The specific interaction of muscle type creatine-kinase (MM-CK) with the myofibrillar M-line was demonstrated by exchanging endogenous MM-CK with an excess of fluorescently labeled MM-CK in situ , using chemically skinned skeletal muscle fibers and confocal microscopy. No binding of labeled MM-CK was noticed at the I-band of skinned fibers, where the enzyme is additionally located in vivo , as shown earlier by immunofluorescence staining of cryosections of intact muscle. However, when rhodamine-labeled MM-CK was diffused into skinned fibers that had been preincubated with phosphofructokinase (PFK), a glycolytic enzyme known to bind to actin, a striking in vivo -like interaction of Rh-MM-CK with the I-band was found, presumably mediated by binding of Rh-MM-CK to the glycolytic enzyme. Aldolase, another actin-binding glycolytic enzyme was also able to bind Rh-MM-CK to the I-band, but formation of the complex occurred preferably at long sarcomere length (>3.0 μm). Neither pyruvate kinase, although known for its binding to actin, nor phosphoglycerate kinase (PGK), not directly interacting with the I-band itself, did mediate I-band targeting of MM-CK. Anchoring of MM-CK to the I-band via PFK, but not so via aldolase, was strongly pH-dependent and occurred below pH 7.0. Labeling performed at different sarcomere length indicated that the PFK/MM-CK complex bound to thin filaments of the I-band, but not within the actomyosin overlap zones. The physiological consequences of the structural interaction of MM-CK with PFK at the I-band is discussed with respect to functional coupling of MM-CK to glycolysis, metabolic regulation and channeling in multi-enzyme complexes. The in situ binding assay with skinned skeletal muscle fibers described here represents a useful method for further studies of specific protein–protein interactions in a structurally intact contractile system under various precisely controlled conditions.

The ratio of mutant to wildtype myosin heavy chain (beta-isoform, beta-MHC) in the soleus muscle of patients with familial hypertrophic cardiomyopathy was determined by a combination of HPLC, mass spectrometry and capillary zone electrophoresis. In two patients, one with a Val 606 Met mutation and another with a Gly 584 Arg mutation, the fraction of mutant beta-MHC was only 12+/-6% and 23+/-0.7% of total beta-MHC, respectively. These results demonstrate the necessity to determine the ratio of mutant to wildtype protein for the interpretation of functional studies on biopsy material from heterozygous patients with an inherited disease.