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⁺.     

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.     

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.     

Pressure-induced dissociation of solubilized sarcoplasmic reticulum ATPase

The effect of hydrostatic pressure on the self-association of sarcoplasmic reticulum ATPase solubilized by nonionic detergent was studied in the pressure range of 1 atm up to 2 kilobars. Polarization of intrinsic tryptophan fluorescence or of fluorescence of a pyrene probe covalently attached to the ATPase was measured. An increase in hydrostatic pressure promoted dissociation of the protein into monomers. For a midpoint dissociation pressure of 1.3 kilobars, the standard volume change in the dissociation reaction was delta Vop = -167 ml/mol. Full reversibility of the pressure effects was shown to occur, as seen by recovery of polarization. An increase in Ca2+ concentration from 50 microM to 5 mM and of pH from 6.9 to 8.6 were found to increase the midpoint dissociation pressure, indicating that these factors stabilize the dimeric state. The hydrolytic activity of the ATPase was measured under pressure. The activity was inhibited by pressure increase. It was found that an irreversible inactivation of the solubilized enzyme occurred during turnover and that increasing pressure added to this instability. Reversibility of the activity was critically dependent on the presence of 10 mM Ca2+ in the assay medium.     

Isolation of subunits from trypsin-cleaved sarcoplasmic reticulum Ca2+ transport adenosine triphosphatase

Summary Controlled tryptic digestion of purified rat skeletal muscle sarcoplasmic reticulum (Ca²⁺ + Mg²⁺)-adenosine triphosphatase yields two products designated Fragments 3a and 3b with molecular weights of 65,000 and 56,000 respectively. The isolation of these products in high yield should facilitate exploration of the molecular characteristics of this adenosine triphosphatase. A simple, rapid method for accomplishing this isolation was developed which provides a high yield and utilizes mild conditions. The fragments obtained by this method were used to determine the phospholipid and sulfhydryl contents of Fragments 3a and 3b. In addition, information was obtained on the orientation of these adenosine triphosphatase components in the enzyme lipoprotein complex.The work was supported in part by Grant #1 P50 HL 19316 from the National Institute of Arthritis and Metabolic Diseases.     

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.     

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.     

Tightly bound calcium of adenosine triphosphatase in sarcoplasmic reticulum from rabbit skeletal muscle

Sarcoplasmic reticulum vesicles were shown to possess a class of tightly bound calcium ions, inaccessible to the chelator, ethylene glycol bis(beta-aminoethyl ether) N,N,N',N'-tetraacetic acid at 0 degrees C or 25 degrees C, amounting to 4.5 nmol/mg of protein (approximately 0.5 mol/mol (Ca2+,Mg2+)-ATPase). The calcium ionophores, A23187 and X537A, induced rapid exchange of tightly bound calcium in the presence of chelator. Chelator alone at 37 degrees C, caused irreversible loss of bound calcium, which correlated with uncoupling of transport from (Ca2+,Mg2+)-ATPase activity. Uncoupling was not accompanied by increased permeability to [14C]inulin. Slow exchange of tightly bound calcium with medium calcium was unaffected by turnover of the ATPase or by tryptic cleavage into 55,000- and 45,000-dalton fragments. Binding studies with labeled calcium suggested that tight binding involves a two-step process: Ca2+ + E in equilibrium K E . Ca2+ leads to E < Ca2+ where E and < Ca2+ represent the ATPase and tightly bound calcium, and K = 1.6 X 10(3) M-1. It is suggested that tightly bound calcium is located in a hydrophobic pocket in, or in close proximity to the ATPase, and, together with tightly bound adenine nucleotides (Aderem, A., McIntosh, D. B., and Berman, M. C. (1979) Proc. Natl. Acad. Sci. U. S. A. 76, 3622-03632), is related to the ability of the ATPase to couple hydrolysis of ATP to vectorial transfer of calcium across the membrane.     

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.     

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.     

The adenosine triphosphatase and calcium ion-transporting activities of the sarcoplasmic reticulum of developing muscle

1. The ATPase (adenosine triphosphatase) specific activity and the total nitrogen content of the myofibrillar fraction per g. wet weight of rabbit longissimus dorsi muscle increased steadily during the late foetal stages and the first few weeks after birth. 2. The ATPase specific activity of the sarcoplasmic-reticular fraction isolated by a sucrose-density-gradient procedure rose to a sharp peak 8-10 days after birth and then declined to the adult value, which was about 25% of the maximum. 3. The peak in ATPase activity was a feature of the sarcoplasmic reticulum isolated from muscle, and the time at which it occurred in relation to birth was related to the degree of development and the activity pattern of the muscle. 4. The peak in ATPase activity of the sarcoplasmic reticulum occurred at an earlier age if newborn animals were made to exercise earlier than was normal. 5. The ;extra' ATPase associated with the sarcoplasmic reticulum and the ability to concentrate Ca(2+) increased in a similar manner over the period of development studied. 6. It is postulated that the Ca(2+)-transport system of the sarcoplasmic reticulum consists of two components, namely the ATPase and the system coupling this enzyme to Ca(2+) transport. During development the ATPase develops first and has almost reached maximum activity in the longissimus dorsi muscle of the rabbit after 8-10 days. Subsequently the activity of the coupling system rises rapidly, leading to an increase in the capacity and efficiency of Ca(2+) transport.     

