Evidence for a role of the sarcoplasmic/endoplasmic reticulum Ca(2+)-ATPase in thapsigargin and Bcl-2 induced changes in Xenopus laevis oocyte maturation.

Thapsigargin (Tg), a selective inhibitor of sarcoplasmic/endoplasmic reticulum Ca(2+)-ATPase (SERCA), causes depletion of intracellular Ca(2+) stores, hence activation of capacitative Ca(2+) entry (CCE). Incubation of Xenopus laevis oocytes with Tg resulted in an increased rate of progesterone-induced meiotic maturation. Non-mitochondrial (45)Ca(2+) uptake by SERCA-containing microsomes prepared from control wild-type oocytes microinjected with sterile water was inhibited essentially 100% by Tg. However, overexpression of Bcl-2, an oncogene known to protect against Tg-induced apoptosis in certain cell types, resulted in only 40% inhibition of microsomal (45)Ca(2+) uptake by Tg while non-inhibited (45)Ca(2+) uptake remained unchanged. Moreover Bcl-2 overexpression also protected against inhibition of CCE. I(Cl(Ca)) was similar in Bcl-2-overexpressing and control oocytes when intracellular Ca(2+) store depletion was induced by microinjection of inositol 1,4,5-trisphosphate (InsP(3)) and other means and when CCE was induced by means independent of SERCA inhibition. Our data indicate that Bcl-2 affects neither the InsP(3) receptor nor Ca(2+) entry itself. At the end of a 24-h period after progesterone addition to the medium, only 25% of Bcl-2-overexpressing oocytes had matured compared to 85% of control oocytes. Our data suggest that SERCA participates in Xenopus oocyte maturation by controlling cytosolic Ca(2+) and/or intracellular Ca(2+) stores, hence CCE. An observed progesterone-dependent protein kinase-catalysed phosphorylation of SERCA is further indication of its role in oocyte maturation.

Interactions of 6-gingerol and ellagic acid with the cardiac sarcoplasmic reticulum Ca2+-ATPase.

The inotropic/lusitropic effects of beta-adrenergic agonists on the heart are mediated largely by protein kinase A (PKA)-catalyzed phosphorylation of phospholamban, the natural protein regulator of the Ca2+ pump present in sarcoplasmic reticulum (SR) membranes. Gingerol, a plant derivative, is known to produce similar effects when tested in isolated cardiac muscle. The purpose of the present study was to compare the effects of gingerol and another plant derivative, ellagic acid, on the kinetics of the SR Ca2+ pump with those of PKA-catalyzed phospholamban phosphorylation to elucidate their mechanisms of Ca2+ pump regulation. As previously demonstrated for PKA, 50 microM gingerol or ellagic acid increased Vmax(Ca) of Ca2+ uptake and Ca2+-ATPase activity assayed at millimolar ATP concentrations in light cardiac SR vesicles. Unlike PKA, which decreases Km(Ca), neither compound had a significant effect on Km(Ca) in unphosphorylated vesicles. However, gingerol increased Km(Ca) in phosphorylated vesicles, in which Ca2+ uptake was significantly increased further at saturating Ca2+ and remained unchanged at subsaturating Ca2+. An inhibition of Ca2+ uptake by gingerol at micromolar MgATP concentrations was overcome with increasing MgATP concentrations. The stimulation of Ca2+ uptake attributable to gingerol in unphosphorylated microsomes at saturating Ca2+ was 30% to 40% when assayed at 0.05 to 2 mM MgATP and only about 12% in phosphorylated microsomes as well as in rabbit fast skeletal muscle light SR. The present results support the view that an ATP-dependent increase in Vmax(Ca) of the SR Ca2+ pump plays an important role in mediating cardiac contractile responses to gingerol and phospholamban-dependent beta-adrenergic stimulation.

Kinetic differences in the phospholamban-regulated calcium pump when studied in crude and purified cardiac sarcoplasmic reticulum vesicles.

