Phosphorylation of a 100 000 dalton component and its relationship to calcium transport in sarcoplasmic reticulum from rabbit skeletal muscle (original) (raw)
Related papers
Calcium transport by cardiac sarcoplasmic reticulum and phosphorylation of phospholamban
Molecular and Cellular Biochemistry, 1982
Active calcium transport by cardiac sarcoplasmic reticulum assumes a central role in the excitation-concentration coupling of the myocardium, in that Ca2+-dependent ATPase (mol.wt. 100 000) of cardiac sarcoplasmic reticulum serves as an energy transducer and a translocator of Ca2+ across the membrane. During the translocation of Ca2+, the ATPase undergoes a complex series of reactions during which the phosphorylated intermediate EP is formed. We documented how the elementary steps of the ATPase reaction are coupled with calcium translocation, and provided evidences to indicate that two key steps of ATPase correspond to the conformational change of the enzyme, and appear to alter the affinity of the enzyme for Ca2+. A line of evidence also indicated that Ca2+-dependent ATPase of cardiac sarcoplasmic reticulum is regulated by a specific protein named phospholamban (mol.wt. 22 000), which serves as a substrate for cyclic AMP-dependent protein kinase. Cyclic AMP-dependent phosphorylation of phospholamban resulted in a marked increase in the rate of turnover of the ATPase, by enhancing the rates of the key elementary steps, i.e. the steps at which the intermediate EP is formed and decomposed. Thus phospholamban is putatively thought to serve as a modulator of Cat2+-dependent ATPase of cardiac sarcoplasmic reticulum. A working model was proposed to interpret the mechanism. Also documented is a possibility that another protein kinase activatable by Ca2+ and calmodulin is functional in regulating the phospholamban-ATPase system, thus suggesting the existence of a dual control system, in which both cyclic AMP- and calmodulin-dependent phosphorylation are in control of the Cat2+-dependent ATPase. Such a control mechanism may provide the interpretation, at the cellular level, that catecholamines exert actions on myocardial contractility. Thus, catecholamine-mediated increases in intracellular cyclic AMP could enhance calcium fluxes across the membrane of sarcoplasmic reticulum, thus resulting in the increased rates of relaxation and, at the same time, the increased rate and extent of contraction. Such a mechanism could also be operational in the tissues, other than the myocardium, in which catecholamines and other hormones serve as the ‘first messenger’, producing intracellular cyclic AMP as the ‘second messenger’.
Biochemistry, 1989
The effects of an antiserum against the 53-kDa glycoprotein (GP-53) of the Sarcoplasmic reticulum (SR) and of monoclonal antibodies against GP-53 on Ca 2+ transport and ATP hydrolysis by SR of rabbit skeletal muscle have been investigated. Preincubation of SR with an antiserum against GP-53 resulted in decreased ATP-driven Ca 2+ transport by the SR but had no effect on Ca 2+-stimulated ATP hydrolysis. Preincubation of SR with preimmune serum had no significant effect on either Ca 2+ transport or Ca 2+-ATPase activity. The effect of anti-GP-53 serum was time and concentration dependent. Preincubation of SR with two monoclonal antibodies against GP-53 had no effect on Ca 2+ transport or on Ca 2+-stimulated ATP hydrolysis. However, preincubation of SR with either monoclonal antibody against GP-53 together with a monoclonal antibody against the Ca 2+-ATPase (at levels which had little effect alone) resulted in markedly decreased rates of Ca 2+ uptake and ATP hydrolysis. Preincubation of SR with anti-GP-53-serum or with monoclonal antibodies, under the same conditions that inhibited Ca 2+ uptake, did not increase the passive permeability of the SR membrane to Ca 2+ , did not decrease the permeability of the SR to oxalate, and did not cause significant proteolysis of the Ca 2+-ATPase. Our results are consistent with the interpretation that GP-53 may modulate the function of the Ca 2+-ATPase of the SR membrane. The sarcoplasmic reticulum (SR 1) of skeletal and cardiac muscle plays a vital role in controlling the cytosolic concentration of Ca 2+ (Hasselbach, 1964; Ebashi et al., 1969; Mac-Lennan, 1970). The Ca 2+-ATPase is an integral protein of the SR membrane that actively transports Ca 2+ from the cytosol to the lumen of the SR (Racker, 1972; Meissner & Fleischer, 1974; Warren et al., 1974). The primary structure of the Ca 2+-ATPase of slow skeletal muscle appears to be identical with that of cardiac muscle but differs slightly from the primary structure of fast skeletal muscle Ca 2+-ATPase (MacLennan et al., 1985; Brandl et al., 1986). The Ca 2+-ATPase of cardiac muscle is regulated by phospholamban, a 22-kDa protein of the SR membrane [reviewed by Tada et al. 1982)]. It has not been established that the Ca 2+-ATPase of skeletal muscle is subject to regulation by another protein, either cytosolic or membrane bound. Michalak et al. (1980) identified GP-53 as an intrinsic protein of the membrane of the SR of skeletal muscle and showed it to be a major component of the SR membrane. Michalak et al. (1980) found GP-53 to be present in a constant molar ratio to the Ca 2+-ATPase: about two GP-53 molecules for every three Ca 2+-ATPase polypeptide chains. Campbell and MacLennan (1981) purified GP-53 and partially characterized it. They found GP-53 to span the SR membrane, with its carbohydrate chains in the SR lumen and much of its protein mass exposed on the cytoplasmic surface of the membrane.
