Ionized and bound calcium inside isolated sarcoplasmic reticulum of skeletal muscle and its significance in phosphorylation of adenosine triphosphatase by orthophosphate.

Calcium loading of skeletal muscle sarcoplasmic reticulum performed passively by incubation with high calcium concentrations (0.5--15 mM) on ice gives calcium loads of 50--60 nmol/mg sarcoplasmic reticulum protein. This accumulated calcium is not released by EGTA [ethyleneglycol bis-(2-aminoethyl)-N,N,N',N'-tetraacetic acid], but almost completely released by ionophore X-537A plus EGTA or phospholipase A plus EGTA treatment and is therefore assumed to be inside the sarcoplasmic reticulum. This calcium is distributed in one saturable and one non-saturable calcium compartment, as derived from the dependence of the calcium load on the calcium concentration in the medium. These compartments are assigned to bound and ionized calcium inside the sarcoplasmic reticulum, respectively. Maximum calcium binding under these conditions was 33 nmol/mg protein with an apparent half-saturation constant of 5,8 nmol/mg free calcium inside, or between 1.2 and 0.6 mM free calcium inside, assuming an average vesicular water space of 5 or 10 microliter/mg protein, respectively. Calcium-dependent phosphorylation of sarcoplasmic reticulum calcium-transport ATPase from orthophosphate depends on the square of free calcium inside, whilst inhibition of phosphorylation depends on the square of free calcium in the medium. Calcium-dependent phosphorylation appears to be determined by the free calcium concentrations inside or outside allowing calcium binding to the ATPase according to the two classes of calcium binding constants for low affinity calcium binding or high affinity calcium binding, respectively. It is further suggested that the saturation of the low-affinity calcium-binding sites of the ATPase facing the inside of the sarcoplasmic reticulum membrane is responsible for the greater apparent orthophosphate and magnesium affinity in calcium-dependent phosphorylation than in calcium-independent phosphorylation from orthophosphate. Maximum calcium-dependent phosphoprotein formation at 20 degrees C and pH 7.0 is about 4 nmol/mg sarcoplasmic reticulum protein.     

Modification of sarcoplasmic reticulum adenosine triphosphatase by adenosine triphosphate magnesium

Lineweaver-Burk plots of Ca²⁺-activated adenosine triphosphatase from rabbit muscle sarcoplasmic reticulum have been determined for a wide range of substrate concentrations. The plots measured at constant Mg²⁺ concentrations are normally nonlinear, but approach linearity either as the sarcoplasmic reticulum ages, or when small quantities of Triton-X100 are added. Titration with N-ethylmaleimide has the same effect on the activity of the ATPase measured either at high or low substrate concentrations. Lineweaver-Burk plots measured under conditions where the Mg²⁺ concentration is varied so as to be always equal to the ATP concentration are linear. These results have been interpreted as evidence that the adenosine triphosphatase has a single active site which uses MgATP as its substrate and which can be modified by free Mg²⁺.     

Cross-linking experiments with the adenosine triphosphatase of sarcoplasmic reticulum.

The proteins of sarcoplasmic reticulum were cross-linked by rapid oxidation of thiol groups with I2. About two-thirds of the thiols were oxidized without any significant cross-linking, implying an extensive formation of intramolecular disulphide bonds. When the thiols were completely oxidized at room temperature a series of oligomers containing up to five molecules were observed, as well as large aggregates which were excluded from the gels. Complete oxidation at -10 degrees C left most of the ATPase (adenosine triphosphatase) as monomer. Similar results were obtained when copper-phenanthroline complexes or dimethyl suberimidate were used as cross-linking reagents. We conclude that most of the cross-linked species arise by linking of randomly colliding ATPase molecules which are present in the membrane at very high concentration.     

Calcium-dependent adenosine triphosphatase activity preservation in isolated sarcoplasmic reticulum.

