Biomedical subjects

J C Perriard

Publications and source records attributed to J C Perriard.

At least 37 records · Page 2Linked to original sources

PubMed1997

Structure-permeation relations of met-enkephalin peptide analogues on absorption and secretion mechanisms in Caco-2 monolayers.

Due to the low effective permeabilities of peptides at many absorption sites, their structure-permeation relations are of high interest. In this work structure-permeation relations of Met-enkephalin analogues are presented using confluent Caco-2 cells as an in vitro permeation model. Four model peptides (Met-enkephalin, [D-Ala2]Met-enkephalin, [D-Ala2]Met-enkephalinamide, and metkephamid) were tested in terms of permeability, lipophilicity, charge, and molecular size. Permeability coefficients (P(eff)) across Caco-2 cells were low, 3.3 x 10(-8) to 9.5 x 10(-8) cm s-1, and were similar to typical paracellular markers. No correlation of permeability and the log(apparent octanol/buffer partition coefficient) was observed. A 40-fold increase of the permeability of metkephamid in the presence of 10 mM EDTA suggested a significant contribution of paracellular transport. Independent support for this conclusion was obtained by visualizing the pathway of the fluorescein isocyanate isomer I 1-metkephamid by confocal laser scanning microscopy (CLSM). The fluorophore-labeled peptide was observed in the intercallular space only. Metkephamid permeabilities were found to be direction-specific. Permeabilities from basolateral to apical (b-to-a) were significantly higher (ca. 4-fold) than in the opposite (a-to-b) direction. The addition of verapamil equalized the permeabilities in the a-to-b and b-to-a directions, suggesting the involvement of a P-glycoprotein-mediated secretion mechanism. Similar observations were obtained with [D-Ala2]Met-enkephalinamide, but not with Met-enkephalin and [D-Ala2]Met-enkephalin. In contrast to the other analogues, metkephamid and [D-Ala2]Met-enkephalinamide are positively charged at neutral pH, as demonstrated by their isoelectric points (pl = 8.6 for [D-Ala2]Met-enkephalinamide and metkephamid and 5.3 for [D-Ala2]Met-enkephalin and Met-enkephalin). The data is in agreement with the literature showing that most compounds secreted by the P-glycoprotein transporter carry a positive charge.

ATP Binding Cassette Transporter, Subfamily B, Mem↗

PubMed1997

Transfected muscle and non-muscle actins are differentially sorted by cultured smooth muscle and non-muscle cells.

We have analyzed by immunolabeling the fate of exogenous epitope-tagged actin isoforms introduced into cultured smooth muscle and non-muscle (i.e. endothelial and epithelial) cells by transfecting the corresponding cDNAs in transient expression assays. Exogenous muscle actins did not produce obvious shape changes in transfected cells. In smooth muscle cells, transfected striated and smooth muscle actins were preferentially recruited into stress fibers. In non-muscle cells, exogenous striated muscle actins were rarely incorporated into stress fibers but remained scattered within the cytoplasm and frequently appeared organized in long crystal-like inclusions. Transfected smooth muscle actins were incorporated into stress fibers of epithelial cells but not of endothelial cells. Exogenous non-muscle actins induced alterations of cell architecture and shape. All cell types transfected by non-muscle actin cDNAs showed an irregular shape and a poorly developed network of stress fibers. beta- and gamma-cytoplasmic actins transfected into muscle and non-muscle cells were dispersed throughout the cytoplasm, often accumulated at the cell periphery and rarely incorporated into stress fibers. These results show that isoactins are differently sorted: not only muscle and non-muscle actins are differentially distributed within the cell but also, according to the cell type, striated and smooth muscle actins can be discriminated for. Our observations support the assumption of isoactin functional diversity.

Actins↗

PubMed1997

Molecular mechanisms of myofibril assembly in heart.

