Purification of mitochondrial creatine kinase. Biochemical and immunological characterization

Mitochondrial creatine kinase was purified from canine myocardium. The preparation exhibited a positively charged isoenzyme free of other creatine kinase isoenzymes and on sodium dodecyl sulfate gel exhibited a single protein band. Amino acid composition showed mitochondrial creatine kinase to be different from that of MM or BB creatine kinase and did not hybridize with the M or B subunits of the cytosolic forms. Antiserum was developed to mitochondrial creatine kinase which did not cross-react with cytosolic creatine kinases. Antiserum to cytosolic creatine kinase exhibited no reaction to mitochondrial creatine kinase. Utilizing the specific antiserum, a radioimmunoassay was developed for the specific detection of mitochondrial creatine kinase. Thus, mitochondrial creatine kinase was purified and shown to be comprised of a unique subunit which is biochemically and immunologically distinct from the cytosolic creatine kinases.     

Purification and localization of brain-type creatine kinase in sodium chloride transporting epithelia of the spiny dogfish, Squalus acanthias.

The targeting of creatine kinase isoenzymes to specific sites within muscle cells provides a system for the regeneration of ATP in situ from ADP and creatine phosphate. We have recently reported the colocalization of brain-type (B) creatine kinase and the nonsarcomeric mitochondrial creatine kinase isoenzymes in the thick ascending limb of the loop of Henle in the rat kidney, suggesting that creatine kinase may regenerate ATP for sodium transport (Friedman, D.L., and Perryman, M.B. (1991) J. Biol. Chem. 266, 22404-22410). In order to test the hypothesis regarding the association of B creatine kinase with sodium transport, we examined the creatine kinase enzymes in the rectal (salt-secreting) gland of the dogfish shark which contains high levels of the Na+/K(+)-ATPase. The creatine kinase isoform composition was determined by non-denaturing electrophoresis, immunoblotting, protein purification, and amino acid sequence analysis. The results demonstrate both B creatine kinase and mitochondrial creatine kinase proteins are present in the rectal gland, an isoform composition which is the same as in the mammalian kidney. By using a combination of chromatographic techniques, shark B creatine kinase was purified to homogeneity and partial sequence data was obtained from two cyanogen bromide peptide fragments. One of these fragments contains the active site and is identical at all sequenced residues with the corresponding region from the echinoderm sperm flagellar creatine kinase, and is 96% homologous with both chicken and rat B creatine kinase subunits. The other fragment corresponds to a region near the N-terminal of mammalian creatine kinases and is 89% homologous with B creatine kinase from chicken. The localization of these isoforms was examined by immunocytochemistry using subunit specific antisera. Mitochondrial creatine kinase and B creatine kinase immunoreactivity are detected in all tubules, and is restricted to the basal region of the cells, which is the site of the Na+/K(+)-ATPase. The conservation of creatine kinase isoform expression in excretory tissue, and the localization of creatine kinase immunoreactivity in the basal region of the tubule cells, demonstrate that subcellular compartmentation of B creatine kinase may underly the functional coupling of creatine kinase activity with sodium transport.     

Expression of creatine kinase isoenzymes in myogenic cell lines.

Investigation of creatine kinase isoenzyme activity in several cloned myogenic cell lines showed differences in B-type subunit expression. In cultures of myoblasts isolated from rat skeletal muscle by selective cell plating and in the cell lines M58 and M41, the activity of the mononucleated cells was of the BB isoenzyme. After cell fusion, MM, MB, and BB isoenzymes were present; the main activity was of the MM isoenzyme. In the myogenic lines L8 and L84, in cultures of mononucleated cells, creatine kinase activity was absent or barely detectable. The high creatine kinase activity after cell fusion was of the MM type. No BB and MB activity was detected in these lines at any stage of differentiation. The difference in expression of creatine kinase isoenzymes seems not to affect the expression of other parameters of differentiation.     

