Phosphorylation of the 100 000 M_r Ca²⁺‐transport ATPase by Ca²⁺ or cyclic AMP‐dependent and ‐independent protein kinases

Surface plasmon resonance studies prove the interaction of skeletal muscle sarcoplasmic reticular Ca2+ release channel/ryanodine receptor with calsequestrin

“…The existence of an endogenous phosphorylation system in the SR lumen has already been reported before (for review [13]): we described ¢rst [14] that calsequestrin can be phosphorylated and the isolated calsequestrin from rabbit skeletal muscle can be obtained in fully or partly phosphorylated forms [15]. Furthermore, two luminally located glycoproteins, sarcalumenin and the histidine-rich Ca 2 binding protein [16^19], can also be phosphorylated, thereby modulating the RyR activity [19,20].…”

“…Calsequestrin was isolated from the protein^glycogen complex as described previously [14,23]. Phosphorylation of calsequestrin by casein kinase II was carried out as reported before [12].…”

Activation of sarcoplasmic reticular Ca2+ transport ATPase by phosphorylation of an associated phosphatidylinositol.

“…The amount of alkylphosphate formed on the isolated Ca2+ transport ATPase is a function of the free Ca2+ concentration; in the presence of micromolar concentrations of Ca2+ it is 2to 3-fold lower than at nanomolar concentrations. This relationship was also observed in the intact SR vesicles (Varsanyi and Heilmeyer, 1981). Previously, we employed phosphorylase kinase, which has been shown to be 1545 Experimental conditions for the assay of both Ca2 + transport ATPase activities were the same as described in Materials and methods.…”

“…In addition to the well known acylphosphate, alkylphosphate is formed on the Ca2 + transport ATPase of fast skeletal muscle sarcoplamsic reticulum (SR) when the enzyme is incubated with ATP/Mg2+; however, this alkylphosphate formation can be observed only under special conditions, namely at high protein and high kinase concentrations (Varsanyi and Heilmeyer, 1981;cf., Varsanyi and Heilmeyer, 1979). By gel electrophoresis in the presence of SDS, the aklylphosphate can be shown to be present on the 100 000 mol.…”

“…Phosphorylase kinase can stimulate Ca2 + transport ATPase activity and Ca2+ uptake into vesicles of fragmented SR . Furthermore, phosphorylase kinase enhances both the steady-state level and the rate of alkylphosphate formation in SR vesicles which concomitantly reduces the steady-state level of the catalytic intermediate, the aspartyl phosphate (Varsanyi and Heilmeyer, 1981). Thus, changes in the activity of the Ca2+ transport ATPase upon phosphorylation could be expected.…”