Phospholamban (PLN) phosphorylation contributes largely to the inotropic and lusitropic effects of beta-adrenergic agonists on the heart. The mechanical effects of PLN phosphorylation on the heart are generally attributed solely to an increase in the apparent affinity of the Ca pump in the sarcoplasmic reticulum (SR) membranes for Ca2+ with little or no effect on Vmax(Ca). In the present report, we compare the kinetic properties of the cardiac SR Ca pump in commonly studied crude microsomes with those of our recently developed preparation of light SR vesicles. We demonstrate that in crude microsomes, the increase in the apparent affinity of the pump for Ca2+ is larger, while the increase in Vmax(Ca) is smaller, than in purified vesicles. The greater phosphorylation-induced increase in apparent Ca2+ affinity in crude microsomes may be further enhanced by an ATP-sensitive inhibitory effect of ruthenium red on the activity of the pump at subsaturating, but not saturating, Ca2+ concentrations as a result of a greater inhibition in unphosphorylated microsomes. Upon increasing the ATP concentration from 1 to 5 mm, an inhibition by 10 micrometer ruthenium red is eliminated in phosphorylated microsomes and reduced in control microsomes. Addition of the phosphoprotein phosphatase inhibitor okadaic acid produces a considerable increase in the phosphorylation-induced effects in both crude and purified microsomes. We conclude that the use of purified cardiac SR vesicles is critical for the demonstration of a major increase in Vmax(Ca) in addition to an increase in the pump's apparent affinity for Ca2+ in response to phosphorylation of PLN by protein kinase A.

Comparison of the kinetic effects of phospholamban phosphorylation and anti-phospholamban monoclonal antibody on the calcium pump in purified cardiac sarcoplasmic reticulum membranes.

Protein kinase A- (PKA-) catalyzed phosphorylation of phospholamban (PLN), the protein regulator of the cardiac Ca pump, mediates abbreviation of systole in response to beta-adrenergic agonists. Investigators previously, however, have been unsuccessful in demonstrating an effect of PLN phosphorylation or anti-PLN monoclonal antibody (mAb), which is considered to mimic phosphorylation's well-known effect on Km(Ca), on microsomal Ca uptake at the (high) Ca2+ concentrations found intracellularly at peak systole. We therefore compared the effects of the catalytic subunit of PKA and anti-PLN mAb on the kinetics of Ca uptake in sucrose gradient-purified cardiac microsomes. Both treatments produced a 33-44% increase in Vmax(Ca) at 25 and 37 degrees C, and an 11-31% decrease in Km(Ca) with comparable changes in Ca2+-ATPase activity. An acceleration of E2P decomposition upon PLN phosphorylation may contribute to the increased Vmax(Ca) of Ca uptake at 25 degrees C but not at 37 degrees C, based on measurement of the kinetics of E2P decomposition and steady-state E2P formation from Pi at different temperatures. Our data document almost identical increases in Vmax(Ca) of microsomal Ca uptake with PLN phosphorylation or addition of anti-PLN mAb and hence provide insight into the kinetic mechanism of PLN's regulation of the cardiac sarcoplasmic reticulum Ca pump protein.

Comparison of the effects of phospholamban and jasmone on the calcium pump of cardiac sarcoplasmic reticulum. Evidence for modulation by phospholamban of both Ca2+ affinity and Vmax (Ca) of calcium transport.

Regulation of the calcium pump of the cardiac sarcoplasmic reticulum by phosphorylation/dephosphorylation of phospholamban is central to the inotropic and lusitropic effects of beta-adrenergic agonists on the heart. In order to study the mechanism of this regulation, we first obtained purified ruthenium red-insensitive microsomes enriched in sarcoplasmic reticulum membranes. The kinetics of microsomal Ca2+ uptake after phospholamban phosphorylation or trypsin treatment, which cleaves the inhibitory cytoplasmic domain of phospholamban, were then compared with those in the presence of jasmone, whose effects on the kinetics of fast skeletal muscle Ca2+-ATPase are largely known. All three treatments increased Vmax (Ca) at 25 degrees C and millimolar ATP; phosphorylation and trypsin decreased the Km (Ca), while jasmone increased it. Trypsin and jasmone increased the rate of E2P decomposition 1.8- and 3. 0-fold, respectively. The effects of phospholamban phosphorylation and jasmone on the Ca2+-ATPase activity paralleled their effects on Ca2+ uptake. Our data demonstrate that phospholamban regulates E2P decomposition in addition to the known increase in the rate of a conformational change in the Ca2+-ATPase upon binding the first of two Ca2+. These steps in the catalytic cycle of the Ca2+-ATPase may contribute to or account for phospholamban's effects on both Vmax (Ca) and Km (Ca), whose relative magnitude may vary under different experimental and, presumably, physiological conditions.