Native mitochondrial creatine kinase forms octameric structures. II. Characterization of dimers and octamers by ultracentrifugation, direct mass measurements by scanning transmission electron microscopy, and image analysis of single mitochondrial creatine kinase octamers

Electron micrographs of negatively stained and metal-shadowed mitochondrial creatine kinase (Mi-CK) molecules purified as described by Schlegel et al. (Schlegel, J., Zurbriggen, B., Wegmann, E., Wyss, M., Eppenberger, H. M., and Wallimann, T. (1988) J. Biol Chem. 263, 16942-16953) revealed a homogeneous population (greater than or equal to 95%) of distinctly sized square-shaped, octameric particles with a side length of 10 nm that frequently exhibited a pronounced 4-fold axis of symmetry. The cube-like molecules consist of four dimers that are arranged around a stain-accumulating central cavity of 2.5-3 nm in diameter. This interpretation is supported by single particle averaging including correlation analysis by computer. Upon prolonged storage or high dilution, the cube-like octamers tended to dissociate into "banana-shaped" dimers. Sedimentation velocity and sedimentation equilibrium experiments yielded an s value of 12.8-13.5 S and an Mr of 328,000 +/- 25,000 for the octameric cubes. An s value of 5.0 S and a Mr of 83,000 +/- 8,000 was found under conditions which revealed banana-shaped dimers. These dimers proved to be very stable, as their dissociation into monomers of 45 kDa (s value = 2.0 S) required 6 M guanidine HCl. Thus, the oligomeric structures observed in the electron microscope are identified as Mi-CK dimers (banana-shaped structures) and cubical Mi-CK octamers assembled from four Mi-CK dimers. The octameric nature of native Mi-CK and the formation of Mi-CK dimers were confirmed by direct mass measurements of individual molecules by scanning transmission electron microscopy yielding a molecular mass of 340 +/- 55 kDa for the octamer and 89 +/- 27 kDa for the dimer. A structural model of Mi-CK octamers and the possible interaction with ATP/ADP-translocator molecules as well as with the outer mitochondrial membrane is proposed. The implications with respect to the physiological function of Mi-CK as an energy-channeling molecule at the producing side of the phosphoryl creatine shuttle are discussed.     

The tryptophan residues of mitochondrial creatine kinase: roles of Trp-223, Trp-206, and Trp-264 in active-site and quaternary structure formation.

The 5 tryptophan residues of chicken sarcomeric mitochondrial creatine kinase (Mib-CK) were individually replaced by phenylalanine or cysteine using site-directed mutagenesis. The mutant proteins were analyzed by enzyme kinetics, fluorescence spectroscopy, circular dichroism, and conformational stability studies. In the present work, Trp-223 is identified as an active-site residue whose replacement even by phenylalanine resulted in > or = 96% inactivation of the enzyme. Trp-223 is responsible for a strong (18-21%) fluorescence quenching effect occurring upon formation of a transition state-analogue complex (TSAC;Mib-CK.creatine.MgADP.NO3-), and Trp-223 is probably required for the conformational change leading to the TSAC-induced octamer dissociation of Mib-CK. Replacement of Trp-206 by cysteine led to a destabilization of the active-site structure, solvent exposure of Trp-223, and to the dissociation of the Mib-CK dimers into monomers. However, this dimer dissociation was counteracted by TSAC formation or the presence of ADP alone. Trp-264 is shown to be located at the dimer-dimer interfaces within the Mib-CK octamer, being the origin of another strong (25%) fluorescence quenching effect, which was observed upon the TSAC-induced octamer dissociation. Substitution of Trp-264 by cysteine drastically accelerated the TSAC-induced dissociation and destabilized the octameric structure by one-fourth of the total free interaction energy, probably by weakening hydrophobic contacts. The roles of the other 2 tryptophan residues, Trp-213 and Trp-268, could be less well assigned.     

Compartmentation of multiple forms of creatine kinase in the distal nephron of the rat kidney

Creatine kinase enzymes are present in tissues such as muscle and brain to interconvert creatine phosphate and ADP, thus providing a system to interconnect energy production and utilization (Bessman, S. P., and Carpenter, C. L. (1985) Annu. Rev. Biochem. 54, 831-862). Creatine kinase isoenzymes in kidney have received little attention since kidney contains relatively low creatine kinase activity compared with muscle and brain and because there is disagreement regarding the identity of the specific isoforms expressed in kidney. Using a combination of chromatographic and immunological techniques, we have identified two isoforms of creatine kinase in rat kidney supernatants, B creatine kinase, and the non-sarcomeric form of the mitochondrial creatine kinase, which represent 82 and 15%, respectively, of the total creatine kinase activity in this tissue. The identity of the non-muscle form of the mitochondrial creatine kinase was confirmed by N-terminal sequence analysis and compared with recently published cDNA sequences (Haas, R. C., and Strauss, A. W. (1990) J. Biol. Chem. 265, 6921-6927). We prepared multiple antisera specific for each isoform using synthetic peptide immunogens based upon nonhomologous regions from the primary sequence of each creatine kinase isoform. Immunocytochemical results demonstrate that both creatine kinase isoforms are colocalized in the inner stripe of the outer medulla in tubules of the distal nephron. A similar distribution of creatine kinase isoforms was obtained when different layers of the renal cortex and medulla were examined for creatine kinase activity and isozyme content using nondenaturing electrophoresis. In general, the distribution of creatine kinase enzymes in kidney corresponds to the regions of greatest ATP utilization, oxygen consumption, and sodium transport. These results suggest a role for creatine kinase enzymes in the coupling of ion transport and oxidative phosphorylation in the distal nephron of the mammalian kidney.     