ATPase activity in sarcoplasmic reticulum vesicles was measured before and after storage for several weeks and under a variety of conditions. Rapid freezing and storage at-80 degrees C provided optimum protection of enzyme activity. Sarcoplasmic reticulum preparations stored at 0 degrees C or frozen slowly and stored at-20 degrees C were not stable. At 0 degrees C sucrose, glycerol, and dithiothreitol had a stabilizing effect while NaCl, dimethylsulfoxide, and antioxidants afforded little or no protection.     

Characterization of cardiac sarcoplasmic reticulum ATP-ADP phosphate exchange and phosphorylation of the calcium transport adenosine triphosphatase.

1. The terminal phosphate of (gamma-32P)ATP is rapidly incorporated into cardiac sarcoplasmic reticulum membranes (0.7--1.3 mumol/g protein) in the presence of calcium and magnesium. Cardiac sarcoplasmic reticulum membranes catalize an ATP-ADP phosphate exchange in the presence of calcium and magnesium. 2. Half-maximum activation of the phosphoprotein formation and ATP-ADP phosphate exchange is reached at an ionized calcium concentration of about 0.3 muM. The Hill coefficients are 1.3. 3. Transphosphorylation and ATP-ADP phosphate exchange require magnesium and are maximally activated at magnesium concentrations close to or equal to the ATP concentration. 4. The phosphoprotein level is reduced to about 45% at an ADP/ATP ratio of 0.1. The rate of calcium-dependent ATP splitting declines, whilst the rate of the calcium-dependent ATP-ADP phosphate exchange increases when the ADP/ATP ratio is varied from 0.1 to 1. The sum of both, the rate of ATP splitting and the rate of ADP-ATP phosphate exchange remains constant. 5. Phosphoprotein formation and ATP-ADP phosphate exchange are not affected by azide, dinitrophenol, dicyclohexyl carbodiimide and oubain, whilst both activities are reduced by blockade of -SH groups localized on the outside of the sarcoplasmic reticulum membrane. 6. The isolated phosphoprotein is acid stable. The trichloroacetic acid denatured 32P-labelled membrane complex is dephosphorylated by hydroxylamine, which might indicate that the phosphorylated protein is an acyl-phosphate. 7. Polyacrylamide gel elctrophoresis (performed with phenol/acetic acid/water) of phosphorylated sarcoplasmic reticulum fractions demonstrates that the 32P-incorporation occurs into a protein of about 100000 molecular weight. 8. It is suggested that the phosphoprotein represents a phosphorylated intermediate of the calcium-dependent ATPase which formation occurs as an early step in the reaction sequence of calcium translocation by cardiac sarcoplasmic reticulum similar as in skeletal muscle.     

Stimulation of adenosine triphosphatase activity of sarcoplasmic reticulum by adenylyl methylene diphosphate.

The effects of adenylyl methylene diphosphate (AMD), a non-hydrolyzable ATP analogue, were examined in sarcoplasmic reticulum vesicles isolated from rabbit skeletal muscle. The Ca2+-dependent APTase activity measured at 5 degrees C and pH 7.0 in 5.2 micrometer [gamma-32P]ATP and in the absence of added alkali metal salts was stimulated by added AMD. The steady state level of phosphoenzyme, however, was not decreased greatly by added AMP under these conditions. The hydrolysis of the phosphoenzyme formed at the steady state in the absence of added alkali metal salts was accelerated by added AMD to an extent that can account for the stimulation of the ATPase activity. At 5 degrees C and pH 7.0 the maximum stimulation of phosphoenzyme hydrolysis by AMD and the Km value for this ATP analogue were 4.3-fold and 40 micrometer, respectively. These results provide further support for our previous conclusion (Shigekawa, M., Dougherty, J.P. and Katz, A.M. (1978) J.Biol. Chem. 253, 1442--1450) that 2 classes of ATP site exist in the calcium pump ATPase in the absence of added alkali metal salts, one being the catalytic site and the other being the regulation site which activates the activity of the catalytic site.     

