Calmodulin-dependent elevation of calcium transport associated with calmodulin-dependent phosphorylation in cardiac sarcoplasmic reticulum

Abstract

The rate of calcium transport by sarcoplasmic reticulum vesicles from dog heart assayed at 25°C, pH 7.0, in the presence of oxalate and a low free Ca 2+ concentration (approx. 0.5 μM) was increased from 0.091 to 0.162 μmol·mg −1·min −1 with 100 nM calmodulin, when the calcium-, calmodulin-dependent phosphorylation was carried out prior to the determination of calcium uptake in the presence of a higher concentration of free Ca 2+ (preincubation with magnesium, ATP and 100 μM CaCl 2; approx. 75 μM free Ca 2+). Half-maximal activation of calcium uptake occurs under these conditions at 10–20 nM calmodulin. The rate of calcium-activated ATP hydrolysis by the Ca 2+-, Mg 2+-dependent transport ATPase of sarcoplasmic reticulum was increased by 100 nM calmodulin in parallel with the increase in calcium transport; calcium-independent ATP splitting was unaffected. The calcium-, calmodulin-dependent phosphorylation of sarcoplasmic reticulum, preincubated with approx. 75 μM Ca 2+ and assayed at approx. 10 μM Ca 2+ approaches maximally 3 nmol/mg protein, with a half-maximal activation at about 8 nM calmodulin; it is abolished by 0.5 mM trifluperazine ∗∗ ∗∗ 10-[3-(4-Methylpiperazin-1-yl)propyl]-2-trifluoromethyl-phenothiazine; trifluoperazine. . More than 90% of the incorporated [ 32P]phosphate is confined to a 9–11 kDa protein, which is also phosphorylated by the catalytic subunit of the cAMP-dependent protein kinase and most probably represents a subunit of phospholamban. The stimulatory effect of 100 nM calmodulin on the rate of calcium uptake assayed at 0.5 μM Ca 2+ was smaller following preincubation of sarcoplasmic reticulum vesicles with calmodulin in the presence of approx. 75 μM Ca 2+, but in the absence of ATP, and was associated with a significant degree of calmodulin-dependent phosphorylation. However, the stimulatory effect on calcium uptake and that on calmodulin-dependent phosphorylation were both absent after preincubation with calmodulin, without calcium and ATP, suggestive of a causal relationship between these processes.