Coupling of creatine kinase to glycolytic enzymes at the sarcomeric I-bandof skeletal muscle: a biochemical study in situ

The specific interaction of muscle type creatine-kinase (MM-CK) with the my ofibrillar M-line was demonstrated by exchanging endogenous MM-CK with an e xcess of fluorescently labeled MM-CK in situ, using chemically skinned skel etal 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 cryosec tions of intact muscle. However, when rhodamine-labeled MM-CK was diffused into skinned fibers that had been preincubated with phosphofructokinase (PF K), a glycolytic enzyme known to bind to actin, a striking in vivo-like int eraction of Rh-MM-CK with the I-band was found, presumably mediated by bind ing of Rh-MM-CK to the glycolytic enzyme. Aldolase, another actin-binding g lycolytic enzyme was also able to bind Rh-MM-CK to the I-band, but formatio n of the complex occurred preferably at long sarcomere length (>3.0 mum). N either pyruvate kinase, although known for its binding to actin, nor phosph oglycerate kinase (PGK), not directly interacting with the I-band itself, d id 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 th e actomyosin overlap zones. The physiological consequences of the structura l 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 channe ling in multi-enzyme complexes. The in situ binding assay with skinned skel etal muscle fibers described here represents a useful method for further st udies of specific protein-protein interactions in a structurally intact con tractile system under various precisely controlled conditions.