-twitch/cardiac isoform of the Ca(2+)-ATPase and phospholamban, which were found to be virtually the same in SR vesicles from the slow-twitch muscle, vastus intermedius; cardiac muscle; and the chronically stimulated fast-twitch muscle, latissimus dorsi. The phospholamban monoclonal antibody 2D12 was added to SR vesicles to evaluate the regulatory effect of phospholamban on calcium uptake. The antibody produced a strong stimulation of calcium uptake into cardiac SR vesicles, by increasing the apparent affinity of the Ca2+ pump for calcium by 2.8-fold. In the SR from the conditioned latissimus dorsi, however, the phospholamban antibody produced only a marginal effect on Ca2+ pump calcium affinity. These different effects of phospholamban on calcium uptake suggest that phospholamban is not tightly coupled to the Ca(2+)-ATPase in SR vesicles from slow-twitch muscles and that phospholamban may have some other function in slow-twitch and chronically stimulated fast-twitch muscle.     

Phospholamban involvement in the maintenance of basal calcium transport in cardiac sarcoplasmic reticulum

Phosphorylation of cardiac sarcoplasmic reticulum membrane vesicles by exogenous c-AMP and c-AMP-dependent protein kinase stimulates calcium uptake and Ca2+-dependent ATP hydrolysis by 40-50% and results in the incorporation of 32P into a 22-KDa protein, phospholamban. Treatment of the membrane with DOC (0.0002% or 5 X 10(-6) M) solubilizes phospholamban from the membrane and induces a 90% inhibition of basal calcium uptake. This inhibition cannot be attributed to an alteration in vesicle integrity or membrane permeability. The (Ca2+ + Mg2+)-ATPase remains associated with the membrane fraction and exhibits optimal levels of Ca2+-stimulated ATP hydrolysis. Phosphorylation prior to DOC treatment allows retention of the phospholamban in the membrane, concomitant with maintenance of the calcium transport activity. The results presented suggest that phospholamban is involved in the maintenance of basal calcium transport function in cardiac sarcoplasmic reticulum and that its phosphorylation stimulates Ca2+ transport.     

Regulation of the skeletal sarcoplasmic reticulum Ca2+ pump by phospholamban in reconstituted phospholipid vesicles

Phospholamban is the regulator of the Ca(2+)-ATPase in cardiac sarcoplasmic reticulum (SR). The mechanism of regulation appears to involve inhibition by dephosphorylated phospholamban, and phosphorylation may relieve this inhibition. Fast-twitch skeletal muscle SR does not contain phospholamban, and it is not known whether the Ca(2+)-ATPase isoform from this muscle may be also subject to regulation by phospholamban in a similar manner as the cardiac isoform. To determine this we reconstituted the skeletal isoform of the SR Ca(2+)-ATPase with phospholamban in phosphatidylcholine proteoliposomes. Inclusion of phospholamban was associated with significant inhibition of the initial rates of Ca2+ uptake at pCa 6.0, and phosphorylation of phospholamban by the catalytic subunit of cAMP-dependent protein kinase reversed the inhibitory effects on the Ca2+ pump. Similar effects of phospholamban were also observed using phosphatidylcholine:phosphatidylserine proteoliposomes, in which the Ca2+ pump was activated by the negatively charged phospholipids (24). Regulation of the Ca(2+)-ATPase appeared to involve binding with the hydrophilic portion of phospholamban, as evidenced by cross-linking experiments, using a synthetic peptide that corresponded to amino acids 1-25 of phospholamban. These findings suggest that the fast-twitch isoform of the SR Ca(2+)-ATPase may be also regulated by phospholamban, although this regulator is not expressed in fast-twitch skeletal muscles.     

