ATP-dependent transport of Ca2+ in myocardium sarcolemma vesicles and its activation by phorbol esters

Highly purified pig myocardium sarcolemma vesicles possess the Ca2+,Mg2+-ATPase activity (4.1 mumol Pi/mg protein/hour) and induce the ATP-dependent accumulation of 45Ca2+ (6.0 nmol/mg protein/min). This reaction is not stimulated by oxalate; Ca2+ are released from the vesicles by saponin and Na+ treatment, which suggests that Ca2+ transport against the concentration gradient is induced by myocardium sarcolemma vesicles and not by sarcoplasmic reticulum fragments. The phorbol ester possessing a biological activity of a growth-promoting factor and activating membrane-bound protein kinase C stimulates the Ca2+,Mg2+-ATPase activity and the ATP-dependent accumulation of Ca2+, whereas its counterpart devoid of biological activity does not influence Ca2+ transport. Polymixin B, a specific inhibitor of protein kinase C, prevents the activating effect of phorbol esters on Ca2+ accumulation inside the vesicles. It is suggested that the ATP-dependent transport of Ca2+ in myocardium sarcolemma is controlled by Ca2+-phospholipid-dependent phosphorylation catalyzed by protein kinase C.     

Mechanism of the stimulation of Ca2+-dependent ATPase of skeletal muscle sarcoplasmic reticulum by protein kinase

Sarcoplasmic reticulum isolated from moderately fast rabbit skeletal muscle contains intrinsic adenosine 3',5'-monophosphate (cAMP)-independent protein kinase activity and a substrate of 100 000 Mr. Phosphorylation of skeletal sarcoplasmic reticulum by either endogenous membrane bound or exogenous cAMP-dependent protein kinase results in stimulation of the initial rates of Ca2+ transport and Ca2+-ATPase activity. To determine the molecular mechanism by which protein kinase-dependent phosphorylation regulates the calcium pump in skeletal sarcoplasmic reticulum, we examined the effects of protein kinase on the individual steps of the Ca2+-ATPase reaction sequence. Skeletal sarcoplasmic reticulum vesicles were preincubated with cAMP and cAMP-dependent protein kinase in the presence (phosphorylated sarcoplasmic reticulum) and absence (control sarcoplasmic reticulum) of adenosine 5'-triphosphate (ATP). Control and phosphorylated sarcoplasmic reticulum were subsequently assayed for formation (5-100 ms) and decomposition (0-73 ms) of the acid-stable phosphorylated enzyme (E approximately P) of Ca2+-ATPase. Protein kinase mediated phosphorylation of skeletal sarcoplasmic reticulum resulted in pronounced stimulation of initial rates and levels of E approximately P in sarcoplasmic reticulum preincubated with either ethylene glycol bis(beta-aminoethyl ether)-N,N'-tetraacetic acid (EGTA) prior to assay (Ca2+-free sarcoplasmic reticulum), or with calcium/EGTA buffer (Ca2+-bound sarcoplasmic reticulum). These effects were evident within a wide range of ionized Ca2+. Phosphorylation of skeletal sarcoplasmic reticulum by protein kinase also increased the initial rate of E approximately P decomposition. These findings suggest that protein kinase-dependent phosphorylation of skeletal sarcoplasmic reticulum regulates several steps in the Ca2+-ATPase reaction sequence which result in an overall stimulation of the active calcium transport observed at steady state.     

Effect of caffeine on kinetics of accumulation and release of Ca2+ by vesicles of the sarcoplasmic reticulum of skeletal muscle

The action of caffeine was studied on the heavy sarcoplasmic reticulum fraction enriched by vesicles derived from terminal cisterns. Caffeine lowers the ATP-dependent accumulation of Ca2+ by vesicles and enhances the first rapid phase of the Ci2+ release from vesicles. The action of caffeine was transient, reversed, Ca2+-dependent. The data obtained suggest that the reduction of ATP-dependent calcium accumulation and enhancement of calcium release by caffeine are mediated by the mechanism of Ca2+-induced Ca2+ release and support the view that caffeine may regulate the equilibrium between open and closed states of Ca2+-channel by increasing the affinity of Ca2+-receptor site of the channel.     

