Coupling of creatine kinase to glycolytic enzymes at the sarcomeric I-band of skeletal muscle: a biochemical study in situ.
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 microm). 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.
Swiss Federal Institute of Technology, Institute of Cell Biology, ETH Zürich. Kraft.Theresia@mh-hannover.de
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Kraft
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Hornemann
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Stolz
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Nier
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Wallimann
D000818:Animals; D003402:Creatine Kinase / Q000378:metabolism* / Q000648:ultrastructure; D005634:Fructose-Bisphosphate Aldolase; D018613:Microscopy, Confocal; D018482:Muscle, Skeletal / Q000378:metabolism* / Q000648:ultrastructure; D010732:Phosphofructokinase-1; D010735:Phosphoglycerate Kinase; D011770:Pyruvate Kinase; D011817:Rabbits; D012518:Sarcomeres / Q000378:metabolism / Q000648:ultrastructure
D010732:Phosphofructokinase-1; D011770:Pyruvate Kinase; D010735:Phosphoglycerate Kinase; D003402:Creatine Kinase; D005634:Fructose-Bisphosphate Aldolase