Functional reconstitution of the cardiac sarcoplasmic reticulum Ca2(+)-ATPase with phospholamban in phospholipid vesicles

The Ca2(+)-ATPase in cardiac sarcoplasmic reticulum (SR) is under regulation by phospholamban, an oligomeric proteolipid. To determine the molecular mechanism by which phospholamban regulates the Ca2(+)-ATPase, a reconstitution system was developed, using a freeze-thaw sonication procedure. The best rates of Ca2+ uptake (700 nmol/min/mg reconstituted vesicles compared with 800 nmol/min/mg SR vesicles) were observed when cholate and phosphatidylcholine were used at a ratio of cholate/phosphatidylcholine/Ca2(+)-ATPase of 2:80:1. The EC50 values for Ca2+ were 0.05 microM for both Ca2+ uptake and Ca2(+)-ATPase activity in the reconstituted vesicles compared with 0.63 microM Ca2+ in native SR vesicles. Inclusion of phospholamban in the reconstituted vesicles was associated with a significant inhibition of the initial rates of Ca2+ uptake at pCa 6.0. However, phosphorylation of phospholamban by the catalytic subunit of the cAMP-dependent protein kinase reversed the inhibitory effect on the Ca2+ pump. Similar findings were observed when a peptide, corresponding to amino acids 1-25 of phospholamban, was used. These findings indicate that phospholamban is an inhibitor of the Ca2(+)-ATPase in cardiac SR and phosphorylation of phospholamban relieves this inhibition. The mechanism by which phospholamban inhibits the Ca2+ pump is unknown, but our findings with the synthetic peptide suggest that a direct interaction between the Ca2(+)-ATPase and the hydrophilic portion of phospholamban may be one of the mechanisms for regulation.     

Phospholamban regulation of cardiac sarcoplasmic reticulum (Ca(2+)-Mg2+)-ATPase. Mechanism of regulation and site of monoclonal antibody interaction.

Monoclonal antibodies raised against canine cardiac sarcoplasmic reticulum phospholamban were used to study the structure-function relationship between phospholamban and the sarcoplasmic reticulum (SR) (Ca(2+)-Mg2+)-ATPase (Suzuki, T., and Wang, J. H. (1986) J. Biol. Chem. 261, 7018-7023). Additional monoclonal antibodies are characterized further. When five of these monoclonal antibodies were assessed for their ability to affect SR Ca2+ uptake three of these antibodies had no effect on SR Ca2+ uptake, whereas the other two monoclonals were able to stimulate SR Ca2+ uptake to levels similar to those caused by phosphorylation of phospholamban at different calcium concentrations. Using synthetic peptides corresponding to various portions of phospholamban in a competitive binding assay, it was possible to map the epitope site of monoclonals which stimulate the (Ca(2+)-Mg2+)-ATPase activity to phospholamban residues 7-16. These results implicate phospholamban residues 7-16 in the regulation of the (Ca(2+)-Mg2+)-ATPase.     

The substitution of calcium for magnesium in H+,K+-ATPase catalytic cycle. Evidence for two actions of divalent cations