Dynamic reversal of enzyme carboxyl group phosphorylation as the basis of the oxygen exchange catalyzed by sarcoplasmic reticulum adenosine triphosphatase.

Millisecond mixing and quenching experiments demonstrate an apparent t1/2 for the labeling of phosphorylated sarcoplasmic reticulum ATPase by 32Pi at pH 6 and 30 degrees C of 30 to 40 ms. Under the same conditions, the rate of exchange of water oxygens with inorganic phosphate (Pi) is about 40 mol of H2O exchanged with Pi per 10(6) g of protein per s. Theoretical equations are developed for the expected 32P-labeling pattern given various comparative rates of flux between Pi and the Michaelis complex and between the Michaelis complex and phosphorylated enzyme. The results show that the rapid reversal of the formation of the phosphorylated enzyme is a major source of the oxygen exchange and are consistent with such reversal being the only source.     

Separation and characterisation of tryptic fragments from the adenosine triphosphatase of sarcoplasmic reticulum.

The two halves of the ATPase, M, 115,000, from sarcoplasmic reticulum produ-ed by limited trypsin treatment have been purified in sodium dodecylsulphate. The fragment of Mr60,000 has been purified by electrophoresis on cellulose acetate slabs and that of Mr 55,000 by gel filtration. The two halves of the 60,000 Mr fragment (Mr33,000 and 24,000) produced by more extensive trypsin treatment have also been purified by gel filtration in sodium dodecylsulphate. The sum of the amino acid analyses of the constituent tryptic fragments is in good agreement with that for the whole ATPase. The amino acid compositions of the two halves of the ATPase were strikingly similar. N-terminal analysis shows that the ATPase and its constituent tryptic polypeptides all possess a single N-terminal alanine implying no further cleavage of the polypeptide by trypsin. Attempts to solubilize selectively the tryptic fragments from the membrane by a variety of denaturing and solubilising agents under a variety of conditions have proved unsuccessful, suggesting that the interaction between the tryptic polypeptides is stronger than between the lipid and the protein. The possibility that the interaction between the tryptic polypeptides includes disulphide bonding has been eliminated.     

Pressure-induced inactivation of sarcoplasmic reticulum adenosine triphosphatase during high-speed centrifugation

Sarcoplasmic reticulum vesicles were found to be highly sensitive to high-speed centrifugation in metal-deprived mediums at low temperature (4 degrees C). The irreversible modifications induced were easily detected from observation of the environment-sensitive spectrum of an iodoacetamide spin-label bound to the ATPase. Centrifugation also resulted in vesicle aggregation and inhibition of calcium transport, ATPase activity, and phosphoenzyme formation. These denaturation-like phenomena were prevented in the presence of sucrose, or by nucleotide binding, or, again, by cation binding to the ATPase high-affinity calcium binding sites and were only present when centrifugation was performed at low temperature. The crucial parameter during this process was found to be the hydrostatic pressure which developed in the centrifuge tube. SR vesicles exposed to 800 bars in a pressure bomb displayed the same features. It is suggested that irreversible denaturation takes place after one or both of the two following well-documented effects of pressure: a rise in the lipid order/disorder transition temperature or dissociation of the oligomeric structure of the calcium pump.     

Isolation and characterization of tryptic fragments of the adenosine triphosphatase of sarcoplasmic reticulum.

