Modification of calcium fluxes by dimethyl sulfoxide and 2-butoxyethanol in sarcoplasmic reticulum vesicles: a possible mechanism for skeletal muscle relaxation induced by dimethyl sulfoxide

In order to investigate the mechanism of skeletal muscle relaxation induced by dimethyl sulfoxide, 2-butoxyethanol and dimethyl sulfoxide were examined for their effects on 1) Ca2+ uptake into and efflux from sarcoplasmic reticulum vesicles prepared from rabbit fast skeletal muscle and crayfish tail muscle by the murexide method, 2) ATPase activities of rabbit reticulum vesicles, 3) the isolated phrenic nerve-diaphragm preparation of the rat and 4) crayfish opener muscle preparation. Ca2+ efflux rate from rabbit reticulum vesicles was markedly decreased with increasing concentrations (5-20% v/v) of dimethyl sulfoxide without affecting the maximum Ca2+ uptake by the reticulum. 2-Butoxyethanol showed quite contrary effects. Dimethyl sulfoxide strongly inhibited the activity of basal ATPase rather than of Ca2+-dependent ATPase. 2-Butoxyethanol did not significantly inhibit the activity of basal ATPase, but markedly increased Ca2+-dependent ATPase activity. Antagonisms between dimethyl sulfoxide and caffeine were demonstrated either in contractions of crayfish opener muscles or in the Ca2+ release from crayfish sarcoplasmic reticulum vesicles. These results indicate a possibility that dimethyl sulfoxide reversibly induces skeletal muscle relaxation mainly in the sarcoplasmic reticulum by means of decreasing the rate and the amount of Ca2+ release from the reticulum.     

Reactivity of sarcoplasmic reticulum from rabbit cardiac muscle with 1-fluoro- and 1,5-difluoro- 2,4-dinitrobenzene

Sarcoplasmic reticulum preparations from rabbit cardiac and fast skeletal muscle react differentially with low concentrations of 1-fluoro- and 1,5-difluoro-2,4-dinitrobenzene. Dinitrophenylation of cardiac sarcoplasmic reticulum by 1-fluoro-2,4-dinitrobenzene is not affected by Ca2+ and is limited to the lipoprotein-lipid region. This contrasts sharply with the predominant Ca2+-dependent dinitrophenylation of the ATPase protein of rabbit skeletal sarcoplasmic reticulum by this reagent. Formation of non-serial high mol. wt. oligomers by 1,5-difluoro-2,4-dinitrobenzene is significantly greater in cardiac than in skeletal vesicles. Substrate MgATP2- does not protect rabbit cardiac sarcoplasmic reticulum ATPase activity or Ca2+ uptake from dinitrophenylation when monofunctional and bifunctional reagents are used. Chemical differences in the overall structure of the two kinds of membrane preparations can be ascertained from a comparison of the effects of Ca2+ and MgATP2- on the reactivity of these reagents.     

Activation of calcium transport in skeletal muscle sarcoplasmic reticulum by monovalent cations.

The rates of calcium transport and Ca2+-dependent ATP hydrolysis by rabbit skeletal muscle sarcoplasmic reticulum were stimulated by monovalent cations. The rate of decomposition of phosphoprotein intermediate of the Ca2+-dependent ATPase of sarcoplasmic reticulum was also increased by these ions to an extent that is sufficient to account for the stimulation of calcium transport and Ca2+-dependent ATPase activity. The order of effectiveness of monovalent cations tested at saturating concentrations in increasing rate of phosphoprotein decomposition is: K+, Na+ greater than Rb+, NH4+ greater than Cs+ greater than Li+, choline+, Tris+.     

ATPase activities, Ca2+ transport and phosphoprotein formation in sarcoplasmic reticulum subfractions of fast and slow rabbit muscles.

