Heterogeneity of Escherichia coli-expressed human muscle creatine kinase.
Creatine kinase (CK) plays an important role in maintaining a constant ATP:ADP ratio during periods of high energy usage. Elevated levels of CK give an early indication of myocardial infarction. The enzyme has four major isozymes with heterogeneity being observed for each of them. In many cases the source of the heterogeneity is unclear. However, some of the isoforms are known to result from exposure to serum proteases, and analysis of the plasma isoforms provides an estimate of the time of onset of the infarction. Somewhat surprisingly, isoelectric focusing (IEF) experiments provided evidence of heterogeneity in human muscle CK (HMCK) expressed in E. coli. To investigate this further, HMCK was purified to apparent homogeneity utilizing Blue Sepharose affinity chromatography and HiPrep Q anion exchange chromatography. Additional purification on a PBE 94 chromatofocusing column resulted in four fractions, three of which, HMCK I - III, were characterized. The three isoforms are all active and have similar kinetic parameters. They exhibited identical bands on SDS PAGE but different anodal mobility on non-denaturing gels. Modification of C-terminal and/or cysteine residues has been ruled out, and deamidation of asparagine or glutamine residue(s) is proposed to be the cause of isoform formation. In addition each of these isoforms showed a similar four-band pattern on a carrier ampholytes-based IEF gel. Two-dimensional IEF analysis showed that an equilibrium was established between the four bands, suggesting that the four components were unstable and generated only when the protein was subjected to IEF.
Wang
Pan-Fen
PF
College of Pharmacy, University of Michigan, Ann Arbor, Michigan 48109, USA.
Kenyon
George L
GL
McLeish
Michael J
MJ
D003001:Cloning, Molecular; D052283:Creatine Kinase, MM Form / Q000235:genetics* / Q000502:physiology; D004926:Escherichia coli; D014644:Genetic Variation; D006801:Humans; D007527:Isoenzymes / Q000235:genetics
D007527:Isoenzymes; D052283:Creatine Kinase, MM Form