Correlation between calmodulin‐dependent increase in the rate of calcium transport and calmodulin‐dependent phosphorylation of cardiac sarcoplasmic reticulum

Abstract

A correlation between the calmodulin-dependent increase in the rate of Ca transport by dog cardiac sarcoplasmic reticulum and calmodulin-dependent phosphorylation was investigated. The dependence of phosphorylation on the total calmodulin concentration at 75 .mu.M and 1 .mu.M free Ca gave apparent calmodulin half-saturation constants Km (CaM) of 9.4 nM and 181 nM, respectively, whilst the apparent Km (CaM) for the rate of calmodulin-stimulated Ca transport carried out at 1 .mu.M Ca, but phosphorylated prior to the Ca uptake at 75 .mu.M or 1 .mu.M Ca were 12.5 nM and 127 nM, respectively. A positive correlation was obtained between calmodulin-dependent increase in the rate of Ca transport and hydroxylamine-insensitive phosphoester formed by the Ca/calmodulin-regulated, membrane-bound protein kinase. More than 90% of incorporated [32P]phosphate is contined to a 26-28-kDa [kilodalton] or 9-11-kDa as determined by polyacrylamide gel electrophoresis following solubilization in sodium dodecyl sulfate at 37.degree. C and at 100.degree. C, respectively, similar to the results obtained by phosphorylation with cAMP-dependent protein kinase. Calmodulin-dependent phosphorylation of the above protein(s) apparently is causally related to the stimulation of the rate of Ca transport by cardiac sarcoplasmic reticulum, which is at least partially due to a shift in the Ca dependence of the rate of Ca transport to lower free Ca concentrations, K(Ca), of 1.25 .mu.M and 0.61 .mu.M in controls and calmodulin-dependent phosphorylation, respectively. Activation of calmodulin-dependent phosphorylation by free Ca at total calmodulin concentrations of 300 nM, 100 nM and 30 nM gave apparent K(Ca) values of 0.83 .mu.M, 1.44 .mu.M and 2.3 .mu.M and Hill coefficients of 4.13, 3.76 and 3.79, respectively, indicating that all 4 Ca binding sites of calmodulin have to be saturated to obtain activation of the Ca/calmodulin-regulated protein kinase. The calmodulin-dependent modulation of Ca transport in vivo is, therefore, determined to great extent by the total calmodulin concentration present in the sarcoplasm.