In order to determine the role of divalent cations in the reaction mechanism of the H+,K+-ATPase, we have substituted calcium for magnesium, which is required by the H+,K+-ATPase for phosphorylation from ATP and from PO4. Calcium was chosen over other divalent cations assayed (barium and manganese) because in the absence of magnesium, calcium activated ATP hydrolysis, generated sufficiently high levels of phosphoenzyme (573 +/- 51 pmol.mg-1) from [gamma-32P]ATP to study dephosphorylation, and inhibited K+-stimulated ATP hydrolysis. The Ca2+-ATPase activity of the H+,K+-ATPase was 40% of the basal Mg2+-ATPase activity. However, the Ca2+,K+-ATPase activity (minus the Ca2+ basal activity) was only 0.7% of the Mg2+,K+-ATPase, indicating that calcium could partially substitute for Mg2+ in activating ATP hydrolysis but not in K+ stimulation of ATP hydrolysis. Approximately 0.1 mM calcium inhibited 50% of the Mg2+-ATPase or Mg2+,K+-ATPase activities. Inhibition of Mg2+,K+-ATPase activity was not competitive with respect to K+. Inhibition by calcium of Mg2+,K+ activity p-nitrophenyl phosphatase activity was competitive with respect to Mg2+ with an apparent Ki of 0.27 mM. Proton transport measured by acridine orange uptake was not detected in the presence of Ca2+ and K+. In the presence of Mg2+ and K+, Ca2+ inhibited proton transport with an apparent affinity similar to the inhibition of the Mg2+, K+-ATPase activity. The site of calcium inhibition was on the exterior of the vesicle. These results suggest that calcium activates basal turnover and inhibits K+ stimulation of the H+,K+-ATPase by binding at a cytosolic divalent cation site. The pseudo-first order rate constant for phosphoenzyme formation from 5 microM [gamma-32P]ATP was at least 22 times slower in the presence of calcium (0.015 s-1) than magnesium (greater than 0.310 s-1). The Ca.EP (phosphoenzyme formed in the presence of Ca2+) formed dephosphorylated four to five times more slowly that the Mg.EP (phosphoenzyme formed in the presence of Mg2+) in the presence of 8 mm trans-1,2-diaminocyclohexane-N,N,N',N'-tetraacetic acid (CDTA) or 250 microM ATP. Approximately 10% of the Ca.EP formed was sensitive to a 100 mM KCl chase compared with greater than 85% of the Mg.EP. By comparing the transient kinetics of the phosphoenzyme formed in the presence of magnesium (Mg.EP) and calcium (Ca.EP), we found two actions of divalent cations on dephosphorylation.(ABSTRACT TRUNCATED AT 400 WORDS)     

Role of phospholamban in regulating cardiac sarcoplasmic reticulum calcium pump

Cardiac sarcoplasmic reticulum plays a critical role in the excitation-contraction cycle and hormonal regulation of heart cells. Catecholamines exert their ionotropic action through the regulation of calcium transport into the sarcoplasmic reticulum. Cyclic 3'-5'-adenosine monophosphate (cAMP) causes the cAMP-dependent protein kinase to phosphorylate the regulatory protein phospholamban, which results in the stimulation of calcium transport. Calmodulin also phosphorylates phospholamban by a calcium-dependent mechanism. We have reported the isolation and purification of phospholamban with low deoxycholate (DOC) concentrations (5 X 10(-6) M). We have also reported the isolation and purification of Ca2+ + Mg2+-ATPase with a similar procedure. Both phospholamban and Ca2+ + Mg2+-ATPase retained their native properties associated with sarcoplasmic reticulum vesicles. Further, we have shown that the removal of phospholamban from membranes of sarcoplasmic reticulum vesicles uncouples Ca2+-uptake from ATPase without any effect on Ca2+ + Mg2+-ATPase activity or Ca2+ efflux. Phospholamban appears to be the substrate for both the Ca2+-calmodulin system and the cAMP-dependent protein kinase system. It is found that the phosphorylation of phospholamban by the Ca2+-calmodulin system is required for the normal basal level of Ca2+ transport, and that the phosphorylation of phospholamban at another site by the cAMP-dependent protein kinase system causes the stimulation of Ca2+-transport above the basal level. The functional effects of the phosphorylation of phospholamban by cAMP-dependent protein kinase system are expressed only after the phosphorylation of phospholamban with Ca2+-calmodulin system. We propose a model for the cardiac Ca2+ + Mg2+-ATPase, whereby the enzyme is normally uncoupled from Ca2+ uptake. The enzyme becomes coupled to Ca2+ transport after the first site of phospholamban is phosphorylated with the Ca2+-calmodulin system. When the second site of phospholamban is phosphorylated with cAMP-dependent protein kinase both Ca2+ transport and ATPase are stimulated and phospholamban becomes inaccessible to DOC solubilization and trypsin.     