Occlusion of calcium in the ADP-sensitive phosphoenzyme of the adenosine triphosphatase of sarcoplasmic reticulum

In order to characterize the form of the phosphorylated Ca2+-ATPase of sarcoplasmic reticulum which occludes the calcium bound in the enzyme (Takisawa, H., and Makinose, M. (1981) Nature (Lond.) 290, 271-273), a kinetic method was developed allowing quantitation of the amount of ADP-sensitive and ADP-insensitive phosphoenzyme. The relationships between occluded Ca2+ in the enzyme and the two forms of phosphoenzyme were studied at various concentrations of CaCl2 and MgCl2. The amount of tightly bound Ca2+ in the phosphoenzyme increases concordantly with the increase in the amount of ADP-sensitive phosphoenzyme, suggesting that occlusion of Ca2+ occurs in the ADP-sensitive phosphoenzyme. These results suggest that 1 mol of ADP-sensitive phosphoenzyme occludes 2 mol of Ca2+. Ca2+ is released from the enzyme under conditions which favor the formation of the ADP-insensitive phosphoenzyme (e.g. 5 mM MgCl2 and 50 microM CaCl2). Ca2+ release correlates approximately with the formation of the ADP-insensitive phosphoenzyme. The simulated time course of Ca2+ release, based on the Ca2+-binding properties of the two forms of phosphoenzyme, shows a good fit with the Ca2+ release curves observed, indicating that the ADP-insensitive phosphoenzyme binds no Ca2+ under these conditions.     

Isolation of subunits from trypsin-cleaved sarcoplasmic reticulum Ca2+ transport adenosine triphosphatase.

Controlled tryptic digestion of purified rat skeletal muscle sarcoplasmic reticulum (Ca2+ + Mg2+)-adenosine triphosphate yields two products designated Fragments 3a and 3b with molecular weights of 65,000 and 56,000 respectively. The isolation of these products in high yield should facilitate exploration of the molecular characteristics of this adenosine triphosphatase. A simple, rapid method for accomplishing this isolation was developed which provides a high yield and utilizes mild conditions. The fragments obtained by this method were used to determine the phospholipid and sulfhydryl contents of Fragments 3a and 3b. In addition, information was obtained on the orientation of these adenosine triphosphatase components in the enzyme lipoprotein complex.     

Effect of urea on the partial reactions and crystallization pattern of sarcoplasmic reticulum adenosine triphosphatase

Steady-state ATPase activity, calcium binding, formation of phosphorylated enzyme intermediate with ATP in the presence of Ca2+, or with Pi in the absence of Ca2+, and association of ATPase molecules into bidimensional crystals, were studied using vesicular fragments of sarcoplasmic reticulum. The vesicles were exposed to increasing concentrations of urea in order to produce stepwise perturbations of protein structure and to test the effect of such perturbations on the partial reactions and crystallization pattern of sarcoplasmic reticulum ATPase. It was found that low concentrations of urea produce specific inhibition of Pi binding and enzyme phosphorylation with Pi (but not with ATP). Intermediate concentrations of urea reduce calcium binding affinity and cooperativity, while the ability of the enzyme to be phosphorylated with ATP and to form dimeric arrays is retained. These observations demonstrate that the sarcoplasmic reticulum ATPase is sensitive to physical perturbations producing specific and reversible changes in the Pi and calcium binding domains. These changes interfere with enzyme turnover, indicating that conformational effects related to binding and dissociation of Pi and calcium are tightly coupled to catalysis and energy transduction. Higher concentrations of urea produce irreversible denaturation, accompanied by total inhibition of calcium binding, enzyme phosphorylation with ATP, and association of ATPase chains in bidimensional crystals. Under these conditions, protein unfolding is manifested by a sharp reduction in the fluorescence of intrinsic tryptophan residues and of a covalently bound probe. These observations suggest that dimeric association and a tendency to form bidimensional crystals correspond to a basic property of the enzyme, which is linked to its native structure and whose character may change in the presence of ligands and/or during the catalytic cycle. On the other hand, the decavanadate-induced crystallization pattern cannot be interpreted in terms of a mechanistic relationship of ATPase dimerization with one of the intermediate states of the catalytic cycle.     

Studies on the structure of the calcium-dependent adenosine triphosphatase from rabbit skeletal muscle sarcoplasmic reticulum

The linear arrangement of the three fragments of Ca²⁺-ATPase from rabbit skeletal muscle sarcoplasmic reticulum with molecular weights of 20,000, 30,000, and 45,000 obtained by limited tryptic hydrolysis was determined by locating the NH₂-terminal acetylated methionyl residue of the original peptide in the M_r = 20,000 fragment. Since both the M_r = 20,000 and 30,000 polypeptides originate from a M_r = 55,000 fragment which is distinct from the M_r = 45,000 polypeptide, the sequence of these three fragments was determined to be 20,000, 30,000, and 45,000. The M_r = 20,000 fragment was further cleaved by cyanogen bromide to yield a M_r = 7,000 COOH-terminal fragment which is relatively hydrophilic. The NH₂-terminal portion is rich in glutamyl residues. The COOH-terminus of the M_r = 30,000 fragment was determined by both digestion with carboxypeptidases and cyanogen bromide cleavage. Using the partial amino acid sequence of the Ca²⁺-ATPase, it was deduced that the active site phosphoaspartyl residue is 154 amino acids from the COOH-terminus of the M_r = 30,000 fragment and hence approximately 35,000 M_r from the NH₂-terminus of the original Ca²⁺-ATPase molecule. Furthermore, it was shown that the two tryptic cleavages of the Ca²⁺-ATPase generating these three large fragments were both single hydrolyses of arginylalanine peptide bonds.