Subfractionation of sarcoplasmic reticulum from fast-twitch and slow-twitch rabbit skeletal muscles was performed on a sucrose density gradient. Vesicle fractions were characterized by: measurement of (Ca2+,Mg2+)-dependent (extra) ATPase, Mg2+-dependent (basal) ATPase, Ca2+ uptake characteristics, polypeptide patterns in sodium dodecylsulphate polyacrylamide gel electrophoreses, phosphoprotein formation and electronmicroscopy of negatively stained samples. In fast-twitch muscle, low and high density vesicles were separated. The latter showed high activity of (Ca2+,Mg2+)-dependent ATPase, negligible activity of Mg2+-dependent ATPase, high initial rate and high capacity of Ca2+ uptake, high amount of phosphorylated 115000-Mr polypeptide, and appeared morphologically as thin-walled vesicles covered with particles of 4 nm in diameter. Low density vesicles had little (Ca2+,Mg2+)-dependent ATPase but high Mg2+-dependent ATPase. Although the initial rate of Ca2+ uptake was markedly lower, the total capacity of uptake was comparable with that of high density vesicles. Phosphorylated 115000-Mr polypeptide was detectable at low concentrations. Instead, 57000 and 47000-Mr polypeptides were characterized as forming stable phosphoproteins in the presence of ATP and Mg2+. Negatively stained, these vesicles appeared to have smooth surfaces. It is suggested that low density vesicles represent a Ca2+ sequestering system different from that of high density vesicles and that Mg2+-dependent (basal) ATPase as well as the 57000 and 47000-Mr polypeptides are part of the Ca2+ transport system within the low density vesicles. According to the results from slow-twitch muscle, Ca2+ sequestration by the sarcoplasmic reticulum functions in this muscle type only through the low density vesicles.     

Monoclonal antibodies to the Ca2+ + Mg2+-dependent ATPase of sarcoplasmic reticulum identify polymorphic forms of the enzyme and indicate the presence in the enzyme of a classical high-affinity Ca2+ binding site

In order to determine whether polymorphic forms of the Ca²⁺ + Mg²⁺-dependent ATPase exist, we have examined the cross-reactivity of five monoclonal antibodies prepared against the rabbit skeletal muscle sarcoplasmic reticulum enzyme with proteins from microsomal fractions isolated from a variety of muscle and nonmuscle tissues. All of the monoclonal antibodies cross-reacted in immunoblots against rat skeletal muscle Ca²⁺ + Mg²⁺-dependent ATPase but they cross-reacted differentially with the enzyme from chicken skeletal muscle. No cross-reactivity was observed with the Ca²⁺ + Mg²⁺-dependent ATPase of lobster skeletal muscle. The pattern of antibody cross-reactivity with a 100,000 dalton protein from sarcoplasmic reticulum and microsomes isolated from various muscle and nonmuscle tissues of rabbit demonstrated the presence of common epitopes in multiple polymorphic forms of the Ca²⁺ + Mg²⁺-dependent ATPase. One of the monoclonal antibodies prepared against the purified Ca²⁺ + Mg²⁺-dependent ATPase of rabbit skeletal muscle sarcoplasmic reticulum was found to cross-react with calsequestrin and with a series of other Ca²⁺-binding proteins and their proteolytic fragments. Its cross-reactivity was enhanced in the presence of EGTA and diminished in the presence of Ca²⁺. Its lack of cross-reactivity with proteins that do not bind Ca²⁺ suggests that it has specificity for antigenic determinants that make up the Ca²⁺-binding sites in several Ca²⁺-binding proteins including the Ca²⁺ + Mg²⁺-dependent ATPase.This paper is dedicated to the memory of Dr. David E. Green.     

Phosphorylation of a 100 000 dalton component and its relationship to calcium transport in sarcoplasmic reticulum from rabbit skeletal muscle

Sarcomplasmic reticulum from rabbit fast skeletal muscle contains intrinsic protein kinase activity (ATP:protein phosphotransferase, EC 2.7.1.37) and a substrate. The protein kinase activity was Mg2+ dependent and could also phosphorylate exogenous protein substrates. Autophosphorylation of sarcoplasmic reticulum vesicles was not stimulated by cyclic AMP, neither was it inhibited by the heat-stable protein kinase inhibitor protein. The phosphorylated membranes had the characteristics of a protein with a phosphoester bond. An average of 73 pmol Pi/mg protein were incorporated in 10 min at 30 degrees C. Addition of exogenous cyclic AMP-dependent protein kinase increased the endogenous level of phosphorylation by 25-100%. Sarcoplasmic reticulum membrane phosphorylation, mediated by either endogenous cyclic AMP-independent or exogenous cyclic AMP-dependent protein kinase, occurred on a 100 000 dalton protein and both enzyme activities resulted in enhanced calcium uptake and Ca2+-dependent ATPase (ATP phosphohydrolase, EC 3.6.1.3), in a manner similar to cardiac microsomal preparations. Regulation of Ca2+ transport in skeletal sarcoplasmic reticulum may be mediated by phosphorylation of a 100 000 dalton component of these membranes.     

