Calcium and phosphate transport in sarcoplasmic reticulum

Stefanova, Helena Ivanova (1989) Calcium and phosphate transport in sarcoplasmic reticulum. University of Southampton, Doctoral Thesis.

Abstract

Proteins involved in transport of Ca²⁺ and P_i (and other anions) in skeletal muscle sarcoplasmic reticulum have been studied. Steady-state kinetic measurements have been used to characterize the dependence of the activity of the (Ca²⁺-Mg²⁺)-ATPase on the concentrations of ATP, Ca²⁺, Mg²⁺, anions and cations. The dependence of ATPase activity on ARTP concentration was found to be complex and was attributed to both catalytic and regulatory effects of ATP. The higher activity of the ATPase observed at high concentrations of ATP was attributed to an increase in the rate of E1'PCa₂ - E2'P transition caused by binding of MgATP. Concentrations of Mg²⁺ greater than those needed to convert all ATP into MgATP were found to be inhibitory, suggesting that binding of Mg²⁺ and ATP might be competitive. The effects of anions and cations on ATPase activity were shown not to follow from simple ionic strength effects, but were attributed to anion and cation binding to the ATPase. The inhibitory effects of anions were found to be in the sequence SCN^{- >} I^{- >} C1^{- >} SO₄^2-> CH₃SO₃^-. Covalent modification of SR vesicles with phenylglyoxal or acetic anhydride were found to decrease accumulation of Ca²⁺. These effects were attributed both to modification of the ATPase and to modification of a P_i transporter in the SR membrane. Higher levels of Ca²⁺ uptake in the presence of P_i, PP_i and oxalate suggested the existence of a distinct anion transporter in the SR membrane. The phosphonocarboxylic acids, 3-phosphonopropionic acid, phenylphosphonic acid and phosphonoacetic acid were found to act as specific competitive inhibitors of P_i and oxalate transport, suggesting they bind at the anion binding site, and that P_i and oxalate are transported on the same transporter. Transport of P_i and oxalate only occurs during active transport of Ca²⁺ and appears to be stimulated by ATP. Addition of phosphonoformic acid increases Ca²⁺ accumulation, so that it is probably transported like P_i. Maleate and succinate are probably transported by a separate transporter since their translocation was not affected by the presence of the phosphonocarboxylic acids. Pretreatment of SR vesicles or purified ATPase with acetic anhydride or phenylglyoxal resulted in inhibition of Ca²⁺ uptake or ATPase activity. ATP partially protected ATPase activity against inhibition. High concentrations of P_i protected the anion transporter against modification. ATP was also found to protect the anion transporter against modification, suggesting an ATP binding site on the anion transporter which could also be modified by acetic anhydride or phenylglyoxal. Acetic anhydride was found to inhibit Ca²⁺ accumulation by vesicles reconstituted with purified ATPase when the enzyme was modified before being reconstituted into phospholipid bilayers containing a high proportion of phosphatidylethanolamine. Modification after reconstitution into vesicles containing high proportion of phosphatidylethanolamine did not affect Ca²⁺ uptake, because acetic anhydride reacted preferentially with the phosphatidylethanolamine.

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