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 β-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°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.

catalyzed phosphorylation of PLN, which leads to increased calcium pump activity, plays a central role in both the lusitropic and inotropic effects of catecholamines on the myocardium. Evidence for this dual role of PLN has been obtained in experiments with cardiomyocytes (3) and with PLN gene-deficient mice, whose hearts exhibited mechanical properties virtually identical to fully catecholamine-stimulated hearts from wild-type mice (4).
Despite major progress in our understanding of the physiological role of PLN, the molecular mechanism of its regulation of the SR calcium pump remains poorly understood. Unphosphorylated PLN is believed to function as a calcium pump inhibitor that becomes inactive upon phosphorylation by PKA (5)(6)(7). Removal of this inhibitory influence is also produced in vitro by proteolytic cleavage of the cytoplasmic domain of PLN (6) or by incubation of SR membranes in the presence of certain polyanions (8) or monoclonal antibodies against PLN (7).
Almost all of the laboratories that have studied the effect of PKA-catalyzed phosphorylation of PLN or trypsin treatment of cardiac microsomes on the kinetics of calcium uptake or Ca 2ϩ -ATPase activity have reported a phosphorylation-induced increase in the apparent equilibrium constant for Ca 2ϩ binding, K m (Ca) (9 -17). Some investigators have concluded that this is the only effect of PLN phosphorylation by PKA (4,(11)(12)(13)(14)(15)(16), while others have reported that V max (Ca) is also increased (2,9). Another laboratory reported that phosphorylation affects V max (Ca) but not K m (Ca) (18). In our studies (9, 10), we interpreted the actions of PLN in terms of effects on different steps in the catalytic cycle of the Ca 2ϩ -ATPase, which may become rate-limiting under different experimental conditions, although rates of individual steps in the catalytic cycle were not measured.
The major elementary steps in the catalytic cycle of the cardiac Ca 2ϩ -ATPase are summarized in Scheme 1, which represents a modification of the models given in Refs. 19 and 20. The enzyme is believed to exist in two major functional states, E 1 and E 2 , whose equilibrium is shifted toward E 1 in the presence of either ATP or Ca 2ϩ . ATP, in the presence of Mg 2ϩ , binds to E 1 with high affinity and accelerates the conversion of E 2 to E 1 and the conformational change involved in the binding of Ca 2ϩ , which also binds to E 1 with high affinity (step 2) (19,21,22). ATP may also bind to E 2 with reduced affinity (step 1a) and accelerate the conversion of E 2 to E 1 , followed by high affinity binding of Ca 2ϩ to the latter (23) (step 2a). After hydrolysis of the ATP and occlusion of Ca 2ϩ (step 3), ADP is released on the cytoplasmic side of the SR membrane (step 4), and Ca 2ϩ is vectorially transferred across the membrane (step 5) and released on its lumenal side (step 6). Finally, E 2 P decomposition and release of inorganic phosphate on the cytoplas-mic side of the membrane return the enzyme to the ligand-free E 2 state (step 7). However, ATP (shown in parentheses) may bind with reduced affinity to the enzyme already at steps 3, 5, and 7 of the cycle and produce forward acceleration of these transitions (20,22).
In the present study, we compare the effects of PLN phosphorylation or trypsin treatment of purified cardiac microsomes on some of the properties of the calcium pump of SR membranes with those produced by the addition of jasmone. Jasmone, a minor component of peppermint oil, has been shown to accelerate E 2 P decomposition (step 7) in fast skeletal muscle SR, causing a decrease in the equilibrium constant for phosphorylation of E 2 by P i (step 7) (24). This effect was attributed to a specific interaction of jasmone with the fast skeletal muscle Ca 2ϩ -ATPase rather than to general perturbation of the phospholipid bilayer of the SR membrane, although an interaction with the Ca 2ϩ -ATPase that involves its relationship to phospholipids cannot be ruled out. Using our newly developed preparation of ruthenium red-insensitive cardiac microsomes enriched in SR membranes, we demonstrate an increase in the rate of E 2 P decomposition of the Ca 2ϩ -ATPase as a result of treatment of these membranes with jasmone or trypsin. This increase may account for or contribute to an observed increase in the V max (Ca) of calcium uptake that is observed with similar treatment or as a result of PKA-catalyzed phosphorylation of the microsomes.

EXPERIMENTAL PROCEDURES
Materials-Cis-jasmone was purchased from Aldrich. PKA was partially purified from bovine heart (25). Ultrapure sucrose was obtained from Schwarz/Mann Biotech, and ruthenium red, the catalytic subunit of bovine heart PKA, and A23187 were from Sigma. 32 P i and [␣-32 P]ATP were obtained from DuPont NEN. Unless otherwise stated, the PKA used was a preparation that we partially purified from bovine heart and dialyzed against 5 mmol/liter histidine-HCl, pH 6.8 (25); the final solution against which the protein kinase was dialyzed was used as a control solution. When the catalytic subunit of PKA was used, a control solution containing all of the salts and other reagents present in the solution in which the enzyme was dissolved was prepared. Enzymes and phospho(enol)pyruvate for the ATPase assay were obtained from Boehringer Mannheim. Cellulose polyethyleneimine-coated plastic thin layer sheets were obtained from J. T. Baker. All other reagents were obtained as previously reported (9,10).