Membrane phosphorylation protects the cardiac sarcoplasmic reticulum Ca(2+)-ATPase against chlorinated oxidants in vitro.

OBJECTIVE: The calcium (Ca) pump of cardiac sarcoplasmic reticulum (SR) membranes is vulnerable to oxidation and hence likely to be damaged by chlorinated compounds, specifically hypochlorite (NaOCl) and monochloramine (NH2Cl), the most potent oxidants produced upon neutrophil activation. This could occur during prolonged ischemia or myocardial infarction when tissue levels of catecholamines are high. Phospholamban (PLN), the phosphorylatable regulator of the Ca pump, plays a central role in the effects of beta-adrenergic agonists on the heart. The purpose of this study was to investigate a possible role of PLN in determining the pump's sensitivity to NaOCl and NH2Cl. METHODS: Ca-uptake and Ca(2+)-ATPase activities in purified phosphorylated and control canine cardiac microsomes, incubated at increasing concentrations of NaOCl or NH2Cl, were related to the extent of PLN phosphorylation by protein kinase A, which was quantitated by PhosphorImager analysis. RESULTS AND CONCLUSIONS: Our data indicate that microsomal phosphorylation protects the Ca pump fully against 10 microM NaOCl or NH2Cl, which inhibit Ca-uptake by 21-41% when assayed at 25 or 37 degrees C and saturating Ca2+ in unphosphorylated microsomes, and protects partially at higher oxidant concentrations. The protective effect of protein kinase A on Ca-uptake is proportional to the amount of phosphorylated PLN. No comparable protection against similar oxidative damage of the Ca pump is observed when light fast skeletal muscle microsomes, which lack PLN, are incubated under conditions favorable for phosphorylation nor when PLN's inhibition of the cardiac Ca pump is relieved by proteolytic cleavage of its cytoplasmic domain. Our findings contribute toward an understanding of possible endogenous protective mechanisms that may promote calcium homeostasis in myocardial cells in inflammatory states associated with neutrophil activation and may suggest an approach toward development of protective strategies against oxidative damage in the heart.

Effects of a nonionic detergent on calcium uptake by cardiac microsomes.

We investigated the effects of the nonionic detergent octaethylene glycol monododecyl ether (C12E8) on the sarcoplasmic reticulum calcium pump in cardiac microsomes in view of its specific effects on different ATP-accelerated steps in the catalytic cycle of the Ca-ATPase in leaky fast skeletal muscle microsomes. At low concentrations of MgATP2- (< 2.5 microM), a nonsolubilizing concentration of added C12E8 (15 microM) increased apparent Vmax(MgATP) of oxalate-facilitated calcium uptake associated with MgATP2- binding to the high affinity catalytic site. An ATP induced acceleration of calcium uptake, attributable to regulatory nucleotide binding, was seen between 2 and 3 microM MgATP2- in both C12E8-treated and control microsomes. These effects of C12E8 are similar to those seen previously with trypsin treatment of microsomes [Lu, Y.-Z., Xu, Z.-C., & Kirchberger, M.A. (1993) Biochemistry 32, 3105-3111]. However, at a saturating Ca2+ between 3 and 10 microM MgATP2-, C12E8 produced a greater reduction in the magnitude of the ATP-induced acceleration of calcium uptake seen with trypsin. At 1 mM MgATP2-, C12E8 and trypsin as well as protein kinase A-catalyzed microsomal phosphorylation all increased the Ca2+ affinity of the pump, but only the latter two treatments significantly increased apparent Vmax(Ca). In fact in trypsin-treated and phosphorylated microsomes, C12E8 reduced Vmax(Ca) to close to the control values; it reduced Vmax(Ca) only slightly in control microsomes. Under our experimental conditions, comparable effects of 15 microM C12E8 on calcium uptake were absent in fast skeletal muscle microsomes, which lack phospholamban.(ABSTRACT TRUNCATED AT 250 WORDS)

Evidence for an effect of phospholamban on the regulatory role of ATP in calcium uptake by the calcium pump of the cardiac sarcoplasmic reticulum.