Mitochondrial creatine kinase from cardiac muscle and brain are two distinct isoenzymes but both form octameric molecules

Mitochondrial creatine kinase (Mi-CK) from chicken cardiac muscle and brain, recently shown to differ in their N-terminal amino acid sequences and to be encoded by multiple mRNAs (Hossle, H.P., Schlegel, J., Wegmann, G., Wyss, M., B?hlen, P., Eppenberger, H. M., Wallimann, T., and Perriard, J.C. (1988) Biochim. Biophys. Res. Commun. 151, 408-416) were separated on two-dimensional nonequilibrium pH-gradient electrophoresis gels and visualized as two distinct protein spots by immunoblotting. Analysis of the two proteins purified by specific elution from Blue-Sepharose with ADP (Wallimann, T., Zurbriggen, B., and Eppenberger, H. M. (1985) Enzyme 33, 226-231) followed by fast protein liquid chromatography cation exchange chromatography showed obvious differences in peptide maps, in immunological cross-reactivity with monoclonal antibodies, and in kinetic parameters. However, even though the two proteins were different, tissue-specific mitochondrial isoforms, both formed regularly-sized, perforated cube-like octameric structures with Mr of 364,000 +/- 25,000 and 352,000 +/- 20,000 for the cardiac and brain isoform, respectively. Electron microscopy of cardiac and brain Mi-CK octamers revealed cube-like molecules with a central cavity or transverse channel filled by negative stain. The octameric molecular structure of Mi-CK isoforms differs from the generally accepted dimeric arrangement of "cytosolic" muscle MM- and brain BB-CK.     

Characterization of a brain particulate bound form of creatine kinase

A bound form of creatine kinase associated with brain particulate was characterized by isoelectric focusing, antigenicity and chromatography and compared to muscle (MM), brain (BB), and heart mitochondrial isoenzymes. On partial purification and isoelectric focusing, the solubilized enzyme has a pl of 7.3, similar to the pl of muscle creatine kinase MM, pl 6.8, but different from brain creatine kinase BB, which precipitates on isoelectric focusing in sucrose or glycerol stabilized media at its calculated pl of 5.6. Gel filtration chromatography of deoxycholate solubilized particulate creatine kinase on Sephadex Gl50 reveals an estimated molecular weight of approximately 80,000 daltons. The brain particulate enzyme is antigenically distinct from both muscle and rat heart mitochondrial creatine kinase isoenzymes but has antigenic similarity with soluble cytoplasmic brain BB. The situation may be analogous to that found with rat heart mitochondria and rat heart cytoplasmic isoenzymes which we have shown to exhibit antigenic similarity even though differences in electrophoretic and amino acid composition have been demonstrated; however, the confident determination that the particulate enzyme is a separate isoenzyme will have to await amino acid analysis.     

Differential effects of peroxynitrite on human mitochondrial creatine kinase isoenzymes. Inactivation,octamer destabilization,and identification of involved residues