Preparation of Microsomes-Crude canine cardiac and fast skeletal muscle microsomes, derived from vastus lateralis muscle of male New Zealand White rabbits, were prepared as described previously (6). The microsomes were purified on a sucrose density gradient as follows. The pellets obtained after the second high speed centrifugation in the preparation of the crude microsomes were suspended with the aid of a Dounce homogenizer in two 6-ml aliquots of buffer G containing 18 mM PIPES-KOH, pH 6.8, and 120 mM KCl, each to a protein concentration not greater than 50 mg/ml. A sucrose step gradient was prepared by loading two or more Beckman SW28 centrifuge tubes as follows: 5 ml of 40% (w/w), 8 ml of 33%, 10 ml of 30%, and 7 ml of 25% sucrose in buffer G. All procedures were carried out in the cold room or in ice except that the concentration of each sucrose solution was checked at 23°C with a refractometer and adjusted prior to chilling. Centrifugation was carried out at 20,000 rpm (70,000 ϫ g max ) for 20 h. The fractions at the density interfaces were collected with the aid of a 5-ml glass syringe and designated F 1 (topmost), F 2 , and F 3 . The sucrose concentration of each of the fractions was adjusted to 12% by the addition of buffer G. Following centrifugation of the fractions in a Beckman Ti52 rotor at 29,000 rpm (101,600 ϫ g max ) for 90 min, the pellets were suspended in buffer B (10 mM HEPES-KOH, pH 6.8, 60 mM KCl, and 1 mM dithiothreitol) at final concentrations ranging from 6.1 to 15.0 mg/ml and stored in liquid nitrogen. Microsomal protein concentrations were determined using the biuret procedure with bovine serum albumin as the standard. The yields of crude and purified cardiac microsomes were approximately 0.9 and 0.1 mg of protein, respectively per gram, wet weight, of left ventricle. The sensitivity of microsomal calcium uptake to 5 M ruthenium red, an inhibitor of the ligand-gated Ca 2ϩ release channel (26), was used as a functional criterion for the absence of these channels. No difference in calcium uptake rates was found in purified microsomes when the assays were run under standard conditions (at 25°C) at 11 M Ca 2ϩ , as described below, or at 37°C in the presence of 5 mM oxalate. Ruthenium red was tested at concentrations ranging from 0.5 to 50 M. Concentrations higher than 5 M produced no further stimulation of calcium uptake in microsomal fractions containing Ca 2ϩ release channels, and concentrations higher than 25 M produced inhibition.
Calcium Uptake-Oxalate-facilitated calcium uptake by microsomes was measured by a filtration procedure described previously (9). In some experiments the microsomes were pretreated with trypsin at a maximally stimulatory concentration of 0.01 mg/ml or trypsin inhibitorinactivated trypsin as a control (9). After the addition of 0.12 mg/ml trypsin inhibitor to the trypsin-treated microsomes, both reaction tubes were kept on ice as aliquots were being removed for assay. The composition of the standard assay mixture was 40 mM histidine-HCl, pH 6.8, at 25°C, 120 mM KCl, 5 mM NaN 3 , 4 mM phosphoenolpyruvate, 0.2 mg/ml pyruvate kinase, 2 mM MgCl 2 , 2.5 mM oxalate-Tris, 1 mM ATP, 0.01 mg/ml microsomes, and a 45 Ca-labeled CaCl 2 -EGTA buffer consisting of 125 M CaCl 2 and different concentrations of EGTA to yield the Ca 2ϩ concentrations specified below. Ca 2ϩ , Mg 2ϩ , and Mg-ATP concentrations were calculated as described previously (27) except that a Ca-EGTA binding constant of 10 6 M Ϫ1 was used. The specific radioactivity of the 45 Ca was about 1.3 ϫ 10 5 Bq/mol at 11 M Ca 2ϩ . When calcium uptake was assayed over a range of Ca 2ϩ concentrations extending from 0.02 to 11 M, the specific radioactivity was progressively decreased from 1.3 ϫ 10 6 Bq/mmol calcium. After 2 and 4 min of incubation, aliquots of the reaction mixture were filtered and processed as described previously (9). Under some conditions producing high calcium uptake rates, samples were removed after 1 and 2 min. Zero time samples were obtained from reaction mixtures from which ATP was omitted. Also, in some experiments, reactions were run at 37°C. The pH of all assay solutions used in this study was adjusted for the temperature at which the assay was run. Under all conditions tested, calcium uptake was linear with time and protein concentration. The optimized kinetic parameters for calcium uptake, measured as a function of either Ca 2ϩ or Mg-ATP concentration, were fit to the Hill equation, V ϭ V max /(1 ϩ (K m (L) /([L]) N ) by a nonlinear least-squares procedure, where L represents either Ca 2ϩ or Mg-ATP and N is the Hill coefficient.
Measurement of calcium uptake by phosphorylated or control microsomes was carried out as follows. The microsomes (13 g of protein/ml) were incubated at 25°C in the standard reaction mixture except that the 45 CaCl 2 -EGTA buffer was omitted and the reaction mixture was present at a 1.35-fold higher concentration and contained 2.7 M cyclic AMP and either PKA at a concentration of 0.30 or 1.0 mg/ml or a control solution (see "Materials"). After a 2-min incubation, an equivalent volume of either distilled water or jasmone was added to a concentration, at this point, of 1.1 mM, and the incubation was continued for an additional 2 min prior to a final addition of 45 Ca 2ϩ to start the calcium uptake reaction. The addition of the Ca 2ϩ buffer diluted the jasmone to 1.0 mM, while both additions reduced the concentrations of the remaining reagents in the calcium uptake reaction mixture to those specified above for the standard assay medium; the final concentrations of cyclic AMP and PKA were 2 M and either 0.30 or 0.75 mg of protein/ml, respectively. Jasmone was added subsequent to the phosphorylation reaction to avoid potential effects of jasmone on this reaction. Previous studies have shown that PLN is the major protein phosphorylated by PKA in crude cardiac microsomes (28), and it was estimated to account for greater than 95% of the phosphate incorporation in our purified microsomes. 2 When the catalytic subunit of PKA was used instead of the holoenzyme, microsomes were incubated in the standard reaction mixture described above but lacking the CaCl 2 -EGTA buffer and including either 150 units/ml of catalytic subunit or an equivalent volume of a control solution (see "Materials"). After incubating the microsomes together with the PKA for 2 min, 2.5 mM oxalate was added to start the calcium uptake reaction.