Lack of effects of calcium X calmodulin-dependent phosphorylation on Ca2+ release from cardiac sarcoplasmic reticulum

Canine cardiac sarcoplasmic reticulum is phosphorylated by an endogenous calcium X calmodulin-dependent protein kinase and phosphorylation occurs mainly on a 27 kDa proteolipid, called phospholamban. To determine whether this phosphorylation has any effect on Ca2+ release, sarcoplasmic reticulum vesicles were phosphorylated by the calcium X calmodulin-dependent protein kinase, while non-phosphorylated vesicles were preincubated under identical conditions but in the absence of ATP to avoid phosphorylation. Both non-phosphorylated and phosphorylated vesicles were centrifuged to remove calmodulin, and subsequently used for Ca2+ release studies. Calcium loading was carried out either by the active calcium pump or by incubation with high (5 mM) calcium for longer periods. Phosphorylation of sarcoplasmic reticulum by calcium X calmodulin-dependent protein kinase had no appreciable effect on the initial rates of Ca2+ released from cardiac sarcoplasmic reticulum vesicles loaded under passive conditions and on the apparent 45Ca2+-40Ca2+ exchange from cardiac sarcoplasmic reticulum vesicles loaded under active conditions. Thus, it appears that calcium X calmodulin-dependent protein kinase mediated phosphorylation of cardiac sarcoplasmic reticulum is not involved in the regulation of Ca2+ release and 45Ca2+-40Ca2+ exchange.     

The effect of ionomycin on calcium fluxes in sarcoplasmic reticulum vesicles and liposomes.

Ionomycin, a recently discovered calcium ionophore, inhibits the ATP-dependent active Ca2+ transport of rabbit sarcoplasmic reticulum vesicles at concentrations as low as 10(-8) to 10(-6) M. The effect is due to an increase in the Ca2+ permeability of the membrane which is also observed on liposomes. The inhibition of Ca2+ uptake is accompanied by an increase in the Ca2+-sensitive ATPase activity of sarcoplasmic reticulum vesicles.     

Effect of Mg ions and spermine on ATP-dependent Ca2+ transport in myometrial intracellular structures. I. Comparative study of Ca2+ accumulation in mitochondria and sarcoplasmic reticulum

In experiments, which were carried out with the use of a radioactive label (45Ca2+) on the suspension of rat uterus myocytes treated by digitonin solution (0.1 mg/ml), influence of Mg ions and spermine on Mg2+, ATP-dependent Ca2+ transport in mitochondria and sarcoplasmic reticulum was investigated. Ca2+ accumulation in mitochondria (1324 +/- 174 pmol Ca2+/10(6) cells for 1 min - the control) was tested as such which was not sensitive to thapsigargin (100 nM) and was blocked by ruthenium red (10 microM). Oxalate-stimulated Ca2+ accumulation in sarcoplasmic reticulum (136 +/- 17 pmol Ca2+/10(6) cells for 1 min - the control) was tested as such which was not sensitive to ruthenium red and was blocked by thapsigargin. It has been shown, that initial speed and level of energy-dependent Ca2+ accumulation in mitochondria considerably exceeded the values of these parameters for sarcoplasmic reticulum Ca2+-accumulation system. Ca2+ accumulation kinetic in mitochondria was characterized by a steady-state phase (for 5-10 min. of incubation) while accumulation kinetic of this cation in sarcoplasmic reticulum corresponded to zero order reaction. Increase of Mg2+ concentration up to 5 mM led to activation of Ca2+-accumulation systems in mitochondria and sarcoplasmic reticulum (values of activation constants K(Mg) for Mg2+ were 2.8 and 0.6 mM, accordingly). Concentration dependence of spermine action on Ca2+ accumulation in mitochondria was described by a dome-shaped curve with a maximum at 1 mM spermine. In case of sarcoplasmic reticulum Ca2+ pump only the inhibition phase was tested at spermine concentration above 1 mM. However values of inhibition constants for both transporting systems were practically identical--5.2 +/- 0.6 and 5.7 +/- 0.7 mM, accordingly. Hence, Mg ions carry out the important role in regulation of energy-dependent Ca2+ transporting systems both in uterus smooth muscle mitochondria and sarcoplasmic reticulum. Spermine acts first of all on mitochondrial calcium uniporter.     