Characterization of the Ca2+- and Mg2+-Dependent ATPases in Electrophorus Electroplax Microsomes

Electrophorus electroplax microsomes were examined for Ca2+- and Mg2+-dependent ATPase activity. In addition to the previously reported low-affinity ATPase, a high-affinity (Ca2+,Mg2+)-ATPase was found. At low ATP and Mg2+ concentrations (200 microM or less), the high-affinity (Ca2+,Mg2+)-ATPase exhibits an activity of 18 nmol Pi mg-1 min-1 with 0.58 microM Ca2+. At higher ATP concentrations (3 mM), the low-affinity Ca2+-ATPase predominates, with an activity of 28 nmol Pi mg-1 min-1 with 1 mM Ca2+. In addition, Mg2+ can also activate the low-affinity ATPase (18 nmol Pi mg-1 min-1). The high-affinity ATPase hydrolyzes ATP at a greater rate than it does GTP, ITP, or UTP and is insensitive to ouabain, oligomycin, or dicyclohexylcarbodiimide inhibition. The high-affinity enzyme is inhibited by vanadate, trifluoperazine, and N-ethylmaleimide. Added calmodulin does not significantly stimulate enzyme activity; rinsing the microsomes with EGTA does not confer calmodulin sensitivity. Thus the high-affinity ATPase from electroplax microsomes is similar to the (Ca2+,Mg2+)-ATPase reported to be associated with Ca2+ transport, based on its affinity for calcium and its response to inhibitors. The low-affinity enzyme hydrolyzes all tested nucleoside triphosphates, as well as diphosphates, but not AMP. Vanadate and N-ethylmaleimide do not inhibit the low-affinity enzymes. The low-affinity enzyme reflects a nonspecific nucleoside triphosphatase, probably an ectoenzyme.     

Effect of ovarian steroids on membrane ATPase activities in microsomes (microsomal fractions) from rat myometrium. Inhibition of a component of the Mg2+-activated ATPase by Ca2+-calmodulin and by oxytocin.

The activities of Mg2+-ATPase (Mg2+-activated ATPase), (Ca2+ + Mg2+)-activated ATPase and (Na+ + K+)-activated ATPase have been determined in microsomes (microsomal fractions) obtained from rat myometrium under different hormonal conditions. Animals were either ovariectomized and treated for a prolonged period of time with 17 beta-oestradiol or progesterone, or myometria were obtained at day 21 of pregnancy. In each case the endometrium was carefully removed. The Mg2+-ATPase consists of two components: an inactivating labile component and a second constant component. The rate of ATP hydrolysis by the labile component of the Mg2+-ATPase declines exponentially as a function of time after adding the membranes to the assay medium; this inactivation is caused by the presence of ATP in the medium. This ATPase activity inhibited by ATP is catalysed by a labile enzyme and hence it gradually diminishes within a few hours, even when the microsomes are kept on ice. This labile component has the highest activity in microsomes from pregnant rats, a lower activity in progesterone-treated rats, and the lowest in 17 beta-oestradiol-treated rats. This component of the Mg2+-ATPase is not affected by 90 nM-oxytocin. The constant component of the Mg2+-ATPase must be ascribed to a different enzyme, which, in contrast with the labile component, is very stable and not affected by the hormonal status of the animal. This constant component of the Mg2+-ATPase is inhibited both by Ca2+-calmodulin, and by oxytocin in microsomes from pregnant and from progesterone-treated animals, whereas such inhibition does not occur in microsomes from 17 beta-oestradiol-treated animals. The activity of the (Na+ + K+)-activated ATPase is not dependent on the hormonal status of the animal. Myometrial microsomes present an ATP-dependent Ca2+ transport, irrespective of the hormonal condition, but only in microsomes obtained from rats treated with 17 beta-oestradiol, can a (Ca2+ + Mg2+)-activated ATPase activity be demonstrated. This activity can be stimulated by calmodulin.     