Ca 2ϩ -ATPase Activity-Ca 2ϩ -ATPase activity was measured at either 25 or 37°C by following the rate of decrease in NADH absorbance at 340 nm in an enzyme-linked assay according to Norby (29) with the following modifications. The reaction mixture contained 40 mM histidine-HCl, pH 6.8; 120 mM KCl; 5 mM NaN 3 ; 2 mM MgCl 2 ; 1 mM ATP; 1 mM phosphoenolpyruvate; 10 units/ml pyruvate kinase; 28 units/ml lactic dehydrogenase; 0.2 mM NADH; either a CaCl 2 -EGTA buffer system, as described above, to yield either 0.32 or 11 M Ca 2ϩ or 10 mM EGTA; either 0.11 mg/ml PKA and 2 M cyclic AMP or control solution (see above); 2.4 g of microsomal protein/ml; and either 1 mM jasmone or an equivalent volume of distilled water. Prior to its addition to the reaction mixture, the A23187 was dissolved in Me 2 SO, which was present at a final concentration of 0.3%. A blank was run with the same reaction mixture except that, instead of microsomes, an equivalent volume of buffer B was present; the blank value was subtracted from all values obtained with microsomes. Rates of ATPase activity were obtained using the Kinetic program in a Shimadzu UV 160U spectrophotometer. The cuvette containing the reaction mixture without the microsomes, jasmone or control solution, and Ca 2ϩ or EGTA was placed in the spectrophotometer, preset at either 25 or 37°C, and after 2 min, the microsomal aliquot or buffer B was added. After an additional 2 min to allow for the phosphorylation or control reaction, jasmone was added, followed by the addition of Ca 2ϩ or EGTA 1 min later, when the Kinetic program was started. The reaction was allowed to proceed for 6 min, which was the length of time necessary for complete temperature equilibration after the various additions to the cuvette, as determined from a negligibly slower rate than during the next 3 min, which was the interval used to obtain the ATPase activity. During this time, the reactions were linear with respect to time and microsomal protein concentration.
Steady-state E 2 P Formation from 32 P i -Prior to measurement of E 2 P formation, microsomes (1.5 mg/ml) that had previously been suspended in KCl-free buffer B were incubated at 25°C for 2 min in 40 mM histidine-HCl, pH 6.8, and 0.01 mg/ml trypsin in the presence (control) and absence of 0.12 mg/ml trypsin inhibitor. After the addition of trypsin inhibitor to the trypsin-treated microsomes, both reaction tubes were kept on ice and used directly for the assay of steady-state E 2 P formation. Our assay was based largely on the findings of Starling et al. (24) with skeletal muscle SR. The microsomes (0.21 mg/ml) were incubated at 23°C in our standard reaction mixture for measuring E 2 P formation, consisting of 50 mM MES-Tris, 5 mM MgCl 2 , 5 mM NaN 3 , 10 mM EGTA-Tris, and 2.0 mM phosphoric acid-Tris ( 32 P i ) at a final pH of 6.8. The specific radioactivity of 32 P i was 3.4 ϫ 10 5 Bq/mol. Assays were also carried out at incubation temperatures of 15 and 37°C. The microsomes were added to the temperature-equilibrated reaction mixture lacking 32 P i and incubated for 2 min, after which time the phosphorylation reaction was started by the addition of 32 P i . Reactions were stopped after 15 s by the addition of 7 volumes of an ice-cold solution containing 1 M perchloric acid, 100 mM orthophosphate, and 20 mM pyrophosphate. The acid-quenched reaction mixtures were each applied with a Pasteur pipette to the center of a prewetted Whatman GF/C filter held in a glass filtration apparatus. Each test tube was washed twice with 3 ml of an ice-cold solution containing 0.25 M perchloric acid, 20 mM orthophosphate, and 20 mM pyrophosphate, and the wash solution was transferred onto the filter. The filter was then washed 10 times with 5 ml of the same solution, and the retained radioactivity was counted by liquid scintillation. Steady-state E 2 P formation is expressed as nmol of E 2 P formed per mg of microsomal protein.
E 2 P Decomposition-The effect of jasmone and trypsin treatment of microsomes on the rate of dephosphorylation of the Ca 2ϩ -ATPase phosphorylated with 32 P i was determined using a Biologic QFM-5 rapid mixing system according to Starling et al. (24) with minor modifications. Purified cardiac microsomes that had been treated with trypsin or trypsin inhibitor-inactivated trypsin, as described above, were centrifuged for 15 min in a Beckman Airfuge at 5°C using an A-100/18 rotor and an air pressure of 27 p.s.i. The pelleted microsomes were resuspended in buffer B and their protein concentration was determined. The pretreated microsomes (4 mg of protein/ml) were incubated in a reaction mixture containing 12.5 mM MES-Tris, pH 6.0, 10 mM EGTA, 2 mM 32 P i (specific radioactivity, 3.4 ϫ 10 5 Bq/mol P i ), 10 mM MgSO 4 , and 14% (v/v) Me 2 SO. One volume of the reaction mixture was mixed with 16 volumes of a chase solution containing 100 mM MES-Tris, pH 7.5, 100 mM KCl, 4 mM MgSO 4 , 5.3 mM ATP, and either 1.0 mM jasmone or distilled water and quenched at different delay times by the addition of 16 volumes of 10.3% trichloroacetic acid to a final concentration of 5%. The acid-quenched samples were applied to Whatman GF/C filters and processed as described above.
Centrifugation of trypsin-treated microsomes and resuspension of the pellet in buffer might result in enrichment of calcium pump sites due to proteolytic cleavage of other membrane proteins. In this case, assuming no loss of Ca 2ϩ -ATPase protein (6), steady-state E 2 P levels in trypsin-treated microsomes would be overestimated relative to control microsomes. The possibility of a direct effect of proteolysis on the Ca 2ϩ -ATPase was tested in microsomes that had been treated with trypsin or trypsin inhibitor-inactivated trypsin (controls) and phosphorylated as described above without prior centrifugation except that the microsomal protein concentration was 1.0 mg/ml and the reagents used for the trypsin and control treatment were present at 67% of their original concentration. No significant difference was detected in mean steady-state E 2 P levels in three different microsome preparations that had been treated with trypsin inhibitor-inactivated trypsin or trypsin under these conditions (1.85 Ϯ 0.21 and 1.79 Ϯ 0.25 (S.E.) nmol/mg of microsomal protein, respectively). Therefore, it was appropriate to normalize the steady-state E 2 P levels in centrifuged, trypsin-treated microsomes to the control values.
