Abstract
Ca2+ uptake and Ca2+-dependent ATP hydrolysis of fast skeletal muscle sarcoplasmic reticulum (SR) are strongly inhibited by trifluoperazine (TFP). Inhibition, which is Ca2+-dependent, is 90% with 14 microM TFP and 0.2 microM Ca2+. TFP interacts strongly, in a Ca2+-dependent way, with two SR proteins, calmodulin and the 53,000-dalton glycoprotein. The two proteins were purified by TFP affinity chromatography. The inhibition of SR activity by TFP was correlated with the interaction of the drug with the glycoprotein, rather than with calmodulin. The main effect was a shift of the (Ca2+-Mg2+)-ATPase from a high to a low affinity form. Calmodulin-dependent phosphorylation of three proteins (Mr = 57,000, 35,000, and 20,000) of the SR membrane of fast skeletal muscle was also demonstrated. Phosphorylation of these three proteins plays no role in the regulation of the active Ca2+-uptake reaction.
Highlights
Ca2+uptake and Ca2+-dependentATP hydrolysis of [7]
The presence of calmodulin has been demonstrated in several membrane systems
The investigation has beenextended to sarcoplasmic reticulum (SR) isolated frommammalian fast skeletal muscle
Summary
TFP and CAPP were generous g&s of Dr Carl Kaiser of Smith, The effects of CAMPand calmodulin on Ca2+transport in cardiac SR'are mediated by specific protein kinases that phosphorylate a minor hydrophobic protein component of the. 20-50 pg of SR protein/ml were incubated for 2 min at room temperature in the presence of 20 mM MOPS, 7.0,80 mM KCI, 5 mM MgC12, 0.5 mM ‘‘CaCl2, and EGTA to yield the desired free Ca” concentration. The basic reaction mixture contained 20 mM MOPS, pH 7.0, 80 mM KCI, 5 mM MgC12, and Ca-EGTA buffers. The phosphoenzyme intermediate steadystate level wasmeasured as previously described [15].Essentially, 100-150 pg of SR protein/ ml were incubated at 0 “C in 80 mM KCI, 5 mM MgC12,20 mM MOPS, pH 7.0, and Ca-EGTA buffers to yield the desired free Ca2+concentration. Brain calmodulin stimulation of the Ca”-dependent ATPase of erythrocyte ghosts was measured with the coupled enzyme assay as described above. Protein concentration was measured according to Lowry et al [21] using bovineserum albumin as a standard
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