caa a22 4500 475744969 CHVBK 20180406123510.0 cr unu---uuuuu 170329e20001001xx s 000 0 eng 10.1023/A:1005623002979 doi (NATIONALLICENCE)springer-10.1023/A:1005623002979 Coupling of creatine kinase to glycolytic enzymes at the sarcomeric I-band of skeletal muscle: a biochemical study in situ [Elektronische Daten] [Theresia Kraft, T. Hornemann, M. Stolz, V. Nier, T. Wallimann] 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. Kluwer Academic Publishers, 2000 Kraft Theresia Molekular- und Zellphysiologie, Medizinische Hochschule Hannover, D-30625, Hannover, Germany aut Hornemann T. Swiss Federal Institute of Technology, Institute of Cell Biology, ETH Zürich, Hönggerberg, CH-8093, Zürich, Switzerland aut Stolz M. Swiss Federal Institute of Technology, Institute of Cell Biology, ETH Zürich, Hönggerberg, CH-8093, Zürich, Switzerland aut Nier V. Molekular- und Zellphysiologie, Medizinische Hochschule Hannover, D-30625, Hannover, Germany aut Wallimann T. Swiss Federal Institute of Technology, Institute of Cell Biology, ETH Zürich, Hönggerberg, CH-8093, Zürich, Switzerland aut Journal of Muscle Research & Cell Motility Kluwer Academic Publishers 21/7(2000-10-01), 691-703 0142-4319 21:7<691 2000 21 10974 https://doi.org/10.1023/A:1005623002979 text/html Onlinezugriff via DOI 1 research-article jats NATIONALLICENCE 856 40 https://doi.org/10.1023/A:1005623002979 text/html Onlinezugriff via DOI NATIONALLICENCE 700 1- Kraft Theresia Molekular- und Zellphysiologie, Medizinische Hochschule Hannover, D-30625, Hannover, Germany aut NATIONALLICENCE 700 1- Hornemann T. Swiss Federal Institute of Technology, Institute of Cell Biology, ETH Zürich, Hönggerberg, CH-8093, Zürich, Switzerland aut NATIONALLICENCE 700 1- Stolz M. Swiss Federal Institute of Technology, Institute of Cell Biology, ETH Zürich, Hönggerberg, CH-8093, Zürich, Switzerland aut NATIONALLICENCE 700 1- Nier V. Molekular- und Zellphysiologie, Medizinische Hochschule Hannover, D-30625, Hannover, Germany aut NATIONALLICENCE 700 1- Wallimann T. Swiss Federal Institute of Technology, Institute of Cell Biology, ETH Zürich, Hönggerberg, CH-8093, Zürich, Switzerland aut NATIONALLICENCE 773 0- Journal of Muscle Research & Cell Motility Kluwer Academic Publishers 21/7(2000-10-01), 691-703 0142-4319 21:7<691 2000 21 10974 Metadata rights reserved Springer special CC-BY-NC licence nationallicence BK010053 XK010053 XK010000 NATIONALLICENCE NATIONALLICENCE NL-springer