Biochimica et Biophysica Acta (BBA) - Molecular Cell Research, 1998
The sarcoplasmic reticulum Ca-ATPase is fully activated when v1 WM [Ca 2 ] saturates the two transport sites; higher [Ca] inhibits the ATPase by competition of Ca-ATP with Mg-ATP as substrates. Here we describe a novel effect of EGTA and other chelators, raising the possibility of an additional activating effect of Ca in the sub-or low WM range. Sarcoplasmic reticulum membranes were isolated from rabbit skeletal muscles. The ATPase activity was measured after incubation at 37³C in 3 mM ATP, 3 mM MgCl 2 , 50 mM MOPS-Tris (pH 7.2), 100 mM KCl, and variable CaCl 2 , EGTA and calcimycin. In the absence of added EGTA and Ca the ATPase activity is high due to contaminant Ca. The determination of the ATPase activity in the presence of increasing amounts of EGTA, without added Ca, yields a decreasing sigmoidal function. K i ranged between 20 and 100 WM, depending on the enzyme concentration. P i production is linear with time for several [EGTA] yielding suboptimal ATPase activities, which are inhibited by thapsigargin. These suboptimal Ca-ATPase activities are inhibited by preincubation of the enzyme in EGTA, at pH 7.2. This effect increases upon increasing EGTA concentration and preincubation time. The inhibitory effect of the previous exposure of the enzyme to EGTA is partially but significantly reverted by increasing [Ca 2 ] during incubations. Calcimycin and EDTA have similar effects as EGTA when added in preincubations. The effect of calcimycin is fully reverted by optimal [Ca 2 ] in incubations. The effects of EGTA, EDTA and calcimycin in preincubation are not additive. The results suggest that an additional calcium, lost during preincubations from a site with affinity near 1 WM, is necessary for full activation of the ATPase. ß
Stimulation of calcium accumulation in cardiac sarcolemma by phosphorylase kinase
Biochemical Journal, 1977
After incubation with phosphorylase kinase, calcium accumulation in cardiac sarcolemma was increased from 65±2 to 95±3nmol/lOmin per mg of protein. Under these conditions, phosphorylase kinase catalysed phosphorylation of membranes. This phosphorylation was hydroxylamine-insensitive, was stimulated by Ca2+ ions and was unaffected by 3': 5 '-cyclic AMP. The regulation of membrane Ca2+ transport by 3': 5'-cyclic AMP-and Ca2+-dependent phosphorylation of membrane proteins has been intensively investigated [see Sulakhe & St. Louis (1976a) for a review]. For example, many investigators have reported stimulation of Ca2+ uptake in cardiac sarcoplasmic reticulum by cyclic AMP-dependent protein kinase (EC 2.7.1.37)-catalysed phosphorylation of a 22000-dalton component in sarcoplasmic reticulum (Kirchberger et al., 1972, 1974; LaRaia & Morkin, 1974; Tada et al., 1975; Schwartz et al., '976). The role of Ca2+-dependent phosphorylase kinase (EC 2.7.1.38) system in muscle glycogenolysis is well established (Krebs, 1972). However, numerous findings suggest that this system may well serve as a link between two cyclic AMP-mediated events in muscle: excitation-contraction coupling and intermediary metabolism [see Entman et al. (1976) for a review]. In this context, the finding by Schwartz et al. (1976) that phosphorylase kinase augmented Ca2+ uptake in sarcoplasmic reticulum is of interest. In the present paper, we describe stimulation of calcium accumulation in cardiac sarcolemma by phosphorylase kinase. We found that phosphorylase kinase catalysed phosphorylation ofcardiac sarcolemma. The phosphorylation was hydroxylamine-insensitive, stimulated by Ca2+ and was unaffected by cyclic AMP. Some ofthese observations have been published in an abstract (Sulakhe & St. Louis, 1976b). Experimental Materials 45CaC12 (20 mCi/mg) and [y-32P]ATP (5-10 Ci/ mmol) were from New England Nuclear, Montreal, Canada; phosphorylase kinase, protein kinase, ATP and cyclic AMP were from Sigma Chemical Co., Vol. 164
Journal of Biological Chemistry
Membrane vesicles capable of energized Ca2+ pumping have been reconstituted from cardiac sarcoplasmic reticulum (SR). Cardiac SR was solubilized with Triton X-100 in a detergent to protein weight ratio of 0.8, and membranous vesicles were reconstituted by removal of detergent with Bio-Beads SM-2 (a neutral porous styrene-divinylbenzene copolymer). The reconstituted vesicles exhibited ATP-dependent oxalate-facilitated Ca2+ accumulation with rates and efficiency comparable to the best reconstituted skeletal muscle preparation (Ca2+-loading rate = 1.65 f 0.31 pmol mg" rnin", Ca2+-activated ATPase activity = 2.39 f 0.25 pmol mg" min", efficiency (Ca2+/ATP) = 0.69 f 0.09). Phospholamban in the reconstituted vesicles was phosphorylated with added catalytic subunit of CAMPdependent protein kinase to almost the same extent as that in original vesicles. However, phosphorylation of phospholamban had no effect on the Ca2+ accumulation of the reconstituted vesicles. This is to be contrasted with a decrease in the half-maximal concentration of Ca2+ for Ca2+ accumulation (KC,) in the original vesicles from 1.35 f 0.08 pM to 0.75 f 0.12 pM by CAMPdependent phosphorylation of phospholamban. On the other hand Kc, for the reconstituted vesicles was about 0.5 p~ and remained unchanged by phosphorylation, indicating that the Ca2+ pump in the reconstituted vesicles is already fully activated. These results suggest that in normal cardiac SR, phospholamban in the dephosphorylated state acts as a suppressor of the Ca2+ pump and that phosphorylation of phospholamban serves to reverse the suppression.
