The mechanism of Ca2+ transport by a Ca2+ pump

Duggleby, Richard Charles (1998) The mechanism of Ca2+ transport by a Ca2+ pump. University of Southampton, Doctoral Thesis.

Record type: Thesis (Doctoral)

Abstract

Skeletal muscle sarcoplasmic reticulum Ca²⁺-ATPase pumps two Ca²⁺ ions across the membrane of the sarcoplasmic reticulum for each molecule of ATP hydrolysed. Recent experiments suggest that transport of Ca²⁺ involves transfer to two bound Ca²⁺ ions from a pair of binding sites exposed to the cytoplasm to a pair of sites exposed to the lumen. The nature of this transfer process was studies using rapid kinetic measurements of phosphorylation-induced release of Ca²⁺. It was found that at alkaline pH values, the rate of release of Ca²⁺ from the phosphorylated ATPase decreases with increasing Mg²⁺ concentration and that at high concentrations of Mg²⁺ release of Ca²⁺ is biphasic. The results are consistent with sequential release of Ca²⁺ from the phosphorylated ATPase. The effect of Mg²⁺ on the rate of release of the first Ca²⁺ could follow from binding to a gating site known to affect the binding of Ca²⁺ to the cytoplasmic sites. To study the order in which Ca²⁺ is transferred between the cytoplasmic and lumenal sites, use was made of the observation that modification of the ATPase with N-ethylmaleimide (NEM) slows the rate of release of Ca²⁺. It was found that the two bound Ca²⁺ ions become randomized during the transfer process. Using fluorescence studies it was found that NEM modification of the ATPase does not affect Ca²⁺ binding to the cytoplasmic sites or phosphorylation of the ATPase by ATP. NEM was found to modify a number of cysteine residues on the ATPase. Modification of the ATPase by NEM in the presence of 4-(bromomethyl)-6,7-dimethoxycoumarin (Br-DMC), a fluorescent probe known to label Cys-344, was also studied. It was found that one of the cysteine residues on the ATPase modified by NEM is Cys-344 and this prevents Br-DMC labelling at this site.