Cause of increase in the efficiency of Ca2+ transport by fragments of sarcoplasmic reticulum from fast skeletal muscles induced by protein kinase

The effect of cAMP-dependent protein kinases from rabbit skeletal muscles on Ca2+ uptake by fragments of skeletal muscle sarcoplasmic reticulum was studied. It was shown that incubation of the sarcoplasmic reticulum fragments with protein kinase increases the rate of Ca2+ uptake without changing the activity of Ca2+-dependent ATPase. This phenomenon is not accompanied by phosphorus incorporation into the protein components of the reticulum membranes. The protein kinase preparation subjected to "self-phosphorylation" is also capable to increase the rate of Ca2+ uptake. Using (14C) -oleic acid, it was shown that the increase of the rate of Ca2+ transport under effects of the "self-phosphorylated" protein kinase occurs due to the binding of free fatty acids present in the sarcoplasmic reticulum membranes. It was found that the effect observed is due to phosphofructokinase (ATP : D-fructose-6-phosphate-1-phosphotransferase) present in the protein kinase preparation.     

Studies on the heterogeneity of sarcoplasmic reticulum vesicles.

Isolated sarcoplasmic reticulum vesicles from rabbit white muscle were separated into a light (15--20% of total microsomes) and a heavy (80--85%) fraction by density gradient centifugation. The ultrastructure, chemical composition, enzymic activities and localization of membrane components in the vesicles of both fractions were investigated. From the following results it was concluded that both fractions are derived from the membranes of the sarcoplasmic reticulum system of the muscle: (i) The protein pattern of both fractions is essentially the same, except for different ratios of acidic, Ca2+-binding proteins. (ii) The 105000 dalton protein of the light fraction cross-reacts immunologically with the Ca2+-dependent ATPase of the heavy fraction. (iii) Ca2+-dependent ATPase, although of different specific activity, is found in both fractions. After rendering the vesicles leaky, specific activities in both fractions reach the same value. The light fraction was found to consist of "inside-out" vesicles by the following criteria: (i) No Ca2+ accumulation can be measured and the Ca2+-dependent ATPase activity is low and variable. (ii) The rate of trypsin digestion is lower and, compared to the heavy microsomes, a different ratio of degradation products is obtained. (iii) The sarcoplasmic reticulum membrane has a highly asymmetrical lipid distribution. This distribution of aminophospholipids is opposite to that in vesicles of heavy fraction. The light sarcoplasmic reticulum fraction has a higher phospholipid to protein ratio than the heavy one. This is consistent with the possibility that the two fractions derive from different parts of the sarcoplasmic reticulum system.     

Effect of heparin modification on its circular dichroism spectrum

The sarcoplasmic reticulum and glycogen pellet derived from rabbit skeletal muscle and the sarcolemma and sarcoplasmic reticulum from pig skeletal muscle contains NAD:dependent mono ADP-ribosyltransferase activity toward the guanidine analog, P- nitrobenzylidine aminoguanidine. No or little activity could be found in the sarcolemma or sarcoplasmic reticulum derived from canine cardiac muscle. Seventy percent of activity extracted from rabbit skeletal muscle is localized in the sarcoplasmic reticulum. The enzyme has a pH optimum of 7.4, and KM of 0.5 mM and 0.35 mM for NAD and p-nitro benzylidine aminoguanidine, respectively. Inorganic phosphate, KCl, and guanidine derivatives inhibit the reaction. Incubation of the sarcoplasmic reticulum or glycogen pellet with (adenylate-32P) NAD or [adenosine-14C(U)]-labeled NAD results in the incorporation of radioactivity into proteins. A large number of proteins are labeled in the sarcoplasmic reticulum fraction. The major labeled band in the glycogen pellet corresponds to a protein of molecular weight of 83 K.     