The effect of total ischemia on the Ca2+-transporting activity of the sarcoplasmic reticulum of the rat myocardium

Different caffeine and calcium concentrations have been studied for their influence on Ca2(+)-pumping function of sarcoplasmic reticulum in a homogenate from control and ischemic rat myocardium. Ca2(+)-transporting system of sarcoplasmic reticulum terminal cisternae membranes from the ischemic myocardium was found to be more sensitive to Ca2+ and caffeine action, inhibiting Ca2+ uptake velocity, as compared to control. This may be one of causes leading to the contractibility disorder under myocardium ischemia.     

The use of a model water-lipid system for the study of the electric function of Ca2+, Mg2+-ATPase of the sarcoplasmic reticulum

The method of dynamic capacity in the model organic phase-water system was used to investigate a possibility of studying the electrical function of Ca2+,Mg2+-ATPase from sarcoplasmic reticulum of the rabbit hind limb skeletal muscles. Decane and decane solution of azolectin were used as an organic phase. It is stated that in the model systems the sarcoplasmic reticulum Ca2+,Mg2+-ATPase did not cause ATP-dependent changes in the boundary Volta potential (delta phi) irrespective of the presence of polyvalent cation chelates in the organic phase. The fragmented sarcoplasmic reticulum is able of realizing Mg-ATP, Ca2+-dependent generation of delta phi only with phospholipids present in the organic phase. It is supposed that generation of delta phi of the fragmented sarcoplasmic reticulum is due to the active transport of calcium ions by the reticulum Ca2+,Mg2+-ATPase.     

Properties of the smooth muscle cell endoplasmic reticulum calcium pump

In experiments with 45Ca2+ conducted on digitonin-treated (0.1 mg/ml) myometrium cells suspension, the properties of ruthenium red-insensitive, oxalate- or phosphate-stimulated and thapsigargin- or cyclopiasonic acid-suppressed Mg2+, ATP-dependent calcium pump of myometrium sarcoplasmic reticulum was studied. The Ca2+ accumulation increased linearly in time up to 10 min, the average initial rate was 80-130 pmol Ca2+/10(6) cells per min. In the presence of 10 mM oxalate the values of the activation constant KMg for Mg2+ and K(m) for ATP were 0.6 and 1.0 mM, respectively. The relative efficiency of the different cations in insuring of the ATP-dependent Ca2+ accumulation was Mg2+ > Mn2+ = Co2+ > Ni2+; the Ca2+ accumulation was not observed in the presence of 3 mM Zn2+ or Cu2+. We observed the suppression of calcium pump activity by different inhibitors such as thapsigargin, cyclopiazonic acid, p-chloromercuribenzoic acid, eosin Y ad Na3 VO4: the values of K0.5 were 2.0 nM, 0.3 microM, 0.6 microM, 0.8 microM and 45 microM respectively. The conclusion was made that suspension of myometrial cells treated with digitonin represent a suitable experimental model for studying the properties of myometrium sarcoplasmic reticulum calcium pump.     

Peroxide modification of skeletal muscle sarcoplasmic reticulum in antioxidant deficiency and under the action of ionol. I. Calcium transport into sarcoplasmic reticulum membranes]

It is shown that in case of antioxidant insufficiency (AOI) activation of NADPH- and ascorbate-dependent lipid peroxidation (LPO) in sarcoplasmic reticulum (SR) of skeletal muscles proceeds 1.7 and 4.1 times faster, respectively. Activation of lipid peroxidation in AOI leads to damage of Ca2+ transport processes in SR of skeletal muscles. Under these conditions ATP-dependent accumulation of 45Ca (by 88%) and Ca(2+)-ATPase (by 14%) activity in SR of skeletal muscles falls. In case of AOI a significant disturbance of passive Ca2+ transport in SR of skeletal muscles takes place, being characterized by an increased passive 45Ca output from vesicles due to breakage of the biomembrane permeability as a result of lipid peroxidation of membranes. Treatment of animals with ionol, a synthetic antioxidant, causes a decrease of activated NADPH- and ascorbate-dependent LPO in SR of skeletal muscles and stabilization of Ca2+ transport processes.     