We investigated the assembly of the first sarcomeres in chicken embryos by confocal microscopy of immunofluorescently stained whole mount rudiments of early chicken hearts isolated around the onset of beating. In embryos with merely 9 somites, myomesin was found to be present in a cross striated pattern, indicating that myomesin is expressed rather early during development. RNA studies confirmed these findings and RT-PCR revealed the presence of myomesin mRNA already in the 7 somite embryo. The expression of myomesin mRNA coding for the skeletal isoform preceded the heart specific transcript. In the adult heart, however, only the heart isoform was detectable. The interaction of myomesin domains with the sarcomere was investigated by transfection of epitope tagged constructs into cultured cardiomyocytes. The second domain of myomesin, an immunoglobulin-like domain, was found to specifically bind to the M-band region of chicken cardiomyocytes. All constructs containing this domain also showed M-band localization. Additionally, constructs consisting of either the second domain of myomesin or the myosin light chain isoform MLC 3f fused to the green fluorescent protein (eGFP) were expressed in rat cardiomyocytes and were found to be distributed in the same manner as the expression constructs tagged only with the much shorter VSV epitope. Transfected cells did not show any alteration in beating activity and no alteration of myofibrillar structure, as judged by simultaneous staining for the Z-disc protein alpha-actinin and other sarcomeric markers. Apparently, the addition of eGFP did not disturb the assembly properties or the function of the two proteins and therefore allowed the easy visualization of assembly and contraction processes directly in the living cardiomyocyte.

Animals↗

PubMed1996

Tissue-specific isoforms of chicken myomesin are generated by alternative splicing.

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.

Alternative Splicing↗

PubMed1996

Evolution of the creative kinases. The chicken acidic type mitochondrial creatine kinase gene as the first nonmammalian gene.

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 (Mia-CK); and mitochondrial sarcomeric, basic type (Mib-CK). We report here the cloning of the chicken Mia-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 Mia-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 Mia-CK transcripts in chicken tissues shows a restricted pattern and therefore does not fulfill all criteria of a housekeeping enzyme.

Amino Acid Sequence↗

PubMed1996

The intracompartmental sorting of myosin alkali light chain isoproteins reflects the sequence of developmental expression as determined by double epitope-tagging competition.

In order to compare within the same cell the various degrees of specificity of myosin alkali light chain (MLC) isoproteins sorting to sarcomeres, a competition assay was established using double epitope tagging. Various combinations of two different MLC isoform cDNAs tagged with either a vesicular stomatitis virus VSV-G (VSV) or a medium T (mT) protein epitope were co-expressed in cultured cardiomyocytes from adult and neonatal rat ventricles. Expressed isoproteins were detected by means of anti-VSV and anti-mT antibodies and their sorting patterns were analyzed by confocal microscopy. The sorting specificity of MLC isoforms to sarcomeric sites was shown to increase in the order MLC3nm, to ML1sa, to MLC1sb, to MLC1f and MLC3f following the sequence of developmental expression. Expressed fast skeletal muscle isoforms (MLC1f and MLC3f) were always localized at the A-bands of myofibrils, while nonmuscle type (MLC3nm) was distributed throughout the cytoplasm. The slow skeletal muscle type (MLC1sa) showed a weak sarcomeric pattern if it was co-expressed with MLC3nm, but it was distributed throughout the cytoplasm when expressed in combination with MLC1f, MLC3f or the slow skeletal/ventricular muscle isoform (MLC1sb). The MLC1sb was localized at the A-bands when it was co-expressed with MLC3nm or MLC1sa, while it was also distributed to the cytoplasm if co-expressed with MLC1f or MLC3f. Further, expression of chimeric cDNAs revealed that the N-terminal lobe of each isoprotein is responsible for the isoform-specific sorting pattern.

Animals↗

PubMed1995

Three-dimensional analysis and visualization of myofibrillogenesis in adult cardiomyocytes by confocal microscopy.

Confocal light microscopy has found its place among the standard analytical tools in cell and molecular biology. When combined with techniques such as immunofluorescence or fluorescent in situ hybridization, the spatial distribution of individual biological components can be traced within cells and tissues and, under certain circumstances, even with living samples. In this article, advanced 3D visualization techniques have been applied to analyze the distribution of myofibrillar proteins in cultured adult rat cardiomyocytes. By combining confocal immunofluorescence microscopy with specially designed three-dimensional visualization, we have obtained images which are similar to those obtained with the scanning electron microscope. The subcellular distribution of proteins expressed after transfection of cDNA is monitored in the cultured heart cells. The expressed proteins are distinguished from their endogenous counterparts by the use of an epitope tagging technique. The described methods are suitable to specifically monitor the behavior of several closely related isoprotein mutants in cell or tissue preparations.

Animals↗

PubMed1995

Equilibration and exchange of fluorescently labeled molecules in skinned skeletal muscle fibers visualized by confocal microscopy.