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.     

Chemical crosslinking and enzyme kinetics provide no evidence for a regulatory role for the 53 kDa glycoprotein of sarcoplasmic reticulum in calcium transport.

m-Maleimidobenzoyl-N-hydroxysuccinimide ester (MBS) was used to cross-link the protein components of rabbit skeletal muscle sarcoplasmic reticulum. Analysis of cross-linked material by SDS-polyacrylamide gel electrophoresis showed that both the (Ca(2+)-Mg2+)-ATPase and the 53 kDa glycoprotein could be cross-linked, since the amount of protein at the locations on the gel corresponding to uncross-linked material was reduced in the presence of 1.0 mM MBS. Cross-linked products of 130 kDa, 200-260 kDa and approx. 300 kDa were identified. Probing the cross-linked products with monoclonal antibodies against ATPase, 53 kDa glycoprotein and calsequestrin revealed no cross-linked products containing the ATPase and either calsequestrin or the 53 kDa glycoprotein over the range of molecular weights examined here. Possible interactions between the ATPase and calsequestrin or the 53 kDa glycoprotein were also investigated by studying the ATPase activity for the purified ATPase and for the ATPase in sarcoplasmic reticulum vesicles made permeable to Ca2+ with A23187. Effects of Ca2+ and ATP on the two systems were indistinguishable, providing no evidence for a major modulatory role of calsequestrin or the 53 kDa glycoprotein on the ATPase.     

Interaction of myotoxin a with the Ca2+-ATPase of skeletal muscle sarcoplasmic reticulum

Myotoxin a is a muscle-damaging toxin isolated from the venom of Crotalus viridis viridis. Its interaction with the Ca2+-ATPase of sarcoplasmic reticulum (SR) vesicles purified from rabbit skeletal muscle was investigated. Myotoxin a inhibited Ca2+ loading and stimulated Ca2+-dependent ATPase without affecting unidirectional Ca2+ efflux. Its action was dose, time, and temperature dependent. Myotoxin a partially blocked the binding of specific anti-(rabbit SR Ca2+-ATPase) antibodies. It is concluded that myotoxin a attaches to the SR Ca2+-ATPase and uncouples Ca2+ uptake from Ca2+-dependent ATP hydrolysis. Myotoxin a also prevented the formation of decavanadate-induced two-dimensional crystalline arrays of the SR Ca2+-ATPase.     

Calmodulin stimulates both adenosine 5'-triphosphate hydrolysis and synthesis catalyzed by a cardiac calcium ion dependent adenosinetriphosphatase

A Ca2+-dependent ATPase purified from a rabbit heart membrane preparation was compared to the Ca2+-dependent ATPase purified from skeletal muscle sarcoplasmic reticulum. The two ATPases display an identical electrophoretic pattern and an identical Ca2+-concentration dependence. However, only the cardiac preparation exhibits a 2-3-fold activation by calmodulin. This effect is best observed when the molar concentrations of calmodulin and ATPase are equivalent and in the presence of high Ca2+ (approximately 10(-5) M) and ATP (approximately 10(-3) M) concentrations. It is demonstrated for the first time that calmodulin stimulates the rate of ATP synthesis, as revealed by an increased production of Pi and a faster ATP in equilibrium Pi exchange, as well as the rate of ATP hydrolysis. It is also demonstrated that calmodulin activation is expressed with purified and detergent-solubilized enzyme in addition to membrane-bound systems. These findings indicate that the effect of calmodulin is an acceleration of the enzyme turnover, due to direct interaction of calmodulin with the enzyme.     

Isolation of calcium pump system and purification of calcium ion-dependent ATPase from heart muscle.

