A Protein that Binds Specifically to the M-Line of Skeletal Muscle is Identified as the Muscle Form of Creatine Kinase (original) (raw)
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The Journal of Cell Biology
Incubation of chicken skeletal muscle fibers with an excess of anti-M-creatine kinase (CK) immunoglobulin G and an excess of anti-M-OK Fab fragments leads to heavy decoration of the M-line (Wallimann, T., D. C. Turner, and H. M. Eppenberger, 1977, J. Cell Biol. 75:297-317) and to removal of the electron-dense M-line structure (Walliman, T., G. W. Pelloni, D. C. Turner, and H. M. Eppenberger, 1978, Proc. Natl. Acad. ScL USA., 75:4296-4300), respectively. On the other hand, incubation with low concentrations of monovalent anti-M-CK Fab did not extract but rather decorated the M-line, giving rise to a distinct two-line staining pattern. A similar double-line staining pattern, although less pronounced, was also observed within the M-line of paraformaldehyde-prefixed myogenic cells, which after permeabilization were incubated with low concentrations of divalent anti-M-CK antibody. In both cases, the two decorated lines appearing in the middle of the A-band were spaced axially 42-44 nm apart and correspond most likely to the two M4 and M4' m-bridge rows described by Sj6str6m and Squire (1977, J. Mol. Biol., 109:49-68; 1977, J. Microscopy., 111:239-278). It is concluded that the muscle-specific form of creatine kinase, MM-CK, contributes mainly to the electron density of these M4 and M4' m-bridges within the M-line structure. This specific labeling pattern is a further demonstration that CK is an integral part of the M-line.
European journal of biochemistry / FEBS, 1981
Purified, homodimeric creatine kinases from chicken were subjected to two-dimensional gel analysis under dissociating conditions. Each of the subunits M-creatine kinase and B-creatine kinase was resolved into a basic and an acidic subspecies with very similar mobilities in the sodium dodecylsulfate dimension. The M-creatine kinase subspecies were found in myogenic cells, fast muscle, slow muscle and the B-creatine kinase subspecies were present in heart, gizzard and brain. The creatine kinase subunits were identified in these tissues by a variety of methods like immunoreplicas of two-dimensional gels, immunoprecipitations, or coelectrophoresis with purified creatine kinase and all gave the same results. In the course of myogenic development in vitro the subspecies were synthesized coordinately and no indication was found for a differential regulation of any of the subspecies of the creatine kinase subunits. No radioactive phosphorus was incorporated into either one of the subspecies...
European Journal of Cell Biology, 1998
Creatine kinaseprotein targetingskeletal muscle associationconfocal microscopysite-directed mutagenesis Muscle-type creatine kinase is known for its unique interaction with the myofibrillar M-band, but the molecular origin for this structural relationship is not well understood. A systematic sequence comparison between the highly homologous cytosolic isoforms, muscle-type and brain-type creatine kinase, yielded two isoenzyme-specific regions in the muscle-type creatine kinases, the M-260 box (residues 258-270) and the M-300 box (residues 300-315). These particular regions were conspicuous for the specific interaction of this CK isoenzyme, but not of brain-type creatine kinase, with the sarcomeric M-band. In situ diffusion assays with fluorescently labeled native, as well as mutated muscle-type creatine kinase variants, were used to study by laser confocal microscopy their association with the M
Functional Equivalence of Creatine Kinase Isoforms in Mouse Skeletal Muscle
Journal of Biological Chemistry, 1997
Creatine kinase (CK) is a highly conserved enzyme abundant in skeletal muscle that has a key role in high energy phosphate metabolism. The localization of the muscle isoenzyme of CK (MM-CK) to the M line and the sarcoplasmic reticulum of myofibrils has been suggested to be important for proper force development in skeletal muscle. The importance of this subcellular compartmentation has not been directly tested in vivo. To test the role of myofibrilar localization of CK, the consequences of a complete CK isoform switch from MM-CK to the brain (BB-CK) isoform, which does not localize to the M line, was studied in transgenic mouse skeletal muscle. In MM-CK knockout mice there are large contractile defects. When MM-CK was replaced by BB-CK, the aberrant contractile phenotypes seen in MM-CK knockout mice were returned to normal despite the lack of myofibrillar localization. These results indicate that CK compartmentation to the myofibril of skeletal muscle is not essential for contractile function and that there is functional equivalence of creatine kinase isoforms in supporting cellular energy metabolism.