Creatine kinase isoenzymes are very susceptible to free radical damage and are inactivated by superoxide radicals and peroxynitrite. In this study, we have analyzed the effects of peroxynitrite on enzymatic activity and octamer stability of the two human mitochondrial isoenzymes (ubiquitous mitochondrial creatine kinase (uMtCK) and sarcomeric mitochondrial creatine kinase (sMtCK)), as well as of chicken sMtCK, and identified the involved residues. Inactivation by peroxynitrite was concentration-dependent and similar for both types of MtCK isoenzymes. Because peroxynitrite did not lower the residual activity of a sMtCK mutant missing the active site cysteine (C278G), oxidation of this residue is sufficient to explain MtCK inactivation. Mass spectrometric analysis confirmed oxidation of Cys-278 and further revealed oxidation of the C-terminal Cys-358, possibly involved in MtCK/membrane interaction. Peroxynitrite also led to concentration-dependent dissociation of MtCK octamers into dimers. In this study, ubiquitous uMtCK was much more stable than sarcomeric sMtCK. Mass spectrometric analysis revealed chemical modifications in peptide Gly-263-Arg-271 located at the dimer/dimer interface, including oxidation of Met-267 and nitration of Trp-268 and/or Trp-264, the latter being a very critical residue for octamer stability. These data demonstrate that peroxynitrite affects the octameric state of MtCK and confirms human sMtCK as the generally more susceptible isoenzyme. The results provide a molecular explanation of how oxidative damage can lead to inactivation and decreased octamer/dimer ratio of MtCK, as seen in neurodegenerative diseases and heart pathology, respectively.     

Identification of a 43-kDa polypeptide associated with acetylcholine receptor-enriched membranes as MM creatine kinase

Creatine kinase isoenzymes from Torpedo californica electric organ, skeletal muscle, and brain were purified and characterized. Torpedo electric organ and skeletal muscle creatine kinase have identical apparent Mr, electrophoretic mobility, and cyanogen bromide fragments. The electrophoretic mobility of the Torpedo creatine kinase was anodal as compared to mammalian MM creatine kinase. No creatine kinase isoenzyme with an electrophoretic mobility similar to mammalian BB creatine kinase was seen in any of the Torpedo tissues examined. Hybridization studies demonstrate the Torpedo electric organ creatine kinase to be composed of identical subunits and capable of producing an enzymatically active heterodimer when combined with canine BB creatine kinase. Creatine kinase from sucrose gradient-purified Torpedo electric organ acetylcholine receptor-rich membranes has an electrophoretic mobility identical with the cytoplasmic isoenzyme and an apparent Mr identical with mammalian MM creatine kinase. Western blot analysis showed Torpedo electric organ skeletal muscle creatine kinase and acetylcholine receptor-enriched membrane creatine kinase reacted with antiserum specific for canine MM creatine kinase. NH2-terminal amino acid sequence determinations show considerable sequence homology between human MM, Torpedo electric organ, chicken MM, and porcine MM creatine kinase. The acetylcholine receptor-associated creatine kinase is, therefore, identical with the cytoplasmic form from the electric organ and is composed of M-subunits.     

Sequence homology and structure predictions of the creatine kinase isoenzymes

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 guamidino 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.Abbreviations GuaK guanidino kinase - CK creatine kinase - B-and M-CK brain and muscle cytosolic CK isoenzyme - Mi-CK mitochondrial CK isoenzyme - ArgK arginine kinase - Cr creatine - PCr phosphorylcreatine - PArg phosphorylarginine     

Velocity of the creatine kinase reaction in the neonatal rabbit heart: role of mitochondrial creatine kinase

To examine the role of changes in the distribution of the creatine kinase (CK) isoenzymes [BB, MB, MM, and mitochondrial CK (mito-CK)] on the creatine kinase reaction velocity in the intact heart, we measured the creatine kinase reaction velocity and substrate concentrations in hearts from neonatal rabbits at different stages of development. Between 3 and 18 days postpartum, total creatine kinase activity did not change, but the isoenzyme distribution and total creatine content changed. Hearts containing 0, 4, or 9% mito-CK activity were studied at three levels of cardiac performance: KCl arrest and Langendorff and isovolumic beating. The creatine kinase reaction velocity in the direction of MgATP production was measured with 31P magnetization transfer under steady-state conditions. Substrate concentrations were measured with 31P NMR (ATP and creatine phosphate) and conventional biochemical analysis (creatine) or estimated (ADP) by assuming creatine kinase equilibrium. The rate of ATP synthesis by oxidative phosphorylation was estimated with oxygen consumption measurements. These results define three relationships. First, the creatine kinase reaction velocity increased as mito-CK activity increased, suggesting that isoenzyme localization can alter reaction velocity. Second, the reaction velocity increased as the rate of ATP synthesis increased. Third, as predicted by the rate equation, reaction velocity increased with the 3-fold increase in creatine and creatine phosphate contents that occurred during development.     

Interfacial behavior of cytoