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Isolation purification and characterization of creatine kinase from bovine cardiac muscle



Isolation purification and characterization of creatine kinase from bovine cardiac muscle



Biochimica et Biophysica Acta 534(1): 38-47



The isolation, and molecular and enzymatic characterization of a homogeneous prpearation of creatine kinase from bovine cardiac muscle is reported, using a low ionic strength extraction of a muscle mince followed by 2 DEAE-cellulose chromatography steps, a procedure designed to release the enzyme bound to the myofibril. The resulting protein, with a specific activity of 55-60 units/mg, was characterized chemically and enzymatically. Its MW deduced by low speed sedimentation equilibrium in a native solvent was 80,000 .+-. 4000, whereas in a denaturing medium of 6 M guanidine hydrochloride, the molecule dissociated into 2 hydrodynamically equivalent polypeptide chains, each of MW 40,000 .+-. 2000. On sodium dodecyl sulfate polyacrylamide gels, the protein moved as a doublet pattern of average MW 40,000, reflecting the presence of 2 subunits of similar but slightly different mass, with the lighter component being present in larger amounts. A similar phenomenon was reported for homogeneous skeletal muscle and chicken breast muscle creatine kinase preparations, isolated by a similar procedure, but here the heavier band dominates, and this is reflected in a slightly higher average MW for the doublet band in these cases (44,000-48,000). The amino acid compositions of the bovine cardiac enzyme is very similar to its skeletal muscle homologue, as are the Km values for the 2 enzymes, with respect to the substrates, creatine phosphate and adenosine diphosphate. Both the cardiac and skeletal enzymes possess appreciable amounts of .beta.-structure, as revealed by circular dichroism measurements, although they differ in their respective absolute contents of apparent .alpha.-helix and .beta.-form. The bovine cardiac enzyme, like its skeletal counterpart, inhibits the ATPase activity of its parent myosin and its enzymatically active subfragments, in decreasing order as one proceeds from myosin through to heavy meromyosin and to subfragment I.

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Accession: 005773119

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PMID: 148914

DOI: 10.1016/0005-2795(78)90473-7


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