Confocal laser fluorescence microscopy was used to study in real time under nearly physiological conditions the equilibration and exchange characteristics of several different fluorescently labeled molecules into chemically skinned, unfixed skeletal muscle fibers of rabbit psoas. The time required for equilibration was found to vary widely from a few minutes up to several days. Specific interactions of molecules with myofibrillar structures seem to slow down equilibration significantly. Time for equilibration, therefore, cannot simply be predicted from diffusion parameters in solution. Specific interactions resulted in characteristic labeling patterns for molecules like creatine kinase (muscle type), pyruvate kinase, actin-binding IgG, and others. For the very slowly equilibrating Rh-NEM-S1, changes in affinity upon binding to actin in the absence of calcium and subsequent slow cooperative activation, beginning at the free end of the filament at the H-zone, were observed. In the presence of calcium, however, binding of Rh-NEM-S1 was homogeneous along the whole actin filament from the very beginning of equilibration. The dissociation properties of the dynamic interactions were analyzed using a chase protocol. Even molecules that bind with rather high affinity and that can be removed only by applying extreme experimental conditions like Rh-phalloidine or Rh-troponin could be displaced easily by unlabeled homologous molecules.

Actins↗

PubMed1995

Dominant negative effect of cytoplasmic actin isoproteins on cardiomyocyte cytoarchitecture and function.

The intracompartmental sorting and functional consequences of ectopic expression of the six vertebrate actin isoforms was investigated in different types of cultured cells. In transfected fibroblasts all isoactin species associated with the endogenous microfilament cytoskeleton, even though cytoplasmic actins also showed partial localization to peripheral submembranous sites. Functional and structural studies were performed in neonatal and adult rat cardiomyocytes. All the muscle isoactin constructs sorted preferentially to sarcomeric sites and, to a lesser extent, also to stress-fiber-like structures. The expression of muscle actins did not interfere with cell contractility, and did not disturb the localization of endogenous sarcomeric proteins. In sharp contrast, ectopic expression of the two cytoplasmic actin isoforms resulted in rapid cessation of cellular contractions and induced severe morphological alterations characterized by an exceptional outgrowth of filopodia and cell flattening. Quantitative analysis in neonatal cardiomyocytes indicated that the levels of accumulation of the different isoactins are very similar and cannot be responsible for the observed isoproteins-specific effects. Structural analysis revealed a remodeling of the cytoarchitecture including a specific alteration of sarcomeric organization; proteins constituting the sarcomeric thin filaments relocated to nonmyofibrillar sites while thick filaments and titin remained unaffected. Experiments with chimeric proteins strongly suggest that isoform specific residues in the carboxy-terminal portion of the cytoplasmic actins are responsible for the dominant negative effects on function and morphology.

Actins↗

PubMed1993

Effect of okadaic acid on protein phosphorylation patterns of chicken myogenic cells with special reference to creatine kinase.

Okadaic acid and other agents affecting cellular phosphorylation and dephosphorylation processes profoundly changed the phosphoprotein pattern of 32Pi-labelled chicken embryonic skeletal muscle cells. The phosphorylation states of proteins in the lower molecular weight range were especially increased. Immunoprecipitation of cellular extracts with anti-creatine kinase antibodies enabled us to identify creatine kinase (CK) phosphoproteins. B-CK was phosphorylated after treating the cultures with 1-oleoyl-2-acetyl-sn-glycerol, dibutyryl-cAMP, okadiac acid and combinations thereof, but not with 1,2-dioleoyl-sn-glycerol. M-CK was also shown to be phosphorylated. The results indicated that in vivo, CK isoforms in muscle are subjected to control mediated by phosphorylation and dephosphorylation processes.

Amino Acid Sequence↗

PubMed1993

Three-dimensional visualization of multi-channel volume data: the amSFP algorithm.

In this paper we present a three-dimensional visualization technique for multi-channel volume data. The technique simulates the physical process of fluorescence, hence its name: achromatic multi-channel simulated fluorescent process (amSFP). The data set is simulated as 3D distribution of different fluorescent dyes, where each channel is represented by a particular type of dye. Apart from the spatial density map, no additional characteristics about the data set have to be defined; no image segmentation is needed prior to visualization. The degree of interaction among the channels in the fluorescence process can be adapted to optimally render specific structures in the image. 3D multi-channel data can be obtained by a three-dimensional imaging device that is able to measure a number of physical quantities at a given location within a specimen. The fluorescence principle, the algorithm, and its implementation are presented. We have used the technique to investigate the relative spatial arrangement of blood vessels and astrocytes in the cat retina. The two components have been stained with different fluorescence dyes and recorded in a confocal light microscope to form a two-channel 3D data set.