Exposure of sarcoplasmic reticulum to trypsin in the presence of 1 M sucrose results in degradation of the Mr = 102,000 ATPase enzyme to two fragments of Mr = 55,000 and 45,000 with subsequent appearance of fragments of Mr = 30,000 and 20,000. These fragments were purified by column chromatography in sodium dodecyl sulfate. Antibodies were raised against the ATPase and the Mr = 55,000, 45,000, and 20,000 fragments. There was no antigenic cross-reactivity between the Mr = 55,000 and 45,000 fragments, indicating that they were derived from a single linear cleavage of the larger enzyme. There was antigenic cross-reactivity between the Mr = 20,000 and 55,000 fragments, indicating an origin of the Mr = 20,000 fragment in the Mr = 55,000 fragment. None of the antibodies inhibited (Ca2+ + Mg2+)-dependent ATPase or Ca2+ transport. The Mr = 20,000 fragment and the Mr = 55,000 fragment were active in Ca2+ ionophore assays. The active site of ATP hydrolysis was labeled with [gamma-32P]ATP and the site of ATP binding was labeled with tritiated N-ethylmaleimide. In both cases radioactivity was found in the intact ATPase and in the Mr = 55,000 and 30,000 fragments, indicating that the Mr = 30,000 fragment was also derived from the Mr = 55,000 fragment. Amino acid composition data showed that the Mr = 45,000 fragment contained about 60% nonpolar and 40% polar amino acids, while the Mr = 55,000 fragment and the Mr = 20,0000 fragment contained about equal amounts of polar and nonpolar amino acids. Studies of the reaction of various antibodies at the external surface of sarcoplasmic reticulum vesicles showed that the ATPase was exposed, whereas calsequestrin and the high affinity Ca2+-binding protein were not. The use of antibodies against the various fragments indicated that the Mr = 55,000 fragment was in large part exposed, whereas the Mr = 20,000 and the 45,000 fragments were only poorly exposed. It is probable that the site of ATP hydrolysis in the Mr = 55,000 fragment is external, whereas the ionophore site is only partially exposed and the Mr = 45,000 fragment is largely buried within the membrane.     

Calcium gradient-dependent and calcium gradient-independent phosphorylation of sarcoplasmic reticulum by orthophosphate. The role of magnesium.

Phosphorylation of the calcium-transport ATPase of skeletal muscle sarcoplasmic reticulum by inorganic phosphate was investigated in the presence or absence of a calcium gradient. The maximum phosphoprotein formation in the presence of a calcium gradient at 20 degrees C and pH 7.0 is approximately 4 nmol/mg sarcoplasmic reticulum protein, but only between 2.4 and 2.8 nmol/mg protein in the absence of a calcium gradient, using Ionophore X-537 A or phospholipase-A-treated sarcoplasmic reticulum vesicles. Maximum phosphoprotein formation independent of calcium gradient at 20 degrees C and pH 6.2 is in the range of 3.6--4 nmol/mg protein. Half-maximum phosphoprotein formation dependent on calcium gradient was achieved with 0.1--0.2 mM free orthophosphate at 10 mM free magnesium or at 0.1--0.2 mM free magnesium at 10 mM free orthophosphate. Phosphoprotein formation independent of calcium gradient is in accordance with a model which assumes, firstly, the formation of a ternary complex of the ATPase protein with orthophosphate and magnesium (E . Pi . Mg) in equilibrium with the phosphoprotein (E-Pi . Mg) and, secondly, an interdependence of both ions in the formation of the ternary complex. The apparent equilibrium constant was 0.6 and the apparent dissociation constants KMg, KMg', KPi and KPi' were 8.8, 1.9, 7.2 and 1.5 mM respectively, assuming a total concentration of the phosphorylation site per enzyme of 7 nmol/mg protein.     

Sequential mechanism of calcium binding and translocation in sarcoplasmic reticulum adenosine triphosphatase