The use of a water-soluble carbodiimide to cross-link cytochrome c to plastocyanin

Canine cardiac sarcoplasmic reticulum is phosphorylated by adenosine 3',5'-monophosphate (cAMP)-dependent and by Ca2+-calmodulin-dependent protein kinases on an Mr 22 000 protein called phospholamban. Both types of phosphorylation are associated with an increase in the initial rate of Ca2+ transport. Thus, phospholamban appears to be a regulator for the calcium pump in cardiac sarcoplasmic reticulum. However, there is conflicting evidence as to the degree of association of the Ca2+-ATPase with its regulator, phospholamban. In this study, we report that phospholamban does not copurify with a Ca2+-ATPase preparation of high specific activity. Although 32P-labeled phospholamban is solubilized in the same fraction as the Ca2+-ATPase from cardiac sarcoplasmic reticulum, it dissociates from the Ca2+ pump during subsequent purification steps. Our isolation procedure results in an increase of over 4-fold in the specific activity of the Ca2+-ATPase, but a decrease of 2.5-fold in the specific activity of 32Pi-phosphoester bonds (pmol Pi/mg). Furthermore, the purified Ca2+-ATPase enzyme preparation is not a substrate for protein kinase in vitro to any significant extent. These data indicate that phospholamban does not copurify with the Ca2+-ATPase from cardiac sarcoplasmic reticulum. Isolation of a Ca2+-ATPase preparation essentially free of phospholamban will aid in future kinetic studies designed to elucidate similarities and differences in the Ca2+-ATPase parameters from cardiac and skeletal muscle (which is known not to contain phospholamban).     

Calmodulin-mediated regulation of calcium transport and (Ca2+ + Mg2+)-activated ATPase activity in isolated cardiac sarcoplasmic reticulum

A severalfold activation of calcium transport and (Ca2+ + Mg2+)-activated ATPase activity by micromolar concentrations of calmodulin was observed in sarcoplasmic reticulum vesicles obtained from canine ventricles. This activation was seen in the presence of 120 mM KCl. The ratio of moles of calcium transported per mol of ATP hydrolyzed remained at about 0.75 when calcium transport and (Ca2+ + Mg2+)-activated ATPase activity were measured in the presence and absence of calmodulin. Thus, the efficiency of the calcium transport process did not change. Stimulation of calcium transport by calmodulin involves the phosphorylation of one or more proteins. The major 32P-labeled protein, as determined by sodium dodecyl sulfate slab gel electrophoresis, was the 22,000-dalton protein called phospholamban. The Ca2+ concentration dependency of calmodulin-stimulated microsomal phosphorylation corresponded to that of calmodulin-stimulated (Ca2+ + Mg2+)-activated ATPase activity. Proteins of 11,000 and 6,000 daltons and other proteins were labeled to a lesser extent. A similar phosphorylation pattern was obtained when microsomes were incubated with cAMP-dependent protein kinase and ethylene glycol bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid. Phosphorylation produced by added cAMP-dependent protein kinase and calmodulin was additive. These studies provided further evidence for Ca2+-dependent regulation of calcium transport by calmodulin in sarcoplasmic reticulum that could play a role in the beat-to-beat regulation of cardiac relaxation in the intact heart.     

Measurement of microsomal ATPase activities: a comparison between the inorganic phosphate-release assay and the NADH-coupled enzyme assay