Efficiency of the ATP Regenerating System-The adequacy of the ATP regenerating system in the calcium uptake assay was checked both at 3 M and 1 mM ATP in the presence and absence of 1 mM jasmone. Microsomes were incubated for 4 min in the standard calcium uptake assay mixture except that nonradioactive calcium was present and enough [␣-32 P]ATP was included to result in 2 ϫ 10 4 cpm in a 1-l aliquot to be applied to a polyethyleneimine-coated plastic thin layer sheet. The reaction tubes were rapidly cooled in ice and centrifuged in an Airfuge as described above. Two 0.5-l aliquots of each supernatant together with 0.5 l of a mixture containing 50 mM each of ATP, ADP, and AMP were spotted onto the thin layer sheet, which was then developed in 0.75 M monobasic potassium phosphate. The spots corresponding to ATP, ADP, AMP, and the origin and solvent front, as visualized under UV light, as well as appropriate blanks, were collected and counted in separate scintillation vials. Results were expressed as the percentage of the total counts found in the spot corresponding to ATP.

RESULTS
Although jasmone has been reported to increase Ca 2ϩ -ATPase activity in fast skeletal muscle SR as a result of an interaction with the calcium pump protein, it was also shown to produce an unexpected 13% decrease in calcium uptake, an observation that remained unexplained (24). Therefore, initially we determined the effects of jasmone on calcium uptake in skeletal muscle and cardiac SR membranes. A concentrationdependent increase in calcium uptake, reaching a maximum of about 45% at 1 mM jasmone, was observed in both types of microsomes when tested under our standard assay conditions (Fig. 1). Jasmone produced no effect on the ATP-regenerating system used in the assay, and the regenerating system was fully effective in maintaining the specified ATP concentrations.
Effect of Jasmone on Calcium Uptake by Trypsin-treated, Phosphorylated, and Control Microsomes-As expected from our previous studies (9), trypsin treatment of cardiac microsomes increased calcium uptake over a range of Ca 2ϩ concentrations extending from 0.02 to 11 M ( Fig. 2A and Table I). Jasmone (1 mM) increased calcium uptake considerably at the highest Ca 2ϩ concentrations tested (Ͼ1 M) with little effect at the lower Ca 2ϩ concentrations. The V max (Ca) of calcium uptake was significantly increased by trypsin treatment and to a greater extent by jasmone. The largest increase in calcium uptake was produced by the combination of trypsin treatment and jasmone, which appeared to act additively. Trypsin treatment of microsomes reduced the K m (Ca) of the calcium pump by 10%, whereas jasmone increased it both in control and trypsintreated microsomes by 71 and 84%, respectively, and unlike trypsin, slightly reduced the Hill coefficient.
Phosphorylation of microsomes increased the V max (Ca) of the calcium pump by 32% and decreased K m (Ca) by 20% (Fig. 2B and Table I). As was observed in trypsin-treated microsomes, jasmone increased both V max (Ca) and K m (Ca) in phosphorylated microsomes and their controls ( Table I). Phosphorylation of our purified microsomes by the isolated catalytic subunit of PKA resulted in an increase in calcium uptake that was virtually identical to the increase obtained with the holoenzyme. At 0.32 M Ca 2ϩ , the rates of calcium uptake by microsomes phosphorylated with the PKA catalytic subunit and by control microsomes were 0.23 Ϯ 0.03 and 0.16 Ϯ 0.02 mol mg Ϫ1 min Ϫ1 (mean Ϯ S.E., n ϭ 3), respectively, compared with rates of 0.22 and 0.15 mol mg Ϫ1 min Ϫ1 , respectively, in microsomes phosphorylated with the holoenzyme (data taken from Fig. 2B), an approximately 45% increase with phosphorylation. At 11 M Ca 2ϩ when the catalytic subunit was used, the calcium uptake rates were 0.54 Ϯ 0.10 and 0.41 Ϯ 0.07 mol mg Ϫ1 min Ϫ1 in phosphorylated and control microsomes, respectively, compared with rates of 0.47 and 0.36 mol mg Ϫ1 min Ϫ1 , respectively, when the holoenzyme was used (data taken from Fig.  2B), both displaying an increase in calcium uptake of approximately 30% with phosphorylation. The differences between the microsomes phosphorylated with the PKA catalytic subunit and their controls were statistically significant at p Ͻ 0.05 when tested by Student's t test for paired variates.
The effects of phosphorylation (or trypsin treatment) on our purified microsome preparations stand in sharp contrast to the results obtained by most laboratories that have utilized crude cardiac microsomes (e.g. Ref. 6), cardiac tissue homogenates (4), reconstituted vesicles containing purified Ca 2ϩ -ATPase and PLN (16), or crude microsomes isolated from noncardiac cells with expressed proteins (11). In these preparations, phosphorylation affects exclusively or predominantly K m (Ca) , while changes in V max (Ca) are generally of small magnitude (6), highly variable among different preparations (cf. Refs. 6 and 9), or absent altogether (e.g. Refs. 4, 11, and 16). Moreover, V max (Ca) can be increased in purified cardiac microsomes by means other than phosphorylation, in this case by jasmone.