Algorithms↗

PubMed1993

Remodelling of cardiomyocyte cytoarchitecture visualized by three-dimensional (3D) confocal microscopy.

The break-down and reassembly of myofibrils in long-term cultures of adult rat cardiomyocytes was investigated by a novel combination of confocal laser scanning microscopy and three-dimensional image reconstruction, referred to as FTCS, to visualize the morphological changes these cells undergo in culture. FTCS is discussed as an alternative imaging mode to low-magnification scanning electron microscopy. The three-dimensional shape of the cells are correlated with the assembly state of myofibrils in different stages. Based on immunofluorescence and confocal laser scanning microscopy it was shown that myofibrils are degraded within a few days after plating and that newly assembled myofibrils are predominantly confined to the continuous area in the perinuclear region close to the membrane in contact with the substratum. The localization of myofibrils along the cell's vertical axis has been investigated both by optical sectioning using confocal light microscopy and by physical sectioning followed by transmission electron microscopy. Based on the distribution of myofibrillar proteins we propose a model of myofibrillar growth locating the putative assembly sites to a region concentric around the nuclei. We provide evidence that the cell shape is dominated by the myofibrillar apparatus.

Animals↗

PubMed1992

Fast myosin light chain expression in chicken muscles studied by in situ hybridization.

We have studied the fiber type-specific expression of the fast myosin light chain isoforms LC 1f, LC 2f, and LC 3f in adult chicken muscles using in situ hybridization and two-dimensional gel electrophoresis. Type II (fast) fibers contain all three fast myosin light chain mRNAs; Types I and III (slow) fibers lack them. The myosin light chain patterns of two-dimensional gels from microdissected single fibers match their mRNA signals in the in situ hybridizations. The results confirm and extend previous studies on the fiber type-specific distribution of myosin light chains in chicken muscles which used specific antibodies. The quantitative ratios between protein and mRNA content were not the same for all three fast myosin light chains, however. In bulk muscle samples, as well as in single fibers, there was proportionally less LC 3f accumulated for a given mRNA concentration than LC 1f or LC 2f. Moreover, the ratio between LC 3f mRNA and protein was different in samples from muscles, indicating that LC 3f is regulated somewhat differently than LC 1f and LC 2f. In contrast to other in situ hybridization studies on the fiber type-specific localization of muscle protein mRNAs, which reported the RNAs to be located preferentially at the periphery of the fibers, we found all three fast myosin light chain mRNAs quite evenly distributed within the fiber's cross-sections, and also in the few rare fibers which showed hybridization signals several-fold higher than their surrounding counterparts. This could indicate principal differences in the intracellular localization among the mRNAs coding for various myofibrillar protein families.

Animals↗

PubMed1992

Molecular analysis of protein sorting during biogenesis of muscle cytoarchitecture.

Isolated, rod-shaped adult rat cardiomyocytes (ARC) were kept in long-term cell cultures and the changes of the cardiomyocyte structure were investigated by confocal microscopy. The cells round up and make contact with the substrate by very flat, foot-like structures. After prolonged culture the amorphous cells regenerate a cardiomyocyte-like cytoarchitecture and myofibrils reemerge. In the perinuclear region myofibrils form continuously while in other cells discontinuous myofibrillogenesis was observed, where short sarcomeric segments occur all over the cytoplasmic space. During the regeneration of myofibrils certain proteins like a smooth muscle actin sort to non sarcomeric region, while myomesin or heart C-protein localize on myofibrils with high specificity. This culture system combined with method of epitope-tagging of contractile proteins are ideally suited to monitor the intracellular localization sites of exogenously introduced constructs to different cytoskeletal, since ARC exhibit at the same time stress fiber-like filaments (SFLF) and nascent myofibrils. The molecular properties of the different members of the myosin light chain isoprotein family were investigated by transfection experiments using epitope-tagged myosin light chain (MLC) cDNA. The sorting of the different types of MLC was shown to be isoprotein specific and with chimeric constructs it was shown that the isoprotein-specific incorporation into myofibrils was dependent on the presence of the middle domain of MLC-1f/3f. These MLC isoproteins can be arranged into a sequence of increasing affinity to myofibrils. A hierarchical order of myofibrillar assembly is postulated based on the association affinity. Similar experiments with constructs containing alpha-cardiac, alpha-smooth muscle and gamma-cytoplasmic actins have shown that expression of epitope-tagged actins in ARC result in different epitope staining patterns. While the alpha-cardiac actin showed a marked preference for sarcomeres, the alpha-smooth muscle isoproteins had an intermediate specificity and could either be preferentially incorporated into stress fiber-like filaments (SFLF) and in some cells to a lesser extent into myofibrils as well. Most striking results were obtained with gamma-cytoplasmic actin carrying a 5 or 11-mer epitope. This actin gave rise to large cells, induced the formation of filopodia filled with the transfected actin and depletion of the transfected actin from the perinuclear myofibrillar region.

