Myofibrillar interaction of cytosolic creatine kinase (CK) isoenzymes: allocation of N-terminal binding epitope in MM-CK and BB-CK.
The molecular origin of the isoenzyme-specific interaction of cytosolic creatine kinase isoenzymes, muscle-type creatine kinase and brain-type creatine kinase, with myofibrillar structures has been studied by confocal microscopy in an functional in situ binding assay with chemically skinned, unfixed skeletal muscle fibers using wild-type and chimeric creatine kinase isoproteins. The specific interaction of both wild-type isoforms with the sarcomeric structure resulted in a stable, isoform-characteristic labeling pattern with muscle-type creatine kinase bound exclusively and tightly to the sarcomeric M-band while brain-type creatine kinase was confined to the I-band region. Chimeric proteins of both muscle-type and brain-type creatine kinases were constructed to localize the corresponding binding domain(s). Exchanged domains included the N-terminal part (residues 1-234), the region containing an isoenzyme 'diagnostic box' (residues 235-285) and the C-terminal part (residues 286-380). The purified recombinant proteins were all fully intact and enzymatically active. All chimeric proteins containing the N-terminal region (amino acid 1-234) of muscle-type or brain-type creatine kinase were always specifically targeted to the sarcomeric M-band or I-band, respectively. We therefore propose that the relevant epitope(s), determining the isoenzyme-specific targeting in skeletal muscle, are entirely located within the N-terminal regions of both cytosolic creatine kinase isoforms.
Stolz
M
M
Swiss Federal Institute of Technology, Institute of Cell Biology, Zürich, Switzerland. stolz@cell.biol.ethz.ch
Wallimann
T
T
D000818:Animals; D001921:Brain / Q000201:enzymology; D002645:Chickens; D003402:Creatine Kinase / Q000737:chemistry / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D004734:Energy Metabolism; D000939:Epitopes / Q000276:immunology; D007527:Isoenzymes; D008958:Models, Molecular; D009132:Muscles / Q000201:enzymology; D016297:Mutagenesis, Site-Directed; D009210:Myofibrils / Q000201:enzymology*; D009928:Organ Specificity; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D012518:Sarcomeres / Q000378:metabolism* / Q000648:ultrastructure
D000939:Epitopes; D007527:Isoenzymes; D011993:Recombinant Fusion Proteins; D003402:Creatine Kinase