Calcium Uptake Assayed at 0 -10 M Mg-ATP-In contrast to its effects on calcium uptake measured at 1 mM Mg-ATP, 1 mM jasmone markedly decreased calcium uptake at 0 -10 M Mg-ATP in both control and trypsin-treated microsomes (Fig. 3). In the presence of jasmone, the apparent V max (Mg-ATP) derived from data in the 0 -2 M Mg-ATP concentration range, which reflects nucleotide binding to the catalytic site, decreased considerably in the control microsomes and to an even greater extent in the trypsin-treated microsomes (Table II). On the other hand, the apparent K m (Mg-ATP) was increased by jasmone in the control but not in the trypsin-treated microsomes. Between 2 and 10 M Mg-ATP, the degree of inhibition by jasmone  ) and absence (q, E) of 2 M cyclic AMP and 0.75 mg/ml PKA with (E, Ç) or without (q, å) 1 mM jasmone prior to the addition of different concentrations of Ca 2ϩ in order to initiate the calcium uptake reaction, as described under "Experimental Procedures." The data shown were obtained in one experiment using a single microsome preparation; essentially similar results were obtained using a different microsome preparation. The optimized apparent kinetic parameters obtained from the data are presented in Table I. decreased in both control and trypsin-treated microsomes, although somewhat more rapidly in the latter, so that at mM Mg-ATP a stimulation was observed (cf. Fig. 2A at 11 M Ca 2ϩ ). In the absence of jasmone, trypsin treatment resulted in stimulation of calcium uptake even at M Mg-ATP concentrations, as reported previously (9). Between 2 and 3 M Mg-ATP calcium uptake was markedly accelerated in both control and trypsin-treated microsomes, previously suggested to be a result of an increase in the rate of the E 2 3 E 1 ATP⅐2Ca conversion caused by binding of regulatory nucleotide to the calcium pump protein (9). Subsequently, this conversion was demonstrated to be regulated by PLN (12). An increase in the concentration of Mg-ATP from 3 to 10 M produced further acceleration of calcium uptake in both control and trypsin-treated microsomes, albeit at a decreased rate.
Ca 2ϩ -ATPase Activity and Calcium Uptake: Temperature Effects-We carried out experiments at 37°C in addition to those at 25°C in order to determine the temperature dependence of some of the calcium pump properties. No correction for the change in temperature is necessary in deriving Ca 2ϩ concentrations, since temperature has been shown not to have a significant effect on the Ca 2ϩ concentration established by a CaCl 2 -EGTA buffer system (30). Therefore, the same CaCl 2 -EGTA buffer systems were used except that the pH was adjusted for the higher temperature. However, the solubility product of calcium oxalate increases with temperature, resulting in higher intralumenal Ca 2ϩ at constant oxalate concentration. Because of uncertainty about the value of the solubility product appropriate for the intralumenal environment, an initial set of experiments was carried out at 2.5 mM oxalate, as before, followed by experiments at 5.0 mM oxalate. In the measurements of ATPase activity below, this problem is avoided altogether by the use of a Ca 2ϩ ionophore.
At 37°C in the presence of 2.5 mM oxalate and 11 M Ca 2ϩ , calcium uptake rates by control microsomes (treated with trypsin inhibitor-inactivated trypsin) were 1.37 Ϯ 0.06 and 1.38 Ϯ 0.07 mol mg Ϫ1 min Ϫ1 in the absence and presence, respectively, of 1 mM jasmone. In trypsin-treated microsomes, the rates were 1.73 Ϯ 0.16 and 1.47 Ϯ 0.13 mol mg Ϫ1 min Ϫ1 , respectively, in the absence and presence of jasmone. Thus, unlike our findings at 25°C, at 37°C jasmone not only failed to stimulate calcium uptake in both control and trypsin-treated microsomes but, in fact, significantly decreased it in the trypsin-treated microsomes (p Ͻ 0.05) by 15%. Trypsin treatment of microsomes, nevertheless, stimulated calcium uptake by 26% in this series of experiments (p Ͻ 0.05).
In order to establish whether protein kinase A-catalyzed phosphorylation of microsomes or jasmone modified calcium uptake via an effect on the calcium pump protein rather than by a general perturbation of membrane permeability, we assayed the Ca 2ϩ -ATPase activity in our purified microsomes under various conditions (Table III). No ATPase activity attributable to the microsomes was detectable in 10 mM EGTA either at 25 or 37°C irrespective of whether the microsomes had been phosphorylated by PKA or incubated under control conditions (see "Experimental Procedures"). This finding as well as a lack of ouabain-sensitive Na ϩ ,K ϩ -ATPase activity 2 suggests an absence of significant contamination of our purified cardiac microsomes by plasma membranes.
At 25°C, phosphorylation increased Ca 2ϩ -ATPase activity  Table II.   TABLE I Kinetic parameters for calcium uptake by trypsin-treated, phosphorylated, and control microsomes measured at 25°C in the presence and absence of 1 mM jasmone Microsomes in group 1 were treated with trypsin or trypsin inhibitor-inactivated trypsin (control) and in group 2 with or without (control) 2 M cyclic AMP and 0.75 mg/ml protein kinase A under conditions favorable for phosphorylation. Calcium uptake by the microsomes was then assayed in the presence and absence of 1 mM jasmone under standard conditions (see "Experimental Procedures"). The values are the means Ϯ S.E. of the optimized parameters that were obtained in separate unweighted fits to the Hill equation of the four data sets shown in Fig. 2A (group 1) or the single data set shown in Figure 2B (group 2). The numbers in parentheses here and in Tables II and IV are the result of separate normalizations using the values for trypsin-treated or phosphorylated microsomes in the absence of jasmone as 100%.