The procedure for the isolation of the highly active fraction of sarcoplasmic reticulum from pigeon and dog hearts is described. The method is based on the partial loading of heart microsomes with calcium and oxalate ions and the precipitation of loaded vesicles in sucrose and potassium chloride concentration gradients. Preparations obtained possess high activity of Ca2+-dependent ATPase and are also able to accumulate up to 10 mumol Ca2+ per mg protein. Purification of sarcoplasmic reticulum membranes is accompanied by a decrease in concentration of cytochrome a+a3 and an increase in the content of [32P]phosphoenzyme. The basic components in "calcium-oxalate preparation" from hearts are proteins with molecular weights of about 100000 (Ca2+-dependent ATPase) and 55000 Calcium-oxalate preparation from pigeon hearts was used for subsequent purification of Ca2+-dependent ATPase. Specific activity of purified enzyme from pigeon hearts is 12-16 mumol Pi/min per mg protein. Enzyme activity of purified Ca2+-dependent ATPase is inhibited by EGTA and is not sensitive to azide, 2,4-dinitrophenol and ouabain. The data obtained demonstrate the similarity of calcium pump systems and Ca2+-dependent ATPases isolated from heart and skeletal muscles.     

The blocking effect of aliphatic hydrocarbons on Ca2+ leakage channels formed by aggregates of Ca2+-ATPase of the sarcoplasmic reticulum

The effects of aliphatic hydrocarbons within the liposomes on the Ca2+ transport function of isolated sarcoplasmic reticulum (SR) membranes of rabbit skeletal muscle, vesiculate preparation of Ca2+ dependent ATPase and proteoliposomes reconstituted from Ca2+-ATPase and egg phosphatidylcholine, were studied. It was shown that liposomes prepared from dipalmitoyl phosphatidylcholine containing aliphatic hydrocarbons increase 2 to 3 times Ca2+ accumulation by Ca2+-dependent ATPase from rabbit skeletal muscle SR. Ca2+ transport by SR vesicles increases in the presence of hydrocarbons by 15--20%. The activating effect of hydrocarbons on Ca2+ transport by proteoliposomes depends on the lipid/protein ratio. The proteoliposomes with a high lipid/protein ratio are practically insensitive to the effects of hydrocarbons. It was suggested that activation of Ca2+ transport by hydrocarbons is due to blocking of Ca2+ leakage channels formed during the aggregation of Ca2+-ATPase molecules. Treatment of membranes by formaldehyde results in the oligomerization of Ca2+-ATPase and decreases 2--4-fold the ATP-dependent accumulation of Ca2+. Subsequent addition of decane restores Ca2+ transport practically completely.     

Influence of monovalent cations on the Ca2+-ATPase of sarcoplasmic reticulum isolated from rabbit skeletal and dog cardiac muscles. An interpretation of transient-state kinetic data

Transient-state kinetics of phosphorylation and dephosphorylation of the Ca2+-ATPase of sarcoplasmic reticulum vesicles from rabbit skeletal and dog cardiac muscles were studied in the presence of varying concentrations of monovalent and divalent cations. Monovalent cations affect the two types of sarcoplasmic reticulum differently. When the rabbit skeletal sarcoplasmic reticulum was Ca2+ deficient, preincubation with K+ (as compared with preincubation with choline chloride) did not affect initial phosphorylation at various concentrations of Ca2+, added with ATP to phosphorylate the enzyme. This is in contrast to preincubation with K+ of the Ca2+-deficient dog cardiac sarcoplasmic reticulum, which resulted in an increase in the phosphoenzyme level. When Ca2+ was bound to the rabbit skeletal sarcoplasmic reticulum, K+ inhibited E - P formation; but under the same conditions, E - P formation of dog cardiac sarcoplasmic reticulum was activated by K+ at 12 microM Ca2+ and inhibited at 0.33 and 1.3 microM Ca2+. Li+, Na+ and K+ also have different effects on E - P decomposition of skeletal and cardiac sarcoplasmic reticulum. The latter responded less to these cations than the former. Studies with ADP revealed differences between the two types of sarcoplasmic reticulum. For rabbit skeletal sarcoplasmic reticulum, 40% of the phosphoenzyme formed was 'ADP sensitive', and the decay of the remaining E - P was enhanced by K+ and ADP. Dog cardiac sarcoplasmic reticulum yielded about 40--48% ADP-sensitive E - P, but the decomposition rate of the remaining E - P was close to the rate measured in the absence of ADP. Thus, these studies showed certain qualitative differences in the transformation and decomposition of phosphoenzymes between skeletal and cardiac muscle which may have bearing on physiological differences between the two muscle types.     

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.     

