Developmental regulation of creatine kinase isoenzymes in myogenic cell cultures from chicken. Biosynthesis of creatine kinase subunits M and B.

295

155

This report describes the biochemical characterization of a novel extracellular matrix component, "myotendinous antigen," which appears early in chick limb morphogenesis at sites connecting developing muscle fibers, tendons, and bone (Chiquet, M., and D. Fambrough, 1984; J. Cell Biol., 98 :1926-1936 . This extracellular matrix antigen is a major component of the secretory proteins released into the medium by fibroblast and muscle cultures ; the soluble form is characterized here. This form of myotendinous antigen is a large glycoprotein complex consisting of several disulfide linked subunits (Mr 150,000-240,000) . The differently sized antigen subunits are related, since they yielded very similar proteolytic cleavage patterns . M1 antibody can bind to the denatured subunits . The antigen subunits, as well as a Mr 80,000 pepsin-resistant antigenic domain derived from them, are resistant to bacterial collagenase . Despite possessing subunits similar in size to fibronectin, myotendinous antigen appears to be both structurally and antigenically unrelated to fibronectin or to other known extracellular matrix components. About seven times more M1 antigen per cell nucleus was released into the medium in fibroblast as compared to muscle cultures . In muscle conditioned medium, myotendinous antigen is noncovalently complexed to very high molecular weight material that could be heavily labeled by [3 H]glucosamine and [358]sulfate . This material is sensitive to chondroitinase ABC and hence appears to contain sulfated glycosaminoglycans . We speculate that myotendinous antigen might interact with proteoglycans on the surface of muscle fibers, thereby acting as a link to tendons .Monoclonal antibody MI recognizes an epitope on an extracellular matrix component that seems to be enriched in tendons, myotendinousjunctions, and in the endomysium at the tips of skeletal muscle fibers (11). This "myotendinous antigen" is, by virtue ofits distribution, a candidate for linking muscle fibers to tendon fascicles and bones, both in the developing and the adult locomotive system of vertebrates. A first step towards assigning a function to the antigen recognized by M 1 antibody is its biochemical characterization, which is the subject ofthis paper. We were able to isolate the antigen, by immunoprecipitation or immunoadsorption, in a soluble form from conditioned media of cells metabolically labeled with radioactive amino acids or sugars. The isolated antigen was compared to other known extracellular matrix components .Since a preparation of type V collagen (44) as an immunogen for generating hybridoma cell lines leading to the isolation of M1 antibody (11), we initially determined whether the antigen was, in fact, type V collagen or another collagenous protein (for reviews, see references 4, 5, 15) . The subunits of the antigen precipitated by M 1 antibody turned out to migrate on SDS acrylamide gels at similar rates as monomers offibronectin, a major extracellular matrix protein (24). Although M 1 antibody and antifibronecti...

“…Immunoprecipitates were centrifuged through the sucrose Triton cushion (10,000 g, 10 min) and the tubes washed as described by Caravatti et al . (6).…”

Section: Methodsmentioning

confidence: 99%

Chick myotendinous antigen. II. A novel extracellular glycoprotein complex consisting of large disulfide-linked subunits.

Chiquet¹,

Fambrough²

1984

295

155

“…BB-CK is the more widely distributed ubiquitous isoform present in brain, smooth muscle, heart and a variety of other tissues (Eppenberger et al, 1967;Trask and Billadello, 1990). All three cytosolic isoforms coexist together only during myogenesis (Caravatti et al, 1979) and to some extent also in mammalian heart, while MB-CK and BB-CK are undetectable in mature skeletal muscle (Turner et al, 1973;Wallimann et al, 1977Wallimann et al, , 1983b. Biochemical fractionation (Wallimann et al, 1977(Wallimann et al, , 1978 and in situ immunolocalization techniques (Wegmann et al, 1992) on skeletal and cardiac muscle have shown that cytosolic MM-CK is not evenly distributed within muscle cells.…”

Section: Introductionmentioning

confidence: 99%

Isoenzyme-Specific Interaction of Muscle-Type Creatine Kinase with the Sarcomeric M-Line Is Mediated by Nh2-Terminal Lysine Charge-Clamps