Microsomes
Jasmone  (Table III), which is close to the K m (Ca) of calcium uptake (see Table I). These increases in ATPase activity are similar to the phosphorylation-or trypsin-induced increases in calcium uptake seen at 25°C in Fig. 2 and Table I. At 37°C, phosphorylation increased calcium uptake and Ca 2ϩ -ATPase activity assayed at 0.32 M Ca 2ϩ by 91 and 100%, respectively, and at 11 M Ca 2ϩ by 21 and 18%, respectively. Comparable increases in calcium uptake activity upon phosphorylation were observed when the assays were carried out at 5.0 mM instead of 2.5 mM oxalate. Therefore, both trypsin treatment and phosphorylation of purified cardiac microsomes enhance calcium pump activity also at 37°C and 11 M Ca 2ϩ under the conditions employed. As expected on the basis of our measurements of calcium uptake, jasmone increased Ca 2ϩ -ATPase activity measured at 25°C and 11 M Ca 2ϩ beyond the increase produced by phosphorylation, whereas at 37°C, jasmone produced a slight decrease in control Ca 2ϩ -ATPase activity (Table  III), which, although only 3%, was statistically significant at the 0.05 level when tested by Student's t test for paired variates. E 2 P Decomposition-Previous studies (9, 31) have implicated PLN in the regulation of E 2 P decomposition, which we then measured. Following 32 P i phosphorylation of trypsin-treated and control microsomes that were resuspended in buffer B (see "Experimental Procedures"), the decomposition of E 2 P was initiated by rapid dilution of the phosphorylated microsomes with a large excess of a higher pH chase solution containing ATP and K ϩ . After preliminary measurements of the E 2 P decomposition rate at various temperatures, a reaction temperature of 15°C was chosen as a consequence of technical constraints imposed by the QFM-5 rapid mixing system. Both jasmone and trypsin treatments of microsomes markedly increased the rate of E 2 P decomposition (Fig. 4). Under all conditions tested, the time course of E 2 P decomposition is described by a single exponential with the rate constants given in the legend to Fig. 4.
Steady-state E 2 P Formation from P i -Since E 2 P decomposition could not be measured above 15°C, we further evaluated this step by measuring the steady-state E 2 P formation from P i . In fast skeletal muscle SR, changes in steady-state E 2 P levels produced by C 12 E 8 and nonylphenol (22,32) reflect changes in E 2 P decomposition rates, which are strongly temperature-dependent (33). An initial series of measurements of steady-state levels of microsomal E 2 P formation from P i was carried out at 23°C in order to establish suitable assay conditions (data not shown). Only a slight decrease in E 2 P formation (about 5%) was observed upon changing the pH from 6.5 to 6.8, and a slight increase of similar magnitude was produced by the addition of 5 mM NaN 3 , which therefore was retained in the reaction mixture. Steady-state levels of E 2 P remained constant when control and trypsin-or jasmone-treated microsomes were incubated over a 10 -60-s time period. A 15-s incubation period was chosen.
Both jasmone and trypsin treatment of cardiac microsomes significantly reduced steady-state E 2 P levels by about 30% when assayed at 23°C under our standard assay conditions (Table IV); the addition of jasmone to trypsin-treated microsomes produced a further 30% reduction, suggesting that at 23°C the effects of trypsin treatment and jasmone act additively. However, when the microsomes were assayed at 37°C, no significant decreases in E 2 P levels were detected as a result of either trypsin treatment or jasmone, whether alone or in combination.
We therefore predicted that the effects of jasmone and trypsin on steady-state E 2 P levels would be even more pronounced at 15°C than at 23°C, since E 2 P decomposition in untreated (control) microsomes becomes slower at lower temperatures and hence would be more susceptible to acceleration. As seen in Table IV, jasmone and trypsin treatment of microsomes each decreased steady-state E 2 P levels to about 55% of the control value, and jasmone added to trypsin-treated microsomes reduced them still further to 42%. Steady-state E 2 P formation from P i in control microsomes remained essentially unchanged in the temperature range tested.

DISCUSSION
In this report evidence is presented for a role of PLN in the regulation of at least two steps in the catalytic cycle of the cardiac SR Ca 2ϩ -ATPase that may contribute to or account for an observed increase in V max (Ca) and decrease in K m (Ca) of microsomal calcium uptake upon PKA-catalyzed PLN phosphorylation ( Fig. 2 and Table I). The first step implicated in this regulation is step 2 or 2a in Scheme 1 or step 1c in Scheme 2, which explicitly lists additional putative substeps in the formation of E 1 ⅐ATP⅐2Ca from E 2 (19,34,35). The second is step 7 or E 2 P decomposition.
Step 1c in Scheme 2, which is shown in the presence of (Mg-)ATP as in Ref. 19, represents a conformational change in the Ca 2ϩ -ATPase protein upon binding of the first of two Ca 2ϩ . Direct evidence for PLN's inhibition of the rate of this conformational change was provided by Cantilina et al. (12), who associated this kinetic effect with a shift in the K m (Ca) of calcium uptake; such a shift was not seen in measurements of equilibrium binding of Ca 2ϩ in the absence of ATP.
Measurement of the rate of E 2 P decomposition in the present study (Fig. 4) indicates that also this step in the cycle can be accelerated by jasmone or removal of PLN's inhibitory influence by trypsin treatment of the microsomes. This observation is consistent with the decrease in steady-state E 2 P levels when the microsomes are incubated in the presence of P i at 15 and 25°C (Table III). This result represents an advance over an earlier study with crude microsomes (36), which showed that Ca 2ϩ -ATPase turnover is accelerated upon microsomal phosphorylation as a result of an increase in "EP" breakdown but did not distinguish between E 1 P and E 2 P. On the other hand, Cantilina et al. (12) failed to observe any increase in the rate of decay of phosphoenzyme formed from ATP as a result of incubation of microsomes in the presence of monoclonal antibody against PLN. The reason for this discrepancy is not readily apparent. Our demonstration of effects of PLN on the rate of E 2 P decomposition and steady-state E 2 P formation from P i TABLE II Apparent kinetic parameters for calcium uptake measured at 0 -2 M Mg-ATP and 25°C in the presence and absence of 1 mM jasmone The values are the optimized apparent parameters obtained by a fit of the data obtained at 0 -2.0 M Mg-ATP (Fig. 3)  provides a possible basis for the increase in V max (Ca) of calcium uptake observed in the present study when the assays were carried out at 25°C. Two major factors have contributed to the contradictory results regarding the effect of PLN on V max (Ca) . First, the use of crude cardiac microsomes prevents detection of high calcium uptake rates because of the presence of calcium release channels that are activated by adenine nucleotides and M Ca 2ϩ concentrations (26). Consequently, in crude microsomes effects of PLN phosphorylation or trypsin treatment on calcium uptake are seen primarily or exclusively at subsaturating Ca 2ϩ concentrations, where the conformational change associated with the binding of the first Ca 2ϩ to E 1 appears to be ratelimiting under commonly used experimental conditions (e.g. Ref. 6). In purified microsomes that are devoid of ruthenium red-sensitive calcium release channels (see "Experimental Procedures"), the major effect of PKA-catalyzed phosphorylation or trypsin treatment of microsomes on the kinetics of calcium uptake at 25°C consists of an increase in V max (Ca) , while the apparent K m (Ca) decreases only slightly (Table I). This difference in the kinetics of calcium uptake is clearly seen in a comparison of crude microsomes with microsomes purified from the same preparations. 2 At 37°C, on the other hand, the major effect of phosphorylation or trypsin treatment of purified microsomes occurs at the lower end of the Ca 2ϩ concentration range, while V max (Ca) is less affected under the conditions employed in this study.
