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

Human myocardial creatine kinase isoenzyme MM is present as a single form in tissue, but upon its release into plasma two additional forms, with faster anodal migration, are apparent on polyacrylamide electrophoresis. We designate the three forms as MM3, MM2, and MM1 in increasing order of anodal mobility. When tissue creatine kinase isoenzyme MM (MM3) is incubated with either carboxypeptidase N or carboxypeptidase B it is converted into the two additional forms, MM2 and MM1. The carboxy terminal amino acid of human, canine, and rabbit tissue MM3 was determined to be lysine, a specific substrate for carboxypeptidases N and B. Evidently the mechanism for the production of multiple forms of creatine MM in human plasma is the hydrolysis of a positively charged C-terminal lysine residue from one M subunit (MM2), followed by hydrolysis of the C-terminal lysine from the other subunit (MM1).

The subforms of creatine kinase (CK; EC 2.7.3.2) in plasma have received recent attention as potential markers for the early diagnosis of acute myocardial infarction. Because changes in CK-MM subforms are not specific for myocardial injury, we developed an assay, based on high-voltage electrophoresis, that is sufficiently sensitive to detect the CK-MB subforms at concentrations substantially below the upper limit of normal (14 U/L). The assay can detect 1.25 U of either MB subform per liter with a precision of 0.20 U/L and gives responses that vary linearly with activity concentration from 0.0 through 30.0 U/L, with an identical signal response for both subforms. When both subforms are present in a serum sample, the assay accurately measures both the relative percentage and the absolute quantity of each: assay activity/known activity was 1.03 for each subform at a total MB subform activity of 5.0 U/L (r = 0.98). Assay time is 25 min, and there is no loss of CK during electrophoresis. Thus, this system can be used to rapidly, sensitively, and precisely quantify the two CK-MB subforms at activities well within the normal reference interval.