Involvement of protein phosphorylation in activation of Ca2+ efflux from sarcoplasmic reticulum
Biochemical Journal, 1991
Preincubation of sarcoplasmic reticulum (SR) membranes with a combination of ATP and NaF resulted in inhibition of Ca2+ accumulation and stimulation of Ca(2+)-ATPase and Ca2+ efflux. Under the same conditions, the activity of the SR phosphoprotein phosphatase was inhibited and the phosphorylation of two polypeptides with apparent molecular masses of 160 and 150 kDa was obtained. The effect of ATP is specific, since the ATP analogue adenosine 5′-[beta gamma-imido]triphosphate did not replace for ATP. In the absence of NaF, ATP was ineffective. The phosphorylation of the 160 kDa and/or 150 kDa proteins and the stimulation of Ca2+ efflux are clearly related. The phosphorylation of both proteins and the increase in Ca2+ efflux show a similar dependence on the concentration of ATP. The level of protein phosphorylation and the stimulation of Ca2+ efflux were also controlled by the NaF concentration which inhibits the phosphatase and of net Ca2+ accumulation, as well as for the stimulation...
Biochemical Journal, 1987
Preincubation of sarcoplasmic reticulum with 1 mM-ATP completely inhibits Ca2l accumulation and stimulates ATPase activity by over 2-fold. This effect of ATP is obtained only when the preincubation is carried out in the presence of Pi, but not with arsenate, chloride or sulphate. The inhibition by ATP of Ca2' accumulation is pH-dependent, increasing as the pH is increased above 7.5. Inhibition of Ca21 accumulation is observed on preincubation with ATP, but not with CTP, UTP, GTP, ADP, adenosine 5'-[fly-methylene]triphosphate or adenosine 5'-[/ly-imido]triphosphate. The presence of Ca2", but not Mg2", during the preincubation, prevents the effect of ATP + Pi on Ca2`accumulation. The ATP + Pi inhibition of Ca2+ accumulation is not due to modification of the ATPase catalytic cycle, but rather to stimulation of a rapid Ca2' efflux from actively or passively loaded vesicles. This Ca2+ effilux is inhibited by dicyclohexylcarbodi-imide. Photoaffinity labelling of sarcoplasmic-reticulum membranes with 8-azido-[a-32P]ATP resulted in specific labelling of two proteins, of approx. 160 and 44 kDa. These proteins were labelled in the presence of Pi, but not other anions. EXPERIMENTAL Materials ATP, ADP, p[NH]ppA, p[CH2]ppA, CTP, UTP, GTP, EGTA and Tricine were obtained from Sigma Chemical Co. 45CaC12 was from The Radiochemical Centre (Amersham, Bucks., U.K.), and [82P]P1 from Nuclear Research Center, Negev, Israel. 8-N3-[X-32P]ATP (6.6 Ci/mmol) was obtained from ICN. [_y32p]_ ATP was synthesized from [32P]P, and ADP by photophosphorylation with lettuce chloroplasts (Avron, 1960) and was purified on Dowex 1-X8 with NaCl as the eluent. Membrane preparation Sarcoplasmic-reticulum vesicles were prepared from rabbit fast-twitch skeletal muscle as described by Camp-Vol. 247 497 Abbreviations used: p[CHJppA, adenosine 5'-[fly-methylene]triphosphate; p[NH]ppA, adenosine 5'-[fly-imido]triphosphate; DDCD, dicyclohexylcarbodi-imide; 8-N3-ATP, 8-azido-ATP; FITC, fluorescein isothiocyanate.