The purpose of this study was to investigate the functional relationship between phospholamban and the nucleotide site of the calcium pump protein of the cardiac sarcoplasmic reticulum. We used control and trypsin-treated cardiac microsomes in which cleavage of the inhibitory cytoplasmic domain of phospholamban is associated with an activation of the calcium pump similar to that produced by protein kinase A catalyzed phospholamban phosphorylation. Phenylglyoxal was shown to inactivate the calcium pump in a pseudo-first-order reaction by binding to a single Arg at the nucleotide binding site. No differences upon trypsin treatment of microsomes were observed in the kinetics of phenylglyoxal inactivation or the ability of millimolar ATP to protect against inactivation. In subsequent kinetic studies, Ca-uptake rates measured at saturating Ca2+ and 5 microM-1 mM MgATP2- were increased 15-32% by trypsin treatment in each of three different microsome preparations. Double-reciprocal plots of the data showed marked downward curvature indicating an acceleratory effect associated with ligand binding to a lower affinity site. At 0.32 microM Ca2+, Ca-uptake rates were lower than at 11 microM Ca2+ but were stimulated to a greater extent by trypsin treatment; control microsomes showed reduced evidence of apparent negative cooperativity. At 0-2 microM MgATP2- and saturating Ca2+, there was a 50% increase in Vmax(app) when the Hill coefficient (N) was 1. At 0-10 microM MgATP2-, second-site binding was evident. At both 0-10 microM and 5 microM-1 mM MgATP2-, trypsin-treated microsomes showed greater activation of Ca uptake attributable to second-site binding than did control microsomes.(ABSTRACT TRUNCATED AT 250 WORDS)

Modulation by polyelectrolytes of canine cardiac microsomal calcium uptake and the possible relationship to phospholamban.

Calcium uptake and (Ca2+ + Mg2+)-ATPase activity in canine cardiac microsomes were found to be stimulated by heparin and various other polyanions. Prior treatment of the microsomes with the ionophores alamethicin or A23187 produced no change in the extent of stimulation of the ATPase activity by heparin yet eliminated net calcium uptake. This finding and a lack of change in the stoichiometric ratio of mol of calcium transported/mol of ATP hydrolyzed (calcium:ATP) suggest that the effect of heparin is on the calcium pump rather than on a parallel calcium efflux pathway. Certain polycationic compounds including poly-L-arginine and histone inhibited both cardiac and fast skeletal muscle microsomal calcium uptake and also produced no change in the stoichiometric ratio of calcium to ATP. Several lines of evidence indicate that the polyanionic compounds tested stimulate calcium uptake by interacting with phospholamban, the putative phosphorylatable regulator of the cardiac sarcoplasmic reticulum calcium pump, whereas polycationic compounds appear to interact with the pump. (i) Heparin stimulated calcium uptake to the same extent as protein kinase A or trypsin, whereas prior phosphorylation or tryptic cleavage of phospholamban from the membrane abolished the stimulatory effect of heparin. (ii) Calcium uptake and (Ca2+ + Mg2+)-ATPase activity in fast skeletal muscle microsomes, which lack phospholamban, were unaffected by heparin. (iii) Purified cardiac (Ca2+ + Mg2+)-ATPase activity was no longer stimulated by heparin yet was still inhibited by polycationic compounds. The heparin-induced stimulation of calcium uptake was dependent on the pH and ionic strength of the heparin-containing preincubation medium, hence electrostatic interactions appear to play a significant role in heparin's stimulatory action. The data are consistent with an inhibitory role of the positively charged cytoplasmic domain of phospholamban with respect to calcium pump activity and the relief of the inhibition upon reduction in phospholamban's positive charge by phosphorylation or binding of polyanions.

Polyphosphoinositide formation in isolated cardiac plasma membranes.