Uptake of calcium by the endoplasmic reticulum of the frog photoreceptor

We studied retinal photoreceptors of Rana pipiens by using techniques designed to investigate calcium localization. Particularly useful were methods in which intracellular sites of calcium uptake were detected by incubation of saponin-treated isolated retinas in calcium-containing media, with oxalate present as a trapping agent. With these procedures, cell compartments accumulate deposits, which can be shown to contain calcium by x-ray microanalysis. Calcium accumulation was prominent in the rough endoplasmic reticulum in the myoid region. In addition, deposits were observed in agranular reticulum and in certain Golgi- associated compartments of the myoid region, in mitochondria, in axonal reticulum, and in agranular reticulum of presynaptic terminals. Calcium was also detected in the endoplasmic reticulum of retinas fixed directly upon isolation, by a freeze-substitution method. The factors influencing accumulation of calcium in the endoplasmic reticulum were evaluated by a semiquantitative approach based on determining the relative frequency of calcium oxalate crystals under varying conditions. Calcium accumulation was markedly enhanced by ATP. Studies with a nonhydrolyzable ATP analogue (adenylyl- imidodiphosphate ) and with inhibitors of the sarcoplasmic reticulum Ca2+-Mg2+ ATPase (mersalyl and tetracaine) indicated that this ATP-dependent calcium uptake reflects an energy-dependent process roughly comparable to that in the sarcoplasmic reticulum.     

The effect of cAMP-dependent protein kinase phosphorylation on the external Ca2+ binding sites of cardiac sarcoplasmic reticulum

Canine cardiac sarcoplasmic reticulum (SR) is known to be phosphorylated by adenosine 3',5'-monophosphate (cAMP)-dependent protein kinase on a 22,000-dalton protein, Phosphorylation is associated with an increase in both the initial rate of Ca2+ uptake and the Ca(2+)-ATPase activity which is partially due to an increase in the affinity of the Ca(2+)-Mg(2+)-ATPase (E) of sarcoplasmic reticulum for calcium. In this study, the effect of cAMP-dependent protein kinase phosphorylation on the binding of calcium to the SR and on the dissociation of calcium from the SR was examined. The rate of dissociation of the E x Ca2 was measured directly and was not found to be significantly altered by cAMP-dependent protein kinase phosphorylation. Since the affinity of the enzyme for Ca2+ is equal to the ratio of the on and off rates of calcium, these results demonstrate that the observed change in affinity must be due to an increase in the rate of calcium binding to the Ca(2+)-Mg(2+)-ATPase of SR. In addition, an increase in the degree of positive cooperativity between the two calcium binding sites was associated with protein kinase phosphorylation.     

Quercetin interaction with the (Ca2+ + Mg2+)-ATPase of sarcoplasmic reticulum

In sarcoplasmic reticulum vesicles or in the (Ca2+ + Mg2+)-ATPase purified from sarcoplasmic reticulum, quercetin inhibited ATP hydrolysis, Ca2+ uptake, ATP-Pi exchange, ATP synthesis coupled to Ca2+ efflux, ATP-ADP exchange, and steady state phosphorylation of the ATPase by inorganic phosphate. Steady state phosphorylation of the ATPase by ATP was not inhibited. Quercetin also inhibited ATP and ADP binding but not the binding of Ca2+. The inhibition of ATP-dependent Ca2+ transport by quercetin was reversible, and ATP, Ca2+, and dithiothreitol did not affect the inhibitory action of quercetin.     