A saturation transfer electron spin resonance study on the break in the Arrhenius plot for the rotational motion of Ca2+-dependent adenosine triphosphatase molecules in purified and lipid-replaced preparations of rabbit skeletal muscle sarcoplasmic reticulum

By means of saturation transfer electron spin resonance spectroscopy the rotational motion of spin-labeled Ca2+-dependent ATPase molecules has been investigated for three kinds of preparations of rabbit skeletal muscle sarcoplasmic reticulum: MacLennan's enzyme (purified ATPase preparation), DOPC- and egg PC-ATPase (purified ATPase preparations in which endogenous lipids are replaced with dioleoyl and egg yolk phosphatidylcholine, respectively). The rotational mobility of the enzyme in these preparations is somewhat lower than that in the intact membrane, probably due to the reduced amount of lipids. For all the preparations, however, the Arrhenius plot for rotational mobility showed a break at about 18 degrees C, the same temperature at which a break in the Arrhenius plot for Ca2+-ATPase activity occurs. This result provides further evidence that the break in the Arrhenius plot is not related to a lipid phase transition but to a change in the physical state of the Ca2+-ATPase molecule existing in fluid lipids.     

The effect of dantrolene on skeletal-muscle sarcoplasmic-reticulum function in malignant hyperpyrexia in pigs.

The effect of the muscle relaxant dantrolene on isolated sarcoplasmic reticulum was studied in control and malignant-hyperpyrexia-susceptible Landrace pigs. The membranes prepared from both sources showed similar Ca2+-dependent ATPase activities, had comparable phospholipid/protein ratios, and their sodium dodecyl sulphate/polyacrylamide-gel patterns were indistinguishable. Membranes from both sources appeared to bind similar amounts of dantrolene. The drug did not stimulate Ca2+-dependent ATPase activity in preparations from either source. The rates of Ca2+ exchange and Ca2+ efflux appeared to be similar in sarcoplasmic reticulum of control and malignant-hyperpyrexia-susceptible pigs. Dantrolene did not affect either the rates or the amount of Ca2+ lost from the vesicles. These results suggest that dantrolene does not directly affect the movement of Ca2+ across the sarcoplasmic-reticulum membrane.     

Ca2+,Mg2+-ATPase of microsomal membranes from bovine aortic smooth muscle. Identification and characterization of an acid-stable phosphorylated intermediate of the Ca2+,Mg2+-ATPase

An acid-stable phosphoprotein was formed in a microsomal membrane fraction isolated from bovine aortic smooth muscle in the presence of Mg2+ + ATP and Ca2+. The microsomes also showed Ca2+ uptake activity. The Ca2+ dependence of phosphoprotein formation and of Ca2+ uptake occurred over the same range of Ca2+ concentration (1-10 microM), and resembled similar findings from rabbit skeletal microsomes. The molecular weight of the phosphorylated protein, estimated by SDS-gel electrophoresis, was approximately 105,000. The phosphoprotein was labile at alkaline pH, and its decomposition was accelerated by hydroxylamine. Half-maximum incorporation of 32P in the presence of 10 microM Ca2+ occurred at 60 nM ATP. The calcium-dependent phosphoprotein formation was not affected by 5 mM NaN3, but was inhibited in a dose-dependent fashion by ADP with a 50% inhibition occurring at 180 microM. Fifty mM MgCl2 was required for the maximal phosphorylation. The rate of phosphoprotein decomposition after adding 2 mM EGTA was accelerated by varying the Mg2+ concentration from 10 microM to 3 mM. Alkaline pH (9.0) slowed the rate of phosphoprotein decay. Optimal Ca2+-dependent phosphoprotein occurred at 15 degrees C over a broad pH range (6.4 to 9.0). The activation energy of EGTA-induced phosphoprotein decomposition was 25.6 kcal/mol between 0 and 16 degrees C and 14.6 kcal/mol between 16 and 30 degrees C. The phosphoprotein formed by aortic microsomes was thus quite similar to the acid-stable phosphorylated intermediate of the Ca2+-transport ATPase of sarcoplasmic reticulum from skeletal and cardiac muscle. These data suggest that the Ca2+-dependent phosphoprotein is a reaction intermediate of the Ca2+,Mg2+-ATPase of the aortic microsomes.