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With increasing SDS/protein ratios, covalent phosphorylation by ATP and Pi is abolished before ATP hydrolysis (Pi production) ceases. We have shown that the SDS-dependent profiles of the decline in covalent phosphorylation by either substrate are virtually identical, reflecting a common mechanism of detergent interaction, while ATP can be hydrolysed via a non-covalent phosphointermediate. Our studies support that the transfer of both terminal Pi from ATP, as well as Pi to its final binding site, is a multistep reaction involving electrostatic interaction with one or more amino acid side chains, including a Lys residue.

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In this investigation low, non-solubilizing concentrations of the strong anionic detergent SDS were used to perturbate the interaction of Ca2+ and Pi with their respective binding domains on the sarcoplasmic reticulum Ca-transport ATPase. Rising SDS concentrations produce a two-step decline of Ca2+-dependent ATP hydrolysis. At pH 6.15, SDS differently affects high affinity Ca2+ binding and phosphorylation by inorganic phosphate and releases the "mutual exclusion" of these two ligand binding steps. The degree of uncoupling is considerably more pronounced in the presence of 20% Me2SO. The reduction of Ca2+ binding by SDS is demonstrated to be a result of decreased affinity of one of the two specific high affinity binding sites and of perturbation of their cooperative interaction. Higher SDS partially restores the original high Ca2+ affinity but not the cooperativity of binding. Phosphorylation exhibits a higher SDS sensitivity than Ca2+ binding: Increasing SDS competitively inhibits and then completely abolishes phosphoenzyme formation. Thus, SDS binds to the phosphorylation domain, evidently involving the Lys352 residue of the ATPase molecule; this is accompanied by a more unspecific concentration-dependent SDS effect, probably mediated by hydrophobic force, which, finally, suppresses phosphorylation. Me2SO does neither qualitatively affect the SDS-dependent chemical properties of the vesicular material nor the SDS-dependent perturbation of the investigated reaction steps.

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The coupling of Ca2+ movements and phosphate fluxes as well as the time-dependent occurrence of sequential reaction intermediates in the forward mode of the Ca,Mg-dependent ATPase reaction have been investigated using leaky vesicles (A23187) in the presence of varying Ca2+, Mg2+, and K+ concentrations. The employed ATP concentration of 2 microM does not allow more than one reaction cycle to occur. The respective fractions of ADP-sensitive and ADP-insensitive phosphoenzyme have been determined. The chosen experimental conditions (0-1 degree C, pH 6.0, absence of solubilizers) allow a prolonged time of observation and exclude interfering alterations of coupling and binding parameters, respectively. It is shown that under the experimental conditions K+ interacts with at least four different reaction steps (phosphoenzyme formation, E1P----E2P transition, E2P hydrolysis, and E2----E1 transformation). Mg2+ represents the sole ionic co-factor for the formation of the substrate MgATP if it is present in high concentrations (5 mM). Additional Ca2+ is bound to the substrate as well as to unspecific sites otherwise occupied by Mg2+ if Mg2+ is reduced to 0.1 mM. In this case the E1P----E2P transition rate (including Ca2+ translocation and Ca2+ release from low-affinity sites) is little diminished. If, in the absence of K+, both Mg2+ and Ca2+ are deficient E2P hydrolysis is vastly retarded. We find Ca2+ release to occur time-coincidently with E1P formation and not concomitantly with the comparably slow appearance of E2P; the molar amount of Ca2+ released, however, rather agreed with that of E2P formed. This suggests that under the prevailing conditions of a high proton concentration, phosphoenzyme states containing occluded Ca2+ or Ca2+ bound to low-affinity sites are transitional and not detectable. Preliminary findings on this subject have been published by us and colleagues from this laboratory [Hasselbach, W., Agostini, B., Medda, P., Migala, A. & Waas, W. (1985) in The sarcoplasmic reticulum calcium pump: Early and recent developments critically overviewed (Fleischer, S. & Tonomura, Y., eds) pp. 19-49, Academic Press, Orlando].

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The interdependence of the competition between Ca2+ and hydrogen ions for the internally located low-affinity Ca2+ binding sites of sarcoplasmic reticulum vesicles and the pH-dependent splitting rate of phosphoenzyme was investigated. Sarcoplasmic reticulum vesicles were preincubated at a selected pH and passive Ca2+ loading, active Ca2+ uptake at the same pH as well as active Ca2+ uptake at a distinct pH (pH-jump method) were observed. In addition, Cai-Cao exchange in the absence and presence of ADP and ATP-ADP exchange were measured. The overall ATP splitting rate was assayed with leaky vesicles in the presence of varied Ca2+ concentration and four different pH. All experiments were carried out at Ca2+ concentrations sufficient to saturate the externally located activating high-affinity binding sites at all pH and in the absence of affecting concentrations of monovalent cations. Active Ca2+ transport (particularly evident applying the pH-jump method) is facilitated at low intravesicular pH, reflecting the favoured Ca2+ release to the intravesicular space, in contrast to the reverse pH-dependence of passive Ca2+ accumulation and the initial rate of Cai-Cao exchange, both favoured by elevated internal Ca2+ binding capacity. The rates of ATP splitting, the continuing slow rate of Cai-Cao exchange, and the ATP-ADP exchange are optimal at an intermediate proton concentration, reflecting the influence of protons on partial reaction steps occurring later in the reaction cycle and the accelerated exchange of Ca2+ at the internal low-affinity sites as well as the establishment of a new pseudo equilibrium between the possible reaction intermediates. The pool of rapidly exchangeable Ca2+ is enlarged whereas the rate of slow exchange is unaltered or diminished (pH 7.8) by ADP.