The specific activity of the Mg2+-ATPase and the (Ca2+ + Mg2+)-ATPase has been measured in a microsomal fraction from pig antral smooth muscle with the phosphate-release assay and the NADH-coupled enzyme assay, and the release of inorganic phosphate as a function of time is compared with the concomitant production of ADP. Both assays are found to overestimate the true Mg2+-ATPase activity. The adenylate kinase inhibitor P1,P5-di(adenosine-5'-)pentaphosphate (Ap5A) reduces the specific activity of the Mg2+-ATPase measured in the NADH-coupled enzyme assay to about half of its original value; however, it does not affect the specific activity of the Mg2+-ATPase in the Pi-release assay. The considerable overestimation of the Mg2+-ATPase activity in the NADH-coupled enzyme assay results from a combined action of an ATP pyrophosphatase (ATP in equilibrium AMP + PPi) and adenylate kinase activity contaminating the microsomes. The adenylate kinase activity in the microsomes catalyses the conversion of AMP formed by the ATP pyrophosphatase together with ATP into two ADP's. Also the phosphate-release assay is prone to an overestimation artefact because an inorganic pyrophosphatase will degrade the pyrophosphate and thus lead to additional Pi-production. Measurements of AMP and NAD+ production by HPLC confirmed our proposed reaction scheme. The same (Ca2+ + Mg2+)-ATPase activity is found in both assays, because the (Ca2+ + Mg2+)-ATPase activity is calculated from the difference in ATPase activity in the presence and absence of Ca2+, so that as a consequence the interfering activities are automatically subtracted.     

Fe2+ and other divalent metal ions uncouple Ca2+ transport from (Ca2+-Mg2+)-ATPase in rat liver plasma membranes

The addition of nanomolar concentrations of free Fe2+, Mn2+, or Co2+ to rat liver plasma membranes resulted in an activation of ATP hydrolysis by these membranes which was not additive with the Ca2+-stimulated ATPase activity coupled to the Ca2+ pump. Detailed analysis showed that, if fact, (i) as for the stimulation of (Ca2+-Mg2+)-ATPase by Ca2+, activation of ATP hydrolysis by Fe2+, Mn3+, or Co2+ followed a cooperative mechanism involving two ions; (ii) two interacting sites for ATP were involved in the activation of both Fe2+- and Ca2+-stimulated ATPase activities; (iii) micromolar concentrations of magnesium caused the same dramatic inhibition of both activities; and (iv) the subcellular distribution of Fe2+-activated ATP hydrolysis activity corresponded to that of plasma membrane markers. This suggests that the (Ca2+-Mg2+)-ATPase might be stimulated not only by Ca2+, but also by Fe2+, Mn2+, or Co2+. However, interaction of (Ca2+-Mg2+)-ATPase with Fe2+, Mn2+, or Co2+ inhibited the Ca2+ pump activity. Furthermore, neither the formation of the phosphorylated intermediate of (Ca2+-Mg2+)-ATPase, nor ATP-dependent (59Fe) uptake could be detected in the presence of Fe2+ concentrations which stimulated ATP hydrolysis. We conclude that: (i) under the influence of certain metal ions, the Ca2+ pump in the liver plasma membrane may be switched to an uncoupled state which displays ATP hydrolysis activity, but does not insure ion transport; (ii) therefore the Ca2+ pump in liver plasma membranes specifically insures Ca2+ transport.     

Ca2+ transport studied with arsenazo III in Tetrahymena microsomes. Effects of calcium ionophore A23187 and trifluoperazine

Transport of Ca2+ in microsomal membrane vesicles of the Tetrahymena has been investigated using arsenazo III as a Ca2+ indicator. The microsomes previously shown to carry a Mg2+-dependent, Ca2+-stimulated ATPase (Muto, Y. and Nozawa, Y. (1984) Biochim. Biophys. Acta 777, 67-74) accumulated calcium upon addition of ATP and Ca2+ sequestered into microsomal vesicles was rapidly discharged by the Ca2+ ionophore A23187. Kinetic studies indicated that the apparent Km for free Ca2+ and ATP are 0.4 and 59 microM, respectively. The Vmax was about 40 nmol/mg protein per min at 37 degrees C. The calcium accumulated during ATP-dependent uptake was released after depletion of ATP in the incubation medium. Furthermore, addition of trifluoperazine which inhibited both (Ca2+ + Mg2+)-ATPase and ATP-dependent Ca2+ uptake rapidly released the calcium accumulated in the microsomal vesicles. These observations suggest that Tetrahymena microsome contains both abilities to take up and to release calcium and may act as a Ca2+-regulating site in this organism.     