A second major factor contributing to the contradictory results among laboratories is the use of different preparations of calcium pump protein and incubation conditions. One of the recent reports that concluded that PLN has no effect on V max (Ca) of calcium uptake was based on the use of microsomes prepared from HEK-293 cells co-expressing the Ca 2ϩ -ATPase protein and PLN (11). These microsomes are crude, postmitochondrial fractions and may contain channels that allow the efflux of transported calcium. A second recent report of a lack of effect of PLN on V max (Ca) was based on experiments utilizing purified Ca 2ϩ -ATPase and PLN reconstituted into phospholipid-containing vesicles (16). Reconstitution of vesicles represents a modification of the native SR membrane that may eliminate factors necessary for normal protein interaction or function. A case in point is the discovery that reconstitution of purified skeletal muscle Ca 2ϩ -ATPase into vesicles containing phosphatidylinositol 4-phosphate leads to a doubling of the rate of E 2 P decomposition with a corresponding decrease in steady-state E 2 P formation from P i , whereas no changes are observed in the phosphorylation of the enzyme by ATP or in the transport step (37). Furthermore, the purification and reconstitution procedures utilized by Reddy et al. (16) rely on the use of nonionic detergents, which have been shown to interfere specifically with the demonstration of changes in the V max (Ca) of the cardiac calcium pump following trypsin treatment when present at micromolar concentrations (9). The use of Bio-Beads by Reddy et al. to remove detergents from the reconstituted vesicles is based on a competition between the Bio-Beads and protein for the detergent and may not be fully effective with cardiac Ca 2ϩ -ATPase when utilizing the procedures that have FIG. 4. Effects of jasmone and trypsin treatment of cardiac microsomes on dephosphorylation of E 2 P. Trypsin-treated (Ç) or trypsin inhibitor-inactivated trypsin-treated (control) (q, å) 32 P-labeled microsomes were mixed in the QFM-5 rapid mixing system with 16 volumes of a chase solution including either 1.0 mM jasmone (å) or distilled water (q, Ç) and, after the indicated incubation times, with 16 volumes of 10.3% trichloroacetic acid, as described under "Experimental Procedures." The 5-s data points were obtained by manually mixing the reactants. To obtain the zero time point(s), the quench solution followed by the chase solution was added manually to the phosphorylated microsomes. Each data point represents an average of duplicate samples. The lines represent single exponential fits with rate constants of 12 (q), 22 (Ç), and 36 (å) s Ϫ1 .

TABLE III
Ca 2ϩ -ATPase activity and calcium uptake in phosphorylated and control microsomes Purified cardiac microsomes were incubated at the indicated temperature in the presence (ϩ) and absence (Ϫ) of 0.11 mg/ml protein kinase A (PKA) and 2 M cyclic AMP under conditions favorable for phosphorylation followed by the addition of either 1 mM jasmone or an equivalent volume of distilled water as indicated. Ca 2ϩ -ATPase and calcium uptake reactions were started by the addition of the indicated concentrations of Ca 2ϩ . Calcium uptake reactions were carried out in the presence of 5.0 mM oxalate-Tris and 0.3 mg/ml PKA, as indicated. The values are the means Ϯ S.E. of three independent experiments with different microsome preparations except as indicated. Differences between the control and each other value under a particular set of conditions are significant at the p Ͻ 0.05 level when tested by Student's t test for paired variates.  (9) and confirmed with purified cardiac microsomes (data not shown) but were absent in fast skeletal muscle microsomes (9). Reddy et al., moreover, observed a slow leak of intravesicular calcium by measuring a decrease in fluorescence signal due to fluo-3 loaded in the microsomes in the presence of calcium, which could contribute to a failure to observe an effect of PLN on V max (Ca) . All of these problems are avoided by the use of purified native membranes. It has been demonstrated in fast skeletal muscle SR that different steps in the catalytic cycle of the Ca 2ϩ -ATPase may become rate-limiting depending on the incubation conditions, including temperature and Ca 2ϩ concentration (22). Thus, at 25°C and saturating Ca 2ϩ , E 2 P decomposition may be ratelimiting in cardiac microsomes, as suggested previously (9). The data in Fig. 4 show that the rate of E 2 P decomposition is increased by jasmone and by trypsin treatment of microsomes, both of which also increase the V max (Ca) of calcium uptake at 25°C ( Fig. 2A and Table I). Further evidence for a rate-limiting role of E 2 P decomposition in enzyme turnover at 25°C in control microsomes comes from a recent finding by Nediani et al. (31), who observed that a purified acylphosphatase increases calcium uptake and Ca 2ϩ -ATPase activity in calf heart microsomes and that this increase is reduced when the microsomes are phosphorylated by PKA. However, the assignment of E 2 P decomposition as the rate-limiting step cannot be made with certainty without measuring the rates of all of the steps in the catalytic cycle.