ATP-dependent phosphate transport in sarcoplasmic reticulum and reconstituted proteoliposomes

During uptake of Ca2+ by rabbit sarcoplasmic reticulum, about 1 mumol of 32Pi was taken up per mumol 45Ca2+ transported. The uptake of Pi was dependent on external Ca2+, Mg2+ and ATP. Intravesicular Ca2+ did not substitute for external Ca2+. In contrast to the accumulation of Ca2+ which was abolished by the ionophore A23187, the uptake of Pi continued to take place provided sufficient Ca2+ was present in the medium. Thus, a Ca2+ gradient did not seem to be required. Similar observations were made with proteoliposomes reconstituted with membrane preparations of sarcoplasmic reticulum and soybean phospholipids. However, when purified Ca2+ -ATPase was used for reconstitution, there was ATP-dependent Ca2+ uptake but no ATP-dependent Pi transport was observed. These data show that the mechanism of Pi transport cannot be a passive movement in response to a Ca2+ gradient but appears to be catalyzed by a specific protein, which is inactivated during purification of the Ca2+ -ATPase. A protein that catalyzes Pi transport in reconstituted vesicles has been solubilized by extraction of sarcoplasmic reticulum with sodium cholate.     

Effect of tetrodotoxin relaxation of cultured skeletal muscle on the sarcoplasmic reticulum Ca2+-transport ATPase

The spontaneous contractions of cultured chick skeletal muscle fibers were abolished by growth of cultures in the presence of tetrodotoxin (TTX). Inhibition of the contractile activity of cultured myofibers was associated with a marked reduction in the rate of azide-insensitive, ATP-dependent Ca2+ uptake by the total particulate fraction of cell homogenates and by purified sarcoplasmic reticulum. Myosin heavy chain (MHC) accumulation and azide-insensitive, ATP-dependent Ca2+ uptake into a total cell membrane fraction were measured simultaneously in the same culture dish. A decrease in the activity of the ATP-dependent Ca2+ uptake system preceded a significant reduction in MHC content of contraction-inhibited cultures. The reduced rate of Ca2+ uptake observed in the sarcoplasmic reticulum from TTX-treated cultures paralleled a decrease in the amount of enzymatically active Ca2+-transport ATPase. The cellular concentration of the ATPase was estimated from a measurement of the concentration of the Ca2+-dependent, hydroxylamine-sensitive, steady state level of phosphorylated intermediate formed in culture microsomes. In contrast to the changes observed in activity of the sarcoplasmic reticulum ATPase and MHC content of TTX-treated cultures, neither the specific activity of creatine kinase nor the accumulation of the MM isoenzyme were affected. It is therefore concluded that the contractile activity of muscle has a selective effect on the maintenance of the adult skeletal muscle phenotype.     

Enhanced Ca2+-induced calcium release by isolated sarcoplasmic reticulum vesicles from malignant hyperthermia susceptible pig muscle

To further define the possible involvement of sarcoplasmic reticulum calcium accumulation and release in the skeletal muscle disorder malignant hyperthermia (MH), we have examined various properties of sarcoplasmic reticulum fractions isolated from normal and MH-susceptible pig muscle. A sarcoplasmic reticulum preparation enriched in vesicles derived from the terminal cisternae, was further fractionated on discontinuous sucrose density gradients (Meissner, G. (1984) J. Biol. Chem. 259, 2365-2374). The resultant MH-susceptible and normal sarcoplasmic reticulum fractions, designated F0-F4, did not differ in yield, cholesterol and phospholipid content, or nitrendipine binding capacity. Calcium accumulation (0.27 mumol Ca/mg per min at 22 degrees C), Ca2+-ATPase activity (0.98 mumol Pi/mg per min at 22 degrees C), and calsequestrin content were also similar for MH-susceptible and normal sarcoplasmic reticulum fraction F3. To examine sarcoplasmic reticulum calcium release, fraction F3 vesicles were passively loaded with 45Ca (approx. 40 nmol Ca/mg), and rapidly diluted into a medium of defined Ca2+ concentration. Upon dilution into 1 microM Ca2+, the extent of Ca2+-dependent calcium release measured after 5 s was significantly greater for MH-susceptible than for normal sarcoplasmic reticulum, 65.9 +/- 2.8% vs. 47.7 +/- 3.9% of the loaded calcium, respectively. The C1/2 for Ca2+ stimulation of this calcium release (5 s value) from MH-susceptible sarcoplasmic reticulum also appeared to be shifted towards a higher Ca2+-sensitivity when compared to normal sarcoplasmic reticulum. Dantrolene had no effect on calcium release from fraction F3, however, halothane (0.1-0.5 mM) increased the extent of calcium release (5 s) similarly in both MH-susceptible and normal sarcoplasmic reticulum. Furthermore, Mg2+ was less effective at inhibiting, while ATP and caffeine were more effective in stimulating, this Ca2+-dependent release of calcium from MH-susceptible, when compared to normal sarcoplasmic reticulum. Our results demonstrate that while sarcoplasmic reticulum calcium-accumulation appears unaffected in MH, aspect(s) of the sarcoplasmic reticulum Ca2+-induced calcium release mechanism are altered. Although the role of the Ca2+-induced calcium release mechanism of sarcoplasmic reticulum in situ is not yet clear, our results suggest that an abnormality in the regulation of sarcoplasmic reticulum calcium release may play an important role in the MH syndrome.     

