Characterization of isoforms of human mitochondrial creatine kinase by isoelectric focusing.
High enzyme activity of mitochondrial creatine kinase (creatine-N-phosphotransferase, mCK, EC 2.7.3.2) was detected in serum from a patient with advanced carcinoma of the rectum and its isoforms were characterized by means of isoelectric focusing (IEF). Three forms of mCK, membrane-bound (pI 6.9-7.0), octameric (pI 7.0-7.9) and dimeric (pI 6.7, 6.8, 6.9 and 7.0), were detected in the fresh serum. These three forms of mCK were converted to five dimeric isoforms, and these were characterized as one reduced form (pI 7.0) and four oxidized (pI 6.6, 6.7, 6.8 and 6.9) forms upon treatment with urea, hydrogen peroxide or 2-mercaptoethanol (2-ME). The C-terminal of the mCKs was concluded to be a lysine residue because the mCKs treated with carboxypeptidase B migrated to positions closer to the anode than did those not treated with carboxypeptidase B. Therefore, four bands were concluded to represent one reduced-delysined isoform (pI 6.4) and three oxidized-delysined isoforms (pI 6.1, 6.2 and 6.3). The broad octameric mCK band disappeared and a narrow band focused at pI 6.8-6.9 appeared upon probable delysination of the mCKs. Thus, the number of lysine residues at the C-terminal of the octamer was concluded to be variable due to variable catalysis by carboxypeptidase N in the plasma. mCKs seemed to be inactivated during conversion from a membrane-bound form to dimeric oxidized-delysined forms via the octameric, dimeric reduced and oxidized forms.
Clinical Laboratories, Kurashiki Central Hospital, Japan.
F
F
Kanemitsu
S
S
Kira
D000230:Adenocarcinoma / Q000201:enzymology; D003402:Creatine Kinase / Q000097:blood / Q000737:chemistry* / Q000302:isolation & purification; D006801:Humans; D007525:Isoelectric Focusing; D007527:Isoenzymes; D008297:Male; D008875:Middle Aged; D008928:Mitochondria / Q000201:enzymology*; D010084:Oxidation-Reduction; D012004:Rectal Neoplasms / Q000201:enzymology
D007527:Isoenzymes; D003402:Creatine Kinase