Molecular and Cellular Biochemistry, 2007
Creatine kinase (CK) (E.C. 2.7.3.2) buffers cellular ATP concentration during fluctuating ATP turnover. Muscle cytosolic CK isoform interacts with various subcellular structures where it is functionally coupled with relevant ATPases. However, how this interaction affects its activity is not known. We have therefore studied the interaction of CK with myofibrils and the role of different conformational states of CK molecule induced by ATP, phosphocreatine, ADP and the ATP-creatine pair. Purified rabbit psoas myofibrils with CK specific activity of 0.4 ± 0.02 IU/mg were used. The exchange rates between the myofibrillar M-band and its surroundings were measured with fluorofore conjugated CK (IAF) by the Fluorescence Lost in Photobleaching (FLIP) method within a very narrow pH range 7.1–7.15. For CK-IAF without docked substrates, the time derivative of the initial loss of the fluorescent signal within the M-band equalled −3.26 at the fifth second and the decrease reached 82% by the 67th second. For CK-IAF with added substrates, the derivatives fell into the range of −0.95 to −1.30, with respective decreases from 16 to 46% at the 67th second. The results show that the substrates slowed down the exchange rate. This indicates that the strength of the bond between CK and the M-band of myofibrils increased.
Monovalent antibodies against MM-creatine kinase remove the M line from myofibrils
Proceedings of the National Academy of Sciences, 1978
Column-purified antibodies against creatine kinase (EC 2.7.3.2) from chicken skeletal muscle (the homodimeric isoenzyme designated MM-CK) bind specifically to the M lines of chicken pectoral muscle myofibrils. Incubation of myofibrils with monovalent Fab' fragments of these antibodies solubilizes most of the myofibril-bound creatine kinase, concomitantly removing most of the electron-dense material from the M lines. This strongly indicates that MM-CK is an integral part of the M-line structure and is consistent with the suggestion that MM-CK molecules form the M bridges that are responsible for the principle M-line substriations.
Journal of muscle research and cell motility, 2000
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...
Activity of creatine kinase in a contracting mammalian muscle of uniform fiber type
Biophysical Journal, 1994
We investigated whether the creatine kinase-catalyzed phosphate exchange between PCr and-yATP in vivo equilibrated with cellular substrates and products as predicted by in vitro kinetic properties of the enzyme, or was a function of ATPase activity as predicted by obligatory "creatine phosphate shuttle" concepts. A transient NMR spin-transfer method was developed, tested, and applied to resting and stimulated ex vivo muscle, the soleus, which is a cellularly homogeneous slowtwitch mammalian muscle, to measure creatine kinase kinetics. The forward and reverse unidirectional CK fluxes were equal, being 1.6 mMs-1 in unstimulated muscle at 220C, and 2.7 mMs-1 at 300C. The CK fluxes did not differ during steady-state stimulation conditions giving a 10-fold range of ATPase rates in which the ATP/PCr ratio increased from approximately 0.3 to 1.6. The observed kinetic behavior of CK activity in the muscle was that expected from the enzyme in vitro in a homogeneous solution only if account was taken of inhibition by an anion-stabilized quaternary dead-end enzyme complex: E-Cr-MgADP-anion. The CK fluxes in soleus were not a function of ATPase activity as predicted by obligatory phosphocreatine shuttle models for cellular energetics.
The role of creatine kinase and arginine kinase in muscle
Biochemical Journal, 1978
Arginine and creatine kinase activities in different muscles are compared with calculated maximum rates of ATP turnover. The magnitude of the kinase activities decreases in the following order: anaerobic muscles and vertebrate skeletal muscles greater than heart muscle greater than insect flight muscle. The maximum activity of phosphagen kinases (i.e. creatine kinase and arginine kinase), in the direction of phosphagen formation, is lower than the calculated maximum rate of ATP turnover in insect flight muscle or rat heart.