Abstrakt: |
The dependence of phosphorylation on the total calmodulin concentration at 75 μM and 1 μM free calcium 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 calcium transport carried out at 1 μM calcium, but phosphorylated prior to the calcium uptake at 75 μM or 1 μM calcium, were 12.5 nM and 127 nM, respectively. A positive correlation was obtained between calmodulin-dependent increase in the rate of calcium transport and hydroxylamine-insensitive phosphoester formed by the calcium/calmodulin-regulated, membrane-bound protein kinase. More than 90% of incorporated [32P]phosphate is confined to a 26–28-kDa or 9–11-kDa protein as determined by polyacrylamide gel electrophosresis following solubilization in sodim dodecyl sulfate 37°C and 100°C, respectively, similar to the results obtained by phosphorylation with cAMP-dependent protein kinase. The data indicate that calmodulin-dependent phosphorylation of the above protein(s) is causally related to the stimulation of the rate of calcium transport by cardiac sarcoplasmic reticulum, which is at least partially due to a shift in the calcium dependence of the rate of calcium transport to lowe free calcium concentrations, K(Ca), of 1.25 μM and 0.61 μM in controls and calmodulin-dependent phosphorylation, respectively. Activation of calmodulin-dependent phsophorylation by free calcium at total calmodulin concentrations of 300 nM, 100 nM and 30 nM gave apparent K(Ca) values of 0.83 μM, 1.44 μM and 2.3 μM and Hill coefficients of 4.13, 3.76 and 3.79, respectively, indicating that all four calcium binding sites of calmodulin have to be saturated to obtain activation of the calcium/calmodulin-regulated protein kinase. The calmodulin-dependent modulation of calcium transport in vivo is, therefore, determined to great extent by the total calmodulin concentration present in the sarcoplasm. [ABSTRACT FROM AUTHOR] |