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.     

A high-affinity, calmodulin-sensitive (Ca2+ + Mg2+)-ATPase and associated calcium-transport pump in the Ehrlich ascites tumor cell plasma membrane

A unique cytoplast preparation from Ehrlich ascites tumor cells (G. V. Henius, P. C. Laris, and J. D. Woodburn (1979) Exp. Cell. Res. 121, 337-345), highly enriched in plasma membranes, was employed to characterize the high-affinity plasma membrane calcium-extrusion pump and its associated adenosine triphosphatase (ATPase). An ATP-dependent calcium-transport system which had a high affinity for free calcium (K0.5 = 0.040 +/- 0.005 microM) was identified. Two different calcium-stimulated ATPase activities were detected. One had a low (K0.5 = 136 +/- 10 microM) and the other a high (K0.5 = 0.103 +/- 0.077 microM) affinity for free calcium. The high-affinity enzyme appeared to represent the ubiquitous high-affinity plasma membrane (Ca2+ + Mg2+)-ATPase (calcium-stimulated, magnesium-dependent ATPase) seen in normal cells. Both calcium transport and the (Ca2+ + Mg2+)-ATPase were significantly stimulated by the calcium-dependent regulatory protein calmodulin, especially when endogenous activator was removed by treatment with the calcium chelator ethylene glycol bis(beta-aminoethyl ether) N,N'-tetraacetic acid. Other similarities between calcium transport and the (Ca2+ + Mg2+)-ATPase included an insensitivity to ouabain (0.5 mM), lack of activation by potassium (20 mM), and a requirement for magnesium. These similar properties suggested that the (Ca2+ + Mg2+)-ATPase represents the enzymatic basis of the high-affinity calcium pump. The calcium pump/enzyme system was inhibited by orthovanadate at comparatively high concentrations (calcium transport: K0.5 congruent to 100 microM; (Ca2+ + Mg2+)-ATPase: K0.5 greater than 100 microM). Upon Hill analysis, the tumor cell (Ca2+ + Mg2+)-ATPase failed to exhibit cooperative activation by calcium which is characteristic of the analogous enzyme in the plasma membrane of normal cells.     

Mechanism of the stimulation of cardiac sarcoplasmic reticulum calcium pump by calmodulin

Calmodulin has been shown to stimulate the initial rates of Ca2+-uptake and Ca2+-ATPase in cardiac sarcoplasmic reticulum, when it is present in the reaction assay media for these activities. To determine whether the stimulatory effect of calmodulin is mediated directly through its interaction with the Ca2+-ATPase, or indirectly through phosphorylation of phospholamban by an endogenous protein kinase, two approaches were taken in the present study. In the first approach, the effects of calmodulin were studied on a Ca2+-ATPase preparation, isolated from cardiac sarcoplasmic reticulum, which was essentially free of phospholamban. The enzyme was preincubated with various concentrations of calmodulin at 0 degrees C and 37 degrees C, but there was no effect on the Ca2+-ATPase activity assayed over a wide range of [Ca2+] (0.1-10 microM). In the second approach, cardiac sarcoplasmic reticulum vesicles were prephosphorylated by an endogenous protein kinase in the presence of calmodulin. Phosphorylation occurred predominantly on phospholamban, an oligomeric proteolipid. The sarcoplasmic reticulum vesicles were washed prior to assaying for Ca2+ uptake and Ca2+-ATPase activity in order to remove the added calmodulin. Phosphorylation of phospholamban enhanced the initial rates of Ca2+-uptake and Ca2+-ATPase, and this stimulation was associated with an increase in the affinity of the Ca2+-pump for calcium. The EC50 values for calcium activation of Ca2+-uptake and Ca2+-ATPase were 0.96 +/- 0.03 microM and 0.96 +/- 0.1 microM calcium by control vesicles, respectively. Phosphorylation decreased these values to 0.64 +/- 0.12 microM calcium for Ca2+-uptake and 0.62 +/- 0.11 microM calcium for Ca2+-ATPase. The stimulatory effect was associated with increases in the apparent initial rates of formation and decomposition of the phosphorylated intermediate of the Ca2+-ATPase. These findings suggest that calmodulin regulates cardiac sarcoplasmic reticulum function by protein kinase-mediated phosphorylation of phospholamban.     

