RECOMBINANT CREATINE-KINASE PROTEINS AND PROPOSED STANDARDS FOR CREATINE-KINASE ISOENZYME AND SUBFORM ASSAYS

We developed standards for creatine kinase (CK; EC 2.7.3.2) assays by expressing human CK cDNAs in COS cells. Cells were transiently transfe cted with full-length cDNAs for CK subunits M and B, individually and in combination; and subsequently, high concentrations of CK activity w ere present in the cell lysate (1.2 U/mg protein). These proteins exhi bited the characteristic isoenzyme-specific electrophoretic mobilities for CK MM and BB isoenzymes. We also produced subforms of CK MM and M B, identical to the modified CK variants produced in plasma after musc le or myocardial injury, by mutating the cDNA for the CK M subunit to delete the carboxy-terminal lysine residue. When this construct was co transfected with the normal cDNAs for CK M and B, five electrophoretic ally distinct CK isoenzymes were detected by nondenaturing electrophor esis: MM3, MM2, MM1, MB2, and MB1. These proteins retained 100% of the ir activity after storage of the cell lysates -20 or 4-degrees-C for 3 months.