Actins↗

PubMed1991

Intracompartmental sorting of essential myosin light chains: molecular dissection and in vivo monitoring by epitope tagging.

The isoprotein-specific intracompartmental sorting of the three essential myosin light chains (LCs), the skeletal muscle LC-1f and LC-3f and the nonmuscle LC-3nm, was investigated. Epitope tagging was used to monitor the intracellular localization to different cytoskeletal structures of the exogenously introduced constructs in adult rat cardiomyocytes (ARCs), which exhibit both stress fibers and regenerating myofibrils. LC-1f and LC-3f bind almost exclusively to the sarcomeric myosin heavy chain (MHC) with high affinity, while the LC-3nm interacts with stress fibers and sarcomeres equally well. Sorting appears to be directed by a hierarchical order of different affinities. Domain mapping by deletion and by construction of a LC-1f/3nm chimera suggests that the LCs are composed of three functionally distinct domains: a basal MHC binding site in the C-terminus; the central part, modulating the preferential interaction with MHC isoforms; and the isoprotein-specific N-terminus of the essential LC, which is probably not involved in the sorting process.

Amino Acid Sequence↗

PubMed1991

Measles virus phosphoprotein retains the nucleocapsid protein in the cytoplasm.

Measles virus (MV) proteins were efficiently expressed in COS and Vero cells from vectors based on the strong cytomegalovirus enhancer-promoter and the simian virus 40 origin of replication. When expressed alone, nucleocapsid protein (N) migrates predominantly into the nucleus whereas phosphoprotein (P) is located in the cytoplasm. Coexpression of N and P proteins results in retention of the N protein in the cytoplasm, as seen also in infected cells. The retention of N protein is due to specific interactions with the P protein since coexpression of N with either the matrix or the hemagglutinin protein had no effect. Mapping of the regions of N-P interactions on P protein revealed that the carboxy-terminal 40% of P was sufficient for specific binding to N; however, the carboxy-terminal 60% of P was required for retention of N in the cytoplasm. Thus, the V and C proteins encoded within the first half of the P gene are not involved in the cytoplasmic retention of N protein. N protein might be fortuitously targeted to the nucleus as a result of its many basic amino acids, presumably destined to interact with the MV genome. However, this set of experiments has allowed to analyze in vivo the interactions between the N and P proteins.

Animals↗

PubMed1991

Developmental regulation of creatine kinase gene expression by myogenic factors in embryonic mouse and chick skeletal muscle.

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.

Animals↗

PubMed1990

Phosphorylation of chicken brain-type creatine kinase affects a physiologically important kinetic parameter and gives rise to protein microheterogeneity in vivo.

In addition to the two monomer subunits of chicken brain-type creatine kinase (B-CK, EC, 2.7.3.2), termed Bb (basic) and Ba (acidic), another subspecies called Bb* was identified by chromatofocussing in the presence of 8 M urea (Quest et al., ). The latter low abundance protein species, isolated from tissue extracts, comigrated on 2D-gels with three minor species (Bb1-3), initially identified in immunoprecipitated, [35S]methionine labeled in vitro translation products of cDNA coding for the basic monomer Bb. During in vitro translation experiments in the presence of [32P]-gamma-ATP, Bb1-3 were labeled while phosphatase treatment eliminated these minor species. It is concluded that Bb* is identical to Bb1-3 and represents phosphorylated derivatives of Bb. B-CK dimer populations from different tissues were separated by ion-exchange chromatography and the Km values of the resulting fractions were determined under phospho-creatine (CP)-limiting conditions. In fractions containing only Bb and Bb* two kinetically different enzyme species were detected (Km values for CP = 1.6 mM and 0.8 mM), while fractions containing B-CK dimers composed of the major Ba and Bb monomers, but no Bb*, were homogeneous in this respect (Km for CP = 1.6 mM). Phosphorylation of Bb to yield Bb* is concluded to reduce the Km of B-CK dimers for CP by about 50%. This Km shift is within the range of CP concentrations found in tissues expressing the B-CK isoform and may therefore be of physiological relevance.

Animals↗