Phosphatidylinositol (PtdIns) and phosphatidylinositol 4-phosphate (PtdIns4P) kinase activities in plasma membranes isolated from canine left ventricle were partially characterized, and their sensitivity to a number of intracellular variables was established. PtdIns and PtdIns4P kinase activities were estimated by the formation of [32P]PtdIns4P and [32P]phosphatidylinositol 4,5-bisphosphate ([32P]PtdIns(4,5)P2), respectively, when membranes were incubated with [gamma-32P]ATP and 0.1% Triton X-100. Unlike [32P]PtdIns4P formation [32P]PtdIns(4,5)P2 formation required exogenous (PtdIns4P) substrate. [32P]PtdIns4P and [32P]PtdIns(4,5)P2 formation were insensitive to Ca2+ at concentrations ranging from 0.1-30 microM. The hydrolysis of [32P]PtdIns4P was less than 15% under standard assay conditions for measuring its formation, and was unaffected by any of the variables tested. The apparent Km of the PtdIns kinase for ATP was 53 +/- 13 (S.E.M.) microM (N = 3). ADP inhibited [32P]PtdIns4P formation competitively with respect to ATP, the Ki being 0.4 mM. The data indicate that ADP is a poor competitive inhibitor of PtdIns kinase at the concentrations which are believed to be present intracellularly normally or which may be attained during mild hypoxia provided ATP levels are maintained in the millimolar range. Hence, any response of the myocardium to alpha-adrenergic hormones during mild hypoxia would be largely unimpaired by effects of Ca2+ on PtdIns and PtdIns(4,5)P2, or of ADP on PtdIns kinase activity.

Differences in calcium uptake in native canine cardiac microsomes are correlated with the ratio of unphosphorylated to phosphorylated phospholamban as determined by Western blot analysis.

Phospholamban (PLM) is detectable by Western blot analysis of canine cardiac microsomes using rabbit antiserum against a peptide containing the 2 to 30 amino acid sequence of PLM. Phosphorylated PLM is distinguishable from the unphosphorylated form by virtue of a reduced electrophoretic mobility. Utilizing digital image analysis to determine relative band densities, it was found that the ratio of unphosphorylated to phosphorylated PLM is correlated with the rate of calcium uptake in 5 preparations of native microsomes (r = 0.94, p less than 0.01). The present analysis may be useful for determining the phosphorylation state of PLM in microsomes obtained from animals in physiological states characterized by impaired sarcoplasmic reticulum calcium pump activity.

Presence of a Ca2+-sensitive CDPdiglyceride-inositol transferase in canine cardiac sarcoplasmic reticulum.

Sarcoplasmic reticulum (SR) and plasma membranes from canine left ventricle were used to evaluate the presence of the enzyme CDPdiglyceride-inositol transferase in these membranes. (K+,-Ca2+)-ATPase activity, a marker for SR, was 79.2 +/- 5.0 (SE) and 11.2 +/- 2.0 mumol.mg-1.h-1 in SR and plasma membrane preparations, respectively, and (Na+,K+)-ATPase activity, a marker for plasma membranes, was 5.6 +/- 1.2 and 99.2 +/- 8.0 mumol.mg-1.h-1, respectively. Contamination of SR and plasma membrane preparations by mitochondria was estimated to be 2% and 8%, respectively, and by Golgi membranes, 0.9% and 1.8%, respectively. Transferase activity, measured at pH 6.8, was 1.32 +/- 0.04 (SE) and 0.28 +/- 0.04 nmol of [3H]phosphatidylinositol ([3H]PtdIns).mg-1.min-1 in three SR and plasma membrane preparations, respectively. The transferase activity detected in the plasma membrane preparation could be accounted for largely, but not entirely, by contaminating SR membranes. The pH optimum for the SR transferase activity was between 8.0 and 9.0; little or no activity was detectable at pH 6.3 and 5.5, the lowest pH tested. Ca2+ inhibited the enzyme, half-maximal inhibition occurring at about 10 microM Ca2+; removal of the Ca2+ by addition of ethylene glycol bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid restored activity. No loss of [3H]PtdIns could be detected when membranes were incubated in the presence or absence of Ca2+. The Ca2+ inhibition of the transferase was noncompetitive with respect to CDP-dipalmitin while that with respect to myo-inositol was slightly noncompetitive at low [Ca2+] and became uncompetitive at higher [Ca2+].(ABSTRACT TRUNCATED AT 250 WORDS)

A regulation of the level of phosphorylated phospholamban by inhibitor-1 in rat heart preparations in vitro.

