Kraft T, Hornemann T, Stolz M, Nier V, Wallimann T
Swiss Federal Institute of Technology, Institute of Cell Biology, ETH Zürich.
J Muscle Res Cell Motil. 2000;21(7):691-703. doi: 10.1023/a:1005623002979.
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.
通过在原位用过量的荧光标记肌型肌酸激酶（MM-CK）替换内源性MM-CK，利用化学去表皮骨骼肌纤维和共聚焦显微镜，证实了肌型肌酸激酶（MM-CK）与肌原纤维M线的特异性相互作用。如先前通过完整肌肉冰冻切片的免疫荧光染色所示，在去表皮纤维的I带未观察到标记的MM-CK结合，而该酶在体内也位于此处。然而，当罗丹明标记的MM-CK扩散到预先与磷酸果糖激酶（PFK，一种已知与肌动蛋白结合的糖酵解酶）预孵育的去表皮纤维中时，发现Rh-MM-CK与I带存在显著的类似体内的相互作用，推测是由Rh-MM-CK与糖酵解酶的结合介导的。醛缩酶，另一种与肌动蛋白结合的糖酵解酶，也能够使Rh-MM-CK结合到I带，但复合物的形成优选在长肌节长度（>3.0微米）时发生。丙酮酸激酶虽然已知能与肌动蛋白结合，但磷酸甘油酸激酶（PGK）不直接与I带本身相互作用，它们都没有介导MM-CK靶向I带。通过PFK将MM-CK锚定到I带，但不是通过醛缩酶，强烈依赖于pH值，且在pH 7.0以下发生。在不同肌节长度进行的标记表明，PFK/MM-CK复合物结合到I带的细肌丝上，但不在肌动球蛋白重叠区域内。关于MM-CK与PFK在I带的结构相互作用的生理后果，从MM-CK与糖酵解的功能偶联、代谢调节和多酶复合物中的通道作用方面进行了讨论。本文所述的去表皮骨骼肌纤维原位结合测定法是在各种精确控制条件下，对结构完整的收缩系统中特定蛋白质-蛋白质相互作用进行进一步研究的有用方法。
