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Creatine Kinase Reaction in
Skinned Rat Psoas Muscle Fibers and Their Myofibrils
M. GREGOR, J. MEJSNAR, A.
JANOVSKÁ, J. ŽURMANOVÁ, O. BENADA1, B. MEJSNAROVÁ2
Department of Physiology and Developmental
Biology, Faculty of Science, Charles University, 1Institute
of Microbiology, Czech Academy of Sciences and 2Cytolab
Ltd., Prague, Czech Republic
Received September 1, 1998
Accepted October 29, 1998
Summary
The aim of this study was to evaluate myofibrillar creatine
kinase (EC 2.7.3.2) activity on the background of the effect of
substrate channeling by myosin ATPase and to compare it with
creatine kinase (CK) activity of whole skinned fibers. In order
to assess CK activity, skinned fibers were prepared from the rat
psoas major muscles defined by light microscopy. The activity in
permeabilized fibers after treatment with saponin, Triton X-100
and Ca2+-free medium reached 2.80, 6.97 and 3.32 m mol ATP min-1
mg-1 protein, respectively, when a coupled enzyme assay system
with external hexokinase and glucose-6-phosphate dehydrogenase
was used. Transmission electron microscopy (TEM) revealed a
possible interference among activities of sarcolemmal,
sarcoplasmic, myofibrillar and mitochondrial CK from persisting
structures. For evaluation of the myofibrillar CK itself, a pure
myofibrillar fraction was prepared. Fraction purity was
confirmed by TEM and by enzymatic assays for marker enzymes. Two
procedures, i.e. the coupled enzyme assay and the evaluation of
phosphocreatine (Pcr) concentration before and after the CK
reaction, were used for measurement of CK activity in this
fraction. The procedures resulted in 3.2 nmol ATP min-1 mg-1
protein and 7.6 nmol PCr min-1 mg-1 protein, respectively. These
alternative approaches revealed a discrepancy between the
reacting portions of Pcr by more than 50 % , which provides
information about the size of the effect, generally described as
substrate channeling.
Key words
Muscle - Skinned fibers - Myofibrils - Creatine kinase -
Substrate channeling
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