The isoenzyme-diagnostic regions of muscle-type creatine kinase, the M-260 and M-300 box, are not responsible for its binding to the myofibrillar M-band.

Muscle-type creatine kinase is known for its unique interaction with the myofibrillar M-band, but the molecular origin for this structural relationship is not well understood. A systematic sequence comparison between the highly homologous cytosolic isoforms, muscle-type and brain-type creatine kinase, yielded two isoenzyme-specific regions in the muscle-type creatine kinases, the M-260 box (residues 258-270) and the M-300 box (residues 300-315). These particular regions were conspicuous for the specific interaction of this CK isoenzyme, but not of brain-type creatine kinase, with the sarcomeric M-band. In situ diffusion assays with fluorescently labeled native, as well as mutated muscle-type creatine kinase variants, were used to study by laser confocal microscopy their association with the M-band of chemically skinned muscle fibers. Neither a set of charge mutants of the M-260 box and/or the M-300 box nor a hybrid construct of both isoforms with the entire C-terminal region derived from the brain-type isoform showed any significant alteration in the in situ M-band-binding properties when compared to the wild-type form of muscle-type creatine kinase. This indicates that in the intact protein of muscle type creatine kinase, these C-terminal isoenzyme-specific regions are not important for M-band interaction and that the actual M-band interaction domain(s) lay mostly within the N-terminal half of the molecule. The highly conserved motives (M-260 box and M-300 box) may serve an isoenzyme-specific purpose yet to be identified.