Occurrence of Heterogenous Forms of the Subunits of Creatine Kinase in Various Muscle and Nonmuscle Tissues and Their Behaviour during Myogenesis

Abstract

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 creatinc kinasc and all gave the same results. In the course of myogcnic 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 thc subspecies, hence phosphorylation could be ruled out as the source of heterogeneity. Furthermore, peptide mapping analysis of partial proteolytic digests did not reveal differences among the subspecies of the same subunit. Not only chicken but also rat creatine kinase displayed this type of heterogeneity. All subspecies were observed after translation of chicken RNA in a cell-free protein-synthesizing system. The heterogeneity probably might best be explained by the existence of multiple, but closely related genes for the creatine kinase subunits.Creatine kinase is a dimeric enzyme that plays a crucial role in the energy metabolism. It is especially characteristic of muscle tissue, but also occurs in other tissues [1,2]. The cytoplasmic nonmitochondrial enzymes are made up from two different kinds of subunits with a M , of 40000, the M-creatine kinase subunit and the B-creatine kinase subunit. MM-creatine kinase is found in appreciable amounts only in adult muscle, whereas BB-creatine kinase is more widely distributed in smooth muscle, chicken heart, brain and a variety of other tissues. During the differentiation of chicken myogenic cells in cultures as well as in embryonic muscle, an isoenzyme transition is observed from BB-creatine kinase to MM-creatine kinase, the form typical for the differentiated state. During this transition the subunits combine at random to yield homodimers and also active heterodimers MBcreatine kinase [3 -51. The subunits forming the active isoenzymes are very likely the products of at least two different genes as indicated by the differences of amino acid composition [6], the lack of immunological cross-reactivity [4,6,7] and differences in the metabolism of the corresponding mRNAs coding for thc two different subunits [S]. In addition M-creatine kinase is found in the M-line of adult skeletal myofibrils as well as in the myofibrils of cultured cells, suggesting a specific property of the MM-creatine kinase dimer whichAhhreviutions. M-creatine kinase and B-creatine kinase. subunit types of creatine kinase; MM-creati...

Biochemical characterization of the mouse muscle-specific enolase: developmental changes in electrophoretic variants and selective binding to other proteins

“…The heterogeneity pattern of the purified mouse β-enolase ( Figure 4D) is very similar to that of β-enolase contained in adult muscle extracts ( Figure 4B). Under these migration conditions, the mouse MCK is also resolved into two major isoproteins ( Figure 4C, M), as has been described in the case of other species [31].…”

Molecular cloning and expression during myogenesis of sequences coding for M-creatine kinase.

“…ic. When the immunoprecipitate was run on a two-dimensional gel, two species oflabeled peptides were found to react with antibody against M-CK as expected (17) but not the radiolabeled contaminating species (background).…”

“…Lanes: 1 and 6, RNA eluted from a membrane containing bound pMCK1 DNA after hybridization with leg muscle poly(A)+RNA; 2 and 7, RNA subjected to the same selection procedure as in lane 1, but membrane-bound pBR322 DNA was used as selecting agent; 3 and 8, no RNA was added to the in vitro translation system; 4 and 9, 50 ng of rabbit reticulocyte RNA enriched for globin; mRNA; 5 and 10, 1 Mg of untreated poly(A)+RNA from chicken leg. (b and c) M-CK region of a two-dimension gel (17). Isoelectric focusing.…”

“…RNA as well as DNA was hybridized at 37-42°C in the presence of formamide and dextran sulfate (24) with 5x SSPE (lx SSPE = 0.18 M NaCl/10 mM NaPO4/1 mM Na2 EDTA, pH 7.0) instead ofstandard saline citrate. After hybridization for [16][17][18] hr, the membranes were washed for 2 hr in 0.3x SSPE containing 0.2% NaDodSO4 at 68°C and then for 1 hr in 0.5 M Na phosphate at pH 7.0 at the same temperature. The washing was repeated until the washes contained negligible radioactivity.…”

“…Chicken RNA was translated and the in vitro translation products were analyzed as described (7). Analysis of immunoprecipitated CK peptides on two-dimensional gels was carried out as described (17).…”