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.     

Effect of insulin secretagogues and potential modulators of secretion on a plasma membrane (Ca2+ + Mg2+)-ATPase activity in islets of Langerhans

Studies were undertaken to determine whether factors which affect insulin secretion may exert their effects by altering the activity of an islet-cell plasma membrane Ca2+ extrusion pump. The insulin secretagogue, D-glucose, and a variety of phosphorylated hexoses, glucose 6-P, glucose 1,6-P, fructose 6-P, and fructose 2,6-P, were evaluated for their effect on an islet-cell plasma membrane (Ca2+ + Mg2+)-ATPase and were found to be ineffective in altering enzyme activity. D-Glucose also did not alter the rate of ATP-dependent Ca2+ uptake into plasma membrane vesicles. Similarly, cAMP, the catalytic subunit of cAMP-dependent protein kinase, arachidonic acid, or prostaglandin E2 did not affect either the plasma membrane (Ca2+ + Mg2+)-ATPase or the rate of ATP-dependent Ca2+ uptake into plasma membrane vesicles. Whereas previous studies have suggested that D-glucose and/or cAMP may inhibit ATPase activities in islets, these results indicate that the agents, i.e., D-glucose and cAMP, which stimulate and/or potentiate insulin secretion from the islet cell, do not modify Ca2+ fluxes by directly regulating the islet-cell plasma membrane (Ca2+ + Mg2+)-ATPase. In contrast, the acidic phospholipids, phosphatidic acid and phosphatidylserine, stimulated the enzyme activity in a concentration-dependent manner whereas phosphatidylcholine had only a minimal effect. The diacylglycerol, dilinolein, stimulated the (Ca2+ + Mg2+)-ATPase activity in the presence of phosphatidylserine, but not in the absence of phospholipids. These effects were independent of phospholipid-stimulated protein phosphorylation in the islet-cell plasma membrane under the conditions of the ATPase assay.     

Calmodulin regulation of Ca2+ transport in human erythrocytes.

Inside-out vesicles of human erythrocytes took up Ca2+ against an electrochemical gradient. This Ca2+ uptake was dependent on ATP and was stimulated by calmodulin. Treatment of vesicles with 1 mM-EDTA exposed an apparent low-CA2+-affinity Ca2+-transport component with Kd of about 100 microM-Ca2+ or more. This was converted into a single high-Ca2+-affinity transport activity of Kd about 2.5 microM-Ca2+ in the presence of 2 micrograms of calmodulin/ml, showing that the decrease in transport activity after EDTA treatment was reversible. Vesicles not extracted with EDTA showed mainly apparent high-Ca2+-affinity kinetics even in the absence of added calmodulin. Trifluoperazine (30 microM) and calmodulin-binding protein (20 micrograms/ml) inhibited about 50% of the high-affinity Ca2+ uptake and (Ca2+ + Mg2+)-ATPase (Ca2+-activated, Mg2+-dependent ATPase) activity of these vesicles, indicating that the vesicles isolated by the procedure used retained some calmodulin from the erythrocytes. Comparison of Ca2+ transport and (Ca2+ + Mg2+)-ATPase activities in inside-out vesicles yielded a variable Ca2+/P1 stoichiometric ratio. At low free Ca2+ concentrations (below 20 micro-Ca2+), a Ca2+/P1 ration of about 2 was found, whereas at higher Ca2+ concentrations the stoichiometry was approx. 1. The stoichiometry was not significantly altered by calmodulin.