Infrared spectroscopic characterization of the structural changes connected with the E1----E2 transition in the Ca2+-ATPase of sarcoplasmic reticulum

The Ca2+-transporting ATPase (EC 3.6.1.38) of sarcoplasmic reticulum alternates between several conformational states during ATP-dependent Ca2+ transport. The E1 conformation is stabilized by 0.1 mM Ca2+ and the E2 conformation by vanadate in a Ca2+-free medium. Fourier transform infrared spectroscopy reveals significant differences between the two states that indicate differences in the protein secondary structure. The two states and the corresponding spectra can be interconverted reversibly by changing the Ca2+ concentration of the medium. The infrared spectral changes indicate the appearance of a new alpha-helical substructure connected with the E1----E2 conversion accompanied by small changes in beta-turns, while the beta-sheet content remains essentially unchanged. There are also differences between the E1 and E2 states in the C = O stretching vibrations of the ester carbonyl groups of phospholipids in intact sarcoplasmic reticulum that are not observed under identical conditions in isolated sarcoplasmic reticulum lipid dispersions. These observations imply an effect of proteins on the structure of the interfacial regions of the phospholipids that is dependent on the conformational state of the Ca2+-ATPase. The CH2- and CH3-stretching frequencies of the membrane lipids are not affected significantly by the E1----E2 transition. The Fourier transform infrared spectra of sarcoplasmic reticulum vesicles in the presence of 20 mM Ca2+ suggest the stabilization of a protein conformation similar to the E2 state except for differences in the behavior of COO- and phospholipid ester C = O groups that may reflect charge effects of the bound Ca2+.     

Effect of disseminated myocardial necrosis on ATPase activity, Ca2+ transport, and lipid peroxidation in cardiac mitochondrial and microsomal membranes

Isoproterenol-induced (5 mg/kg) disseminated necrosis of the rabbit myocardium led to a decrease in the efficiency of calcium pump of sarcoplasmic reticulum fragments. This was shown by the reduced Ca/ATP ratio, as well as by Ca2+ and Ca2+ ATPase accumulation rate. In these conditions, calcium transport to mitochondria increased. Lipid peroxidation plays a definite role in the impairment of membrane permeability since the concentration of malonic dialdehyde rises in microsomal and mitochondrial fractions.U     

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.     

Isolation of highly active preparations of sarcoplasmic reticulum and Ca2-dependent ATPase from cardiac muscle]

A microsomal preparation with a high ability for Ca2+ uptake has been isolated from pigeon heart. A method of further purification of Ca2+-accumulating system of heart, based on the ability of sarcoplasmic reticulum for the energy-dependent Ca2+ accumulation in the presence of oxalate, has been developed. Upon centrifugation in the gradient of sucrose and KCl concentration the fragments of sarcoplasmic reticulum, rendered "heavy" by calcium oxalate, can be separated from foreign cell membranes. The main component of heart "calcium pump" is Ca2+-dependent ATPase (making up to about 50% of all proteins of the purified reticulum), having a molecular weight of 100.000--105.000. Specific activity of heart Ca2+-ATPase as well as the ability of purified heart sarcoplasmic reticulum for Ca2+ uptake are only slightly less than those of the skeletal muscle reticulum. The data obtained suggest that heart sarcoplasmic reticulum may be efficient for providing heart muscle relaxation.