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The activity of the calcium transport systems in the sarcoplasmic reticulum membranes operating in the forward and reverse modes depends on the presence of magnesium ions. When the system operates as an NTP-driven calcium pump, magnesium enters the reaction chain chelated with NTP. Magnesium is a component of the NDP-sensitive phosphoprotein species involved in the NDP-NTP exchange reaction. When the system operates in the reverse mode as a calcium efflux-driven NDP synthetase, magnesium together with inorganic phosphate rapidly forms the initial phosphorylated intermediate. The stability of the magnesium-phosphoprotein complex is considerably enhanced by an existing calcium gradient. Magnesium as well as calcium affects the partitioning of the energy in the reaction sequence.

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Initial Ca2+ transport and phosphoprotein formation of the sarcoplasmic reticulum membrane with GTP were investigated in a comparative study. While saturation of the high-affinity sites for Ca2+ binding and transporting as well as for GTP binding on the external surface of the membrane resulted in Ca2+ transport and phosphoprotein formation in a molar ratio of 2, the variation of the concentrations of the two reactants yielded ratios between 1.7 and 5.7. The ratios varied with a similar dependence on the concentrations of Ca2+ and GTP, except at 500 microM Ca2+, if the reaction was started by Ca2+ instead of GTP but the overall rates decreased. 1 mM DL-propranolol in the preincubation medium selectively inhibited Ca2+ transport but had no effect on initial phosphoprotein formation. These observations indicate that:L (a) phosphorylation of one enzyme molecule induces Ca2+ transport by a variable but limited number of neighbouring molecules, (b) not all Ca2+ bound is essential for phosphorylation but can be transported in parallel, (c) Ca2+ bound to low-affinity sites occupied at 500 microM Ca2+ in the reaction medium is also transported initially, (d) the accessibility of the high-affinity Ca2+ binding sites for DL-propranolol differs, (e) DL-propranolol interacts with Ca2+ binding and transporting sites only in that conformation of the enzyme that can be phosphorylated by the nucleotide.

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Experiments were undertaken to determine if drugs (verapamil, propranolol, and methylprednisolone sodium saccinate) that protect the fine ultrastructure of heart muscle against damage caused by hypoxia, protect mitochondrial function. Mitochondrial function was assessed in terms of oxidative phosphorylating and Ca2 +-accumulating activities. Isolated rabbit hearts were used, and hypoxic conditions induced by reducing the perfusate PO2 from 80.8 to 0.80 kPa (600 to 6 mmHg). The drugs were either added at the start of the hypoxic perfusion or (verapamil and propranolol) the rabbits were pretreated with them. Verapamil, propranolol and, to a lesser extent, methylprednisolone sodium succinate, provided protection evidenced by the maintainance of near normal mitochondrial oxidative phosphorylating and Ca2 +-accumulating activities after 60 min hypoxic perfusion. When added directly to mitochondria isolated from hypoxic-perfused muscle, the drugs had no effect.

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The possibility that the cardiac SR undergoes developmental changes at about the time of birth, and that these changes affect its ability to accumulate Ca2+ and to hydrolyse ATP has been studied. SR-rich microsomal fractions were prepared from heart muscle excised from foetal guinea pigs and rabbits 1 day before their anticipated date of birth, and from 1 day old and adult animals. For control purposes microsomes were also prepared from the relevant maternal stock animals. One day before birth the cardiac microsomes of the foetal but not of the maternal animals exhibited a decreased ability to accumulate Ca2+ by uptake but not by the binding process, and a decreased ability to hydrolyse ATP. This reduction in ATPase activity involved both the Ca2+-dependent and the Ca2+-independent ATPase enzymes. One day after birth the Ca2+-accumulating activity of the neonatal microsomes had increased, that of the rabbit via an increase in Ca2+ uptake and that of the guinea pig by an increase in Ca2+ binding. These changes were accompanied by an increase in the activity of the Ca2+-dependent ATPase. The results are interpreted to mean that the cardiac SR changes at about the time of birth, and that although the pattern of these changes may be species specific they result in an increase in the Ca2+-accumulating activity of the SR.

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The effect of acetaldehyde, the hepatic metabolite of alcohol, on the functioning of cardiac muscle was investigated at the subcellular level. Concentrations of acetaldehyde that occur in plasma failed to alter either the microsomal Ca2+-accumulating and ATPase activity or the Ca2+-accumulating activity of the mitochondria. These same concentrations of acetaldehyde inhibited the Ca2+-dependent myofibrillar ATPase.

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