| Summary: | The purpose of this study was to determine if the muscle isoform of the energy
transmitting kinase; Creatine Phosphokinase (CPK - EC N° 2.7.3.2) MM-isozyme, is a
substrate for calcium activated neutral protease (CANP - EC N° 3.4.22.17). 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. === Education, Faculty of === Kinesiology, School of === Graduate
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