At 0 -2 M ATP (saturating Ca 2ϩ and 25°C), calcium uptake is decreased by jasmone and increased by trypsin treatment of cardiac microsomes (Fig. 3). Since the conformational transition of the ATPase associated with the binding of the first of the two Ca 2ϩ bound (step 1c in Scheme 2), which was shown to be regulated by PLN (12), is sensitive to even M concentrations of ATP (19,32), this step is likely to be rate-limiting under the experimental conditions used to obtain the data shown in Fig.   3. We propose that it is this step that is inhibited by jasmone, which could account for the observed inhibition in calcium transport. An inhibition of this step by both PLN and jasmone (Table II) could explain the marked increase in K m (Mg-ATP) observed with jasmone (Table II), since less enzyme would be available in the E 1 conformation for Mg-ATP binding. The known acceleratory action of jasmone on the Ca 2ϩ transport step and E 2 P decomposition (24) (steps 5 and 7, respectively, in Scheme 1) would accentuate the proposed rate limitation at step 1c in Scheme 2 and produce an apparent increase in K m (Mg-ATP) without affecting equilibrium binding of Mg-ATP. A further acceleration of E 2 P decomposition as a result of trypsin treatment (Fig. 4) would be expected to increase the apparent K m (Mg-ATP) still further except for the fact that the same step we propose to be inhibited by jasmone, namely step 1c in Scheme 2, is increased by trypsin (as demonstrated with phosphorylation (12)), thereby producing an apparent K m (Mg-ATP) that is closer to the control value (Table II).
The proposed inhibition by jasmone of the conformational transition associated with the binding of the first Ca 2ϩ by the ATPase (step 1c in Scheme 2) can also account for the increase in apparent K m (Ca) in control, trypsin-treated, and phosphorylated microsomes in the presence of jasmone (Table I). Further evidence consistent with this proposed assignment comes from the data shown in Fig. 9 of Starling et al. (24), indicating that jasmone does not perturb the pH dependence of the fluorescence of 4-nitrobenzo-2-oxa-1,3-diazole-labeled Ca 2ϩ -ATPase, which is believed to monitor the E 1 7 E 2 equilibrium in the absence of Ca 2ϩ . Since M Mg-ATP is known to accelerate the E 2 3 E 1 and E 1 ⅐1Ca 3 E 1 Ј⅐1Ca transitions (19,32), the proposed inhibition of the latter step by jasmone may be much reduced but not eliminated at higher Mg-ATP concentrations (see below).
At 37°C and millimolar ATP on the other hand, both V max (Ca) of calcium uptake (estimated at 11 M Ca 2ϩ ; see "Results") and the steady-state level of E 2 P formation (Table  IV) are unaffected by jasmone. Although the rate of E 2 P decomposition was too rapid to measure with the QFM-5 rapid mixing system at 37°C, the steady-state E 2 P formation from P i , which reflects changes in E 2 P decomposition relative to its formation (24,32), was unaffected by jasmone, trypsin, or both (Table IV). Trypsin treatment or phosphorylation, however, produced a significant increase in calcium uptake (see "Results") or Ca 2ϩ -ATPase activity (Table III), respectively, when measured at 37°C. Thus, increasing the temperature from 25 to 37°C may shift the putative rate-limiting step from strongly temperature-dependent E 2 P decomposition (33) to the next slowest step in the cycle, which has a weaker temperature dependence and is blocked by PLN. In this case, candidates for the next slowest step in the cycle are the E 1 ⅐Ca 1 3 E 1 Ј⅐1Ca transition (step 1c or 1cЈ in Scheme 2) or the E 1 P⅐2Ca 3 E 2 P transition (steps 5 and 6 in Scheme 1). The latter step was suggested by Hughes et al. (39) to be regulated by PLN on the SCHEME 2

TABLE IV
Effect of temperature on steady-state E 2 P formation from 32 P i in control and trypsin-treated microsomes Microsomes suspended in KCl-free buffer B were treated with trypsin inhibitor-inactivated trypsin (control) or 0.01 mg/ml trypsin for 2 min at 25°C. Steady-state E 2 P formation from 32 P i was then measured in the presence and absence of 1 mM jasmone at the indicated temperatures under standard assay conditions (see "Experimental Procedures"). The values are the means Ϯ S.E. obtained in three different experiments with different microsome preparations. Differences between values identified by the same superscript are significant at a p level of Ͻ0.05 when tested by Student's t test for paired (d), unpaired (e, f), or both paired and unpaired (a-c, g) variates. basis of a study with a synthetic peptide analog of PLN. The proposed inhibition by jasmone of the E 2 3 E 1 P⅐2Ca transition or, specifically, the E 1 ⅐Ca 1 3 E 1 Ј⅐1Ca transition (12) could explain the 15% inhibition of calcium uptake that was produced by jasmone in trypsin-treated microsomes (see "Results") and the smaller yet statistically significant decrease in Ca 2ϩ -ATPase activity (Table IV), although other interpretations are possible. Thus, at 37°C, when whole cycle turnover is accelerated by temperature, trypsin treatment, or phosphorylation (Table III) and the stimulatory actions of jasmone on E 2 P decomposition ( Fig. 4 and Table IV) and on the E 1 P 3 E 2 P transition (24), the proposed inhibition by jasmone of the E 1 ⅐Ca 1 3 E 1 Ј⅐1Ca transition, as discussed further below, may again become apparent. Multiple additional factors other than the ones explored in the present study, such as pH and the intralumenal environment, are likely to impinge upon the different steps in the catalytic cycle of the Ca 2ϩ -ATPase and affect their contribution to rate limitation under physiological conditions. However, the potential for regulation of both V max (Ca) and K m (Ca) of the SR calcium pump by PLN that we have demonstrated is likely to be of physiological significance in the event of attainment of saturating intracellular Ca 2ϩ concentrations at peak systole as a result of strong ␤-adrenergic stimulation of the heart or under pathologic conditions involving calcium overload. Under such conditions, an effect of ␤-adrenergic agonist-induced PLN phosphorylation on both kinetic parameters will ensure abbreviation of systole at high heart rates, promote greater filling of SR Ca 2ϩ stores (40), and increase the rate of myocardial relaxation to promote diastolic filling.