Untitled

Abstract

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.

The Generation of the Oxidized Form of Creatine Kinase Is a Negative Regulation on Muscle Creatine Kinase

“…Domains 7-8 of myomesin play a crucial role in the interaction with CK, and it has also been demonstrated that this interaction is not strictly species-specific (8,11,31). Since there is no published cDNA sequence of rabbit muscle myomesin, human myomesin fragment (hMy7-8) was cloned, expressed and purified to test the binding affinity of R-CK and O-CK with M-line protein.…”

The regulation of the interaction between F-actin and muscle fructose 1,6-bisphosphatase

“…Preparation of skeletal muscle fibres from rat psoas muscle and chemical permeabilization of the membranes were carried out according to the method described by Kraft et al [16]. The skinned skeletal muscle fibres were mounted on microscopic slides (in chambers made with PAP PEN, without covering the chambers) and incubated overnight at 4 • C in a drop (100 L) of the relaxing solution (10 mM imidazole, 2 mM magnesium chloride, 1 mM EGTA, 1 mM ATP, 20 mM creatine phosphate, 2 mM dithiothreitol, 106 mM potassium propionate; pH 7.5, T = 4 • C) containing 0.2 mg/mL of FITC-FBPase and then with 2 g/mL of rhodamine-phalloidin (for 1 h at room temperature).…”

Impaired Intracellular Energetic Communication in Muscles from Creatine Kinase and Adenylate Kinase (M-CK/AK1) Double Knock-out Mice

“…Glycolytic and Guanylate Metabolism in MAK ϭ/ϭ MuscleStudies in AK1 or M-CK single mutant mice and cell model systems suggest a compensatory interchange between the AK/CK circuits and the glycolytic machinery for energy production (4,6,16,33,39,48). We determined here that 18 O-metabolic labeling of Glc-6-P by hexokinase, the initial step in the glycolytic cascade, was significantly increased from 8.1 Ϯ 0.9% (n ϭ 6) in the wild-type to 18.9 Ϯ 0.9% (n ϭ 6) in MAK ϭ/ϭ…”

“…The interaction between AK-CK and glycolytic phosphotransfer systems is critical for energy supply and local ATP regeneration in different cell types (4,6,16,33,39,48,53). In muscles deficient in both AK1 and M-CK, glycolysis is probably the only remaining major cytosolic phosphotransfer circuit.…”