Hornemann¹,

Stolz²,

Wallimann³

2000

Creatine kinase (CK) is located in an isoenzyme-specific manner at subcellular sites of energy production and consumption. In muscle cells, the muscle-type CK isoform (MM-CK) specifically interacts with the sarcomeric M-line, while the highly homologous brain-type CK isoform (BB-CK) does not share this property. Sequence comparison revealed two pairs of lysine residues that are highly conserved in M-CK but are not present in B-CK. The role of these lysines in mediating M-line interaction was tested with a set of M-CK and B-CK point mutants and chimeras. We found that all four lysine residues are involved in the isoenzyme-specific M-line interaction, acting pair-wise as strong (K104/K115) and weak interaction sites (K8/K24). An exchange of these lysines in MM-CK led to a loss of M-line binding, whereas the introduction of the very same lysines into BB-CK led to a gain of function by transforming BB-CK into a fully competent M-line–binding protein. The role of the four lysines in MM-CK is discussed within the context of the recently solved x-ray structures of MM-CK and BB-CK.

“…The enzyme is a dimer of two subunits which exist as a nonskeletal muscle form, brain creatine kinase (BB-CK), in embryonic muscle and in nonmuscle cells like brain. Upon muscle differentiation the synthesis of the M-CK subunit is induced and BB-CK is gradually replaced by the skeletal muscle-specific enzyme MM-CK (8,14,40,41,51). Although myofibers that differentiate in culture have been shown to contain all three isoenzymes, MM-CK, MB-CK and BB-CK (40,51) only MM-CK appears to be localized and specifically bound to the M-band of the myofibril (53).…”

mentioning

confidence: 99%

Intracellular targeting of isoproteins in muscle cytoarchitecture.

Schäfer

Perriard

1988

Abstract. Part of the muscle creatine kinase (MM-CK) in skeletal muscle of chicken is localized in the M-band of myofibrils, while chicken heart cells containing myofibrils and BB-CK, but not expressing MM-CK, do not show this association. The specificity of the MM-CK interaction was tested using cultured chicken heart cells as "living test tubes" by microinjection of in vitro generated MM-CK and hybrid M-CK/B-CK mRNA with SP6 RNA polymerase. The resulting translation products were detected in injected cells with isoprotein-specific antibodies. M-CK molecules and translation products of chimeric cDNA molecules containing the head half of the B-CK and the tail half of the M-CK coding regions were localized in the M-band of the myofibrils. The tail, but not the head portion of M-CK is essential for the association of M-CK with the M-band of myofibrils. We conclude that gross biochemical properties do not always coincide with a molecule's specific functions like the participation in cell cytoarchitecture which may depend on molecular targeting even within the same cellular compartment. IN most cells of higher organisms isoforms, representing closely related proteins with conserved amino acid sequences and often similar functional properties are the products of multigene families. Distinct isoforms may exist as related proteins in different tissues, be developmentally regulated within a single tissue or even coexist within a single cytoplasmic compartment. In part divergence in protein sequence among isoforms may serve to target a given isoprotein to its appropriate compartment within the cell. Specific sequences, often located at the amino terminus, have been found to be responsible for the extracellular export of many secretory proteins (5) or the intracellular targeting of proteins destined for intracellular compartments like mitochondria, chloroplasts or the nucleus (11,17,26).Here we examine the molecular basis for the localization of muscle creatine kinase (MM-CK) ~ within the M-band of the muscle myofibril (50,54,55,56). Creatine kinase is the major enzyme ensuring energy production in the muscle cell. The enzyme is a dimer of two subunits which exist as a nonskeletal muscle form, brain creatine kinase (BB-CK), in embryonic muscle and in nonmuscle cells like brain. Upon muscle differentiation the synthesis of the M-CK subunit is induced and BB-CK is gradually replaced by the skeletal muscle-specific enzyme 14,40,41,51). Although myofibers that differentiate in culture have been shown to contain all three isoenzymes, MM-CK, MB-CK and BB-CK (40, 51) only MM-CK appears to be localized Beat W. Sch~ifer's present address is Department of Pharmacology, Stanford University, Stanford, CA 94305. Abbreviations used in this paper:B-CK, creatine kinase subunit; BB-CK, brain creatine kinase; CMF, calcium-and magnesium-free; MB-CK heterodimeric creatine kinase; M-CK, creatine kinase subunit; MM-CK, muscle creatine kinase. and specifically bound to the M-band of the myofibril (53). This result can be explained in two ways. ...