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Creatine phosphokinase: in vitro activity modification and proteolysis with calpain Arthur, Gavin D.


The purpose of this study was to determine if the muscle isoform of the energy transmitting kinase; Creatine Phosphokinase (CPK - EC N° MM-isozyme, is a substrate for calcium activated neutral protease (CANP - EC N° CPK activity was measured under three different conditions: (1) control assay; (2) with 5mM Ca²⁺,(5x10⁻³Ca²⁺j; (3) with 5mM Ca²⁺ and a range of CANP amounts from 10 to lOOug. 5mM Ca²⁺ consistently caused significant inhibition of the CPK activity to 36% of control (p <0.05). In the presence of 5mM Ca²⁺ and lOug of CANP, CPK activity was not significantly different from the control activity. With 27ug CANP there was a slight but significant activation of CPK to 123.18 + 12.9% above the control activity (p <0.05). As the amount of CANP present was increased to 54, 67, 84 and lOOug, the CPK activity was reduced to 56.96 + 0,31%, 50.46 + 2.65%, 36.06 + 0.5%, and 2.08 + 2.56% respectively. SDS-PAGE showed that significant proteolysis of CPK occurred with a range of CANP from 10 to 3Oug. Densitometric scanning of the CPK band and the 28kDa CANP subunit showed that proteolysis of CPK was dependent on the amount of CANP present. The proteolysis of CPK resulted in the formation of two large fragments. The molecular weight of these proteolytic fragments were estimated to be 38 and 35kDa. The results of this study show that CPK is a substrate for CANP in vitro and that minor proteolysis results in activation of CPK, while increased proteolysis results in loss of activity.

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