pH-Induced changes in the reactions controlled by the low- and high-affinity calcium(2+)-binding sites in sarcoplasmic reticulum

Abstract

The effect of pH on the Ca2+-binding sites of high and low affinity, located, respectively, on the outer and inner surfaces of the sarcoplasmic reticulum membrane [rabbit skeletal muscle], was investigated using intact and leaky sarcoplasmic reticulum vesicles. With the use of intact vesicles, different pH profiles of membrane phosphorylation and rates of nucleoside triphosphate hydrolysis were obtained depending on the assay temperature, on the Ca2+ concentration, and on whether ATP or ITP was used as substrate. The different pH profiles were related to the amount of Ca2+ accumulated by the vesicles, i.e., to different degrees of saturation of the inner, low-affinity Ca2+-binding site. With the use of leaky vesicles, the saturation of the 2 Ca2+-binding sites was controled more precisely since the Ca2+ concentration on both sides of the membrane was equal to the Ca2+ concentration of the assay medium. Using leaky vesicles and measuring the rates of nucleotide hydrolysis, nucleotide-phosphate exchange and membrane phosphorylation by nucleotide as an indication of the degree of saturation of the Ca2+-binding sites, the affinity of both the high- and low-affinity sites increased 3-4 orders of magnitude when the pH of the assay medium was increased from 6.1-8.65.