Calcium-binding and calcium-uptake by cardiac microsomes: A kinetic analysis

Abstract

Cardiac microsomes, which contain membrane fragments derived from the sarcoplasmic reticulum, can remove Ca 2+ from solution by two kinetically dissimilar mechanisms. Ca-binding, which occurs in the presence of ATP, accounts for the rapid sequestration of all smamounts of Ca 2+. Ca-uptake, which is seen when a calcium-precipitating agent such as oxalate is included, permits much larger amounts of Ca 2+ to be sequestered, though more slowly. The [Ca 2+] dependence of these two processes was examined in the range of [Ca 2+] between 1 × 10 −7 m to 1 × 10 −5 m. Ca-binding of microsomes prepared in sucrose (suc-microsomes) and in dilute buffers (H 2O-microsomes) exhibited saturation kinetics. The number of sites ( n) was approximately 13 nmol per mg protein for suc-microsomes and 45 nmol per mg protein for H 2O-microsomes. The affinity constant for suc-microsomes was approximately 1.2 × 10 6 m −1 (half saturation at [Ca 2+] = 8 ¢ 10 −7 m-Ca 2+) and approximately 2 × 10 6 m −1 (half saturation at [Ca 2+] = 5 × 10 −7 m) for H 2O-microsomes. Ca-uptake did not show saturation kinetics. The rate of Ca-uptake (approximately 0.04 μmol/min/mg protein per μ m Ca 2+ for both types of microsomes) increased linearly with increasing [Ca 2+] within this range of [Ca 2+]. Ca-binding is thus shown kinetically to involve a finite number of Ca-transport sites, whereas Ca-uptake, from a kinetic standpoint, resembles a non-facilitated diffusion process, possibly driven by an electrochemical gradient across the vesicular membrane that results from ATP hydrolysis. The rate limiting step in Ca-uptake does not involve the high affinity sites defined in the studies of Ca-binding.