Cooperative calcium binding (apparent Kd = 1.04 X 10(-6) M) to the ATPase of sarcoplasmic reticulum vesicles occurs with a maximal stoichiometry of 2 mols of divalent cation/mol of enzyme in the absence of ATP. The bound calcium is distributed into two pools which undergo fast or slow isotopic exchange, respectively. The two pools retain a 1:1 molar ratio under various conditions and are both located within a protein crevice, as suggested by their cooperative interaction and exchange kinetics. Following enzyme phosphorylation by ATP, both pools of bound calcium are "internalized" (cannot be displaced by quench reagents). If following 45Ca2+ binding, isotopic dilution is obtained in the medium by adding 40Ca2+ with ATP, internalization of both pools of bound 45Ca2+ (2 mol/mol of phosphoenzyme) is still observed within the first enzyme cycle. When the cycle is reversed by addition of excess ADP soon after ATP, only half of the internalized 45Ca2+ is released from the enzyme into the medium outside the vesicles, while the other half remains with the vesicles. If half of the bound 45Ca2+ is exchanged (fast exchange) with 40Ca2+ previous to the addition of ATP, none of the remaining 45Ca2+ is released outside the vesicles upon reversal of the enzyme cycle. Therefore, the pool of bound calcium which undergoes slower exchange with the outside medium, is the first to be released inside the vesicles upon enzyme phosphorylation. A sequential mechanism of calcium binding and translocation is proposed, that accounts for binding cooperativity and exchange kinetics, presteady state transients following addition of ATP, and the Ca2+ concentration dependence of ATPase activity in steady state.     

The binding of lipid to the lipid-free adenosine triphosphatase protein of sarcoplasmic reticulum.

Dinitrophenylated dipalmitoyl phosphatidylethanolamine and its lyso derivative have been shown to bind to the lipid-free ATPase protein derived from the sarcoplasmic reticulum. The binding of these lipids is accompained by the quenching of up to 95% of the tryptophyl fluorescence of the protein. This effect is reversed by 9-10 mM deoxycholate. The solubility of the lipid-free ATPase protein in the absence of deoxycholate and the solubility of submillimolar concentrations of the dinitrophenylated monopalmitoyl phosphatidylethanolamine anion in aqueous media allowed binding experiments using this lipid ligand to be carried out in a simple buffer system. It is shown that in the case of this lipid the initial phase of the binding process displays an apparent positive co-operatively. Data from the second phase in the saturation of the protein with this lipid is consistent with binding to independent, equivalent, non-interacting sites with a microscopic (intrinsic) association constant of 1.63 x 10(6) M-1, the fluorescence being quenched in the geometric fashion. Altogether a total of about 15 molecules of this lipid may be bound by the protein.     

Transient state kinetic effects of calcium ion on sarcoplasmic reticulum adenosine triphosphatase.

A rapid mixing technique was used to investigate the effects of Ca2+ ion on the kinetics of ATP hydrolysis by sarcoplasmic reticulum vesicles. "Basic" ATPase measured in the absence of Ca2+ showed an initial burst of inorganic phosphate production. Similarities in the transient state kinetic properties of basic and "extra" or Ca2+-dependent ATPase suggest that the two activities represent a single enzyme species. At low concentrations of Ca2+ (less than 10(-6) M) the time course of the partial reactions of extra ATPase appeared to fit a simple scheme in which the acid-stable, phosphorylated enzyme (E approximately P) breaks down directly to inorganic phosphate and free enzyme. A similar mechanism seemed to apply to moderate levels of ATP and high external concentrations of Ca2+ known to inhibit transport activity. In the intermediate range of Ca2+ concentrations inorganic phosphate production was resolved into two phases consisting of a fast initial rate (burst) and slow steady state. Acid-stable phosphorylated protein showed a transient decay which coincided with the appearance of the burst. This behavior is consistent with a scheme in which E approximately P breaks down to an acid-labile or noncovalent intermediate state (E-P). A slow secondary increase in phosphorylation followed the transient decay in E approximately P. This late phase of protein labeling was eliminated following pretreatment with Triton X-100, sodium oxalate, or diethyl ether which decrease or prevent the formation of a transport gradient. An analysis of the dependence of the steady state level of phosphorylation and rate of inorganic phosphate production on Ca2+ concentration indicated that the phosphorylation mechanism involves interaction of two Ca2+ ions with the enzymatic carrier. The pathway by which E approximately P breaks down, i.e. whether it goes to E + Pi or E-P, may depend on the extent to which these sites are occupied by Ca2+. The transport of Ca2+ is discussed in terms of a flip-flop mechanism in which E approximately P and E-P represent high and low affinity Ca2+ binding states occurring in separate halves of an enzyme dimer.