EFFECTS OF ADENINE-NUCLEOTIDES ON CALCIUM-BINDING BY RAT-HEART SARCOPLASMIC-RETICULUM

Summary: The purpose of the present study was to investigate the interactions between ATP, ADP and calcium binding by rat heart sarcoplasmic reticulum vesicles (SR), and to re-evaluate the assay method used to study calcium binding. Calcium binding or transport was studied by the Millipore filtration method. Rat heart SR has an unusually high Mg2+ stimulated ATPase activity (1.37±0.16 μmol Pi per min per mg at 25°C) so that previous incubation with ATP in calcium binding studies releases ADP and Pi. By maintaining ATP at high and ADP at low concentrations with an ATP- regenerating system (phosphoenolpyruvate and pyruvate kinase), calcium binding capacity was increased by two to three times that of a non-ATP-regenerating system and there was a direct relationship between the amount of Ca-binding and SR protein concentration. When Ca2+ and Mg2+concentrations were controlled and ATP and ADP concentrations were varied independently the initial rate of Ca-binding was inhibited 25% by 1 mmol·litre-1 ADP and 48% by 3mmol·litre-1 ADP. ATP limited the initial rate of Ca-binding only at ATP levels below 2mmol·litre-1. At low ATP concentrations Ca-release was observed. However, in the presence of an ATP-regenerating system no Ca-release was observed, even at low concentrations of ATP.This study shows that ADP is an inhibitor of Ca-binding by rat heart SR. However, the possibility that high ADP concentrations in the presence of Pi from ATP hydrolysis, could facilitate calcium release cannot be excluded. In addition to the possible physiological importance, these effects must be regarded when assaying rat cardiac SR calcium binding.