Inhibitor-1 following phosphorylation by protein kinase A inhibits phosphoprotein phosphatase-1. We have found that in the rat heart inhibitor-1 is present only in the cytosolic fraction and that its phosphorylation in ventricular slices was increased by isoproterenol but not by isoproterenol and propranolol together. Cardiac microsomal phosphoprotein phosphatase activity, with added phosphorylase a as the substrate, was inhibited 33% by phosphorylated inhibitor-1. Phosphorylated inhibitor-1 decreased the dephosphorylation by exogenous phosphoprotein phosphatase-1 of phospholamban present in the sarcoplasmic reticulum membranes. These results suggest an interaction of cytoplasmic inhibitor-1 with either cytoplasmic or membrane-bound phosphoprotein phosphatase-1 with a subsequent effect on the level of phosphorylated phospholamban and the probable involvement of this interaction in the cardiac response to beta-adrenergic hormones.

Proteolytic activation of the canine cardiac sarcoplasmic reticulum calcium pump.

Mild trypsin treatment of canine cardiac microsomes consisting largely of sarcoplasmic reticulum vesicles produced a severalfold activation of oxalate-facilitated calcium uptake. The increase in calcium uptake was associated with an increase in ATP hydrolysis. Proteases other than trypsin were also effective although to a lesser degree. Trypsin produced a shift of the Ca2+ concentration dependency curve for calcium uptake toward lower Ca2+ concentrations, which was almost identical with that produced by phosphorylation of microsomes by cyclic AMP dependent protein kinase when the trypsin and the protein kinase were present at maximally activating concentrations. The Hill numbers (+/- SD) of the Ca2+ dependency after treatment of microsomes with trypsin (1.5 +/- 0.1) or protein kinase (1.7 +/- 0.1) were similar and were not significantly different from those for untreated control microsomes (1.6 +/- 0.1 and 1.8 +/- 0.1, respectively). Autoradiograms of sodium dodecyl sulfate-polyacrylamide electrophoretic gels indicate that 32P incorporation into phospholamban (Mr 27.3K) or its presumed monomeric subunit (Mr 5.5K) was markedly reduced when trypsin-treated microsomes were incubated in the presence of cyclic AMP dependent protein kinase and [gamma-32P]ATP compared to control microsomes incubated similarly but pretreated with trypsin inhibitor inactivated trypsin. The activation of calcium uptake by increasing concentrations of trypsin was paralleled by the reduction of phosphorylation of phospholamban. Trypsin treatment of microsomes previously thiophosphorylated in the presence of cyclic AMP dependent protein kinase and [gamma-35S]thio-ATP did not result in a loss of 35S label from phospholamban, which suggests that phosphorylation of phospholamban protects against trypsin attack.(ABSTRACT TRUNCATED AT 250 WORDS)

Evidence for catecholamine-stimulated adenylate cyclase activity in frog and rabbit corneal epithelium and cyclic AMP-dependent protein kinase and its protein substrates in frog corneal epithelium.

Evidence was obtained for catecholamine-stimulated adenylate cyclase activity in particulate fractions of frog and rabbit corneal epithelium. Epinephrine (10(-5)M) stimulated adenylate cyclase by 22 and 53% in the frog and rabbit, respectively. The corresponding changes were statistically significant (P less than 0.01) when the data was analyzed using paired variates. Preincubation with 10(-4)M propranolol eliminated any stimulatory effect by 10(-5)M isoproterenol. Adenylate cyclase activity derived from either source was activated several fold by either 10 mM NaF or 10(-5)MGpp (NH)p. Soluble fractions of homogenized frog corneal epithelium contained cyclic AMP-dependent protein kinase activity which was half-maximally stimulated by about 6 nM cyclic AMP. Evidence was also obtained for the presence of protein substrates of cyclic AMP dependent protein kinase in frog corneal epithelium. With exogenous cyclic AMP and protein kinase, a rapid 32P labelling of proteins having approximate molecular weights of 56, 46, 23 and 21 K was obtained with sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis. A less marked and slower increase in phosphoprotein formation was observed when corneal membranes were incubated with cyclic AMP in the absence of added protein kinase.

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.