Comparisons of the protein sequences and gene structures of the known creatine kinase isoenzymes and other guanidino kinases revealed high homology and were used to determine the evolutionary relationships of the various guanidino kinases. A 'CK framework' is defined, consisting of the most conserved sequence blocks, and 'diagnostic boxes' are identified which are characteristic for anyone creatine kinase isoenzyme (e.g. for vertebrate B-CK) and which may serve to distinguish this isoenzyme from all others (e.g. from M-CKs and Mi-CKs). Comparison of the guanidino kinases by near-UV and far-UV circular dichroism further indicates pronounced conservation of secondary structure as well as of aromatic amino acids that are involved in catalysis.

Comparisons of the protein sequences and gene structures of the known creatine kinase isoenzymes and other guanidino kinases revealed high homology and were used to determine the evolutionary relationships of the various guanidino kinases. A 'CK framework' is defined, consisting of the most conserved sequence blocks, and 'diagnostic boxes' are identified which are characteristic for anyone creatine kinase isoenzyme (e.g. for vertebrate B-CK) and which may serve to distinguish this isoenzyme from all others (e.g. from M-CKs and Mi-CKs). Comparison of the guanidino kinases by near-UV and far-UV circular dichroism further indicates pronounced conservation of secondary structure as well as of aromatic amino acids that are involved in catalysis. (Mol Cell Biochem 133/134: 245-262, 1994).

Myomesin is a high molecular weight protein that is present in the M-band of all fiber types of cross-striated skeletal muscle and heart. We have isolated two cDNAs encoding tissue-specific isoforms of chicken myomesin with calculated molecular masses of 174 kDa in skeletal muscle and 182 kDa in heart. Distinct sequences are found at the 3-end of the two cDNAs, giving rise to different C-terminal domains. Partial analysis of the gene structure has shown that in chicken, both isoforms are generated by alternative splicing of a composite exon. Amino acid sequences show that the main body of myomesin consists of five fibronectin type III (class I motifs) and seven immunoglobulin-like domains (class II motifs). An identical structure was found in M-protein and human 190K protein (the human counterpart of chicken myomesin), and a comparable domain arrangement occurs in the M-band-associated protein skelemin. We postulate that myomesin, M-protein, and skelemin belong to the same subfamily of high molecular weight M-band-associated proteins of the immunoglobulin superfamily and that they probably have the same ancestor in evolution.

The B isoform of creatine kinase (BCK), which is expressed at a high level in embryonic neural tissues, is also expressed abundantly in developing striated muscle and is an early marker for skeletal myogenesis. Using isoform-specific 35S-labeled antisense cRNA probes for in situ hybridization, we have detected BCK mRNAs in embryonic mouse and chick myotomes, the first skeletal muscle masses to form in developing embryos. These transcripts are detectable as soon as myotomes are morphologically distinguishable. BCK is expressed at high levels in both skeletal and cardiac muscle in mouse and chick embryos. In the mouse, BCK transcript levels fall of rapidly in striated muscle shortly after the onset of MCK gene expression. The M isoform of creatine kinase (MCK), the striated muscle-specific isoform, is expressed later than BCK. In the mouse, BCK transcripts are expressed in myotomes at 8.5 days post coitum (p.c.), but MCK transcripts are not detected before 13 days p.c. In the chick, BCK mRNAs are present at Hamburger-Hamilton stage 13, but MCK mRNAs are not detected before stage 19. We have compared the patterns of expression of the CK genes with those of myogenic differentiation factor genes, which are thought to regulate skeletal muscle-specific gene expression. In the chick, both CMD1, first detected at stage 13, and myogenin, first detected at stage 15, are present prior to MCK, which begins to be expressed at stage 19. Unlike the mouse embryo, CMD1, the chick homologue of MyoD1, is expressed before chick myogenin. In the mouse, myogenin, first detected at 8.5 days p.c., is expressed at the same time as BCK in myotomes. Both myogenin and MyoD1, which begins to be detected two days later than myogenin, are expressed at least two days before MCK. It has been proposed that the myogenic factors, MyoD1 and myogenin, directly regulate MCK gene expression in the mouse by binding to its enhancer. However, our results show that MCK transcripts are not detected until well after MyoD1 and myogenin mRNAs are expressed, suggesting that these factors by themselves are not sufficient to initiate MCK gene expression.

In both mammals and birds, the creatine kinase (CK) family consists of four types of genes: cytosolic brain type (B-CK); cytosolic muscle type (M-CK); mitochondrial ubiquitous, acidic type (Mi a -CK); and mitochondrial sarcomeric, basic type (Mi b -CK). We report here the cloning of the chicken Mi a -CK cDNA and its gene. Amino acid sequences of the mature chicken Mi-CK proteins show about 90% identity to the homologous mammalian isoforms. The leader peptides, however, which are isoenzyme-specifically conserved among the mammalian Mi-CKs, are quite different in the chicken with amino acid identity values compared with the mammalian leader peptides of 38.5-51.3%.The chicken Mi a -CK gene spans about 7.6 kilobases and contains 9 exons. The region around exon 1 shows a peculiar base composition, with more than 80% GC, and has the characteristics of a CpG island. The upstream sequences lack TATA or CCAAT boxes and display further properties of housekeeping genes. Several transcription factor binding sites known from mammalian Mi-CK genes are absent from the chicken gene. Although the promoter structure suggests a ubiquitous range of expression, analysis of Mi a -CK transcripts in chicken tissues shows a restricted pattern and therefore does not fulfill all criteria of a housekeeping enzyme.A sufficient capacity and balanced regulation of "high energy phosphate" supply and turnover is essential for the proper function of any cell. Large amounts of energy-rich phosphagens can be found in many cells or tissues throughout the animal kingdom. In all vertebrates and also in some invertebrates this phosphagen is phosphorylcreatine (PCr) 1 (1). PCr and ADP are the products of the reversible transfer of ␥-phosphate groups from ATP to creatine, catalyzed by the creatine kinases (CKs). Two fundamental types of CKs can be found in vertebrates: cytosolic and mitochondrial CKs. The subcellular localization, the biochemical and kinetic data, and the loss of flagellar motility in spermatozoa upon inhibition of the CK system and other data (for review, see Ref.2) led to the suggestion of a metabolic PCr circuit with PCr as a transport and storage form of high energy phosphate. The PCr circuit connects sites of high energy phosphate production (glycolysis and oxidative phosphorylation) with those of high energy phosphate consumption. At the producing end of the circuit, CK is thought to have privileged access to ATP generated either by glycoclysis or by oxidative phosphorylation in the mitochondrial matrix and uses this ATP to generate PCr. At the receiving end, CK is functionally coupled to various ATPases (for instance myosin ATPase of myofibrils), which use the ATP generated in the reverse CK reaction.In chicken there are five different CK subunits known so far; three are found in the cytosol, two in mitochondria. The cytosolic subunits are called M-CK (muscle) and B a -CK and B b -CK (brain, more acidic and more basic, respectively) and can dimerize with each other (3-5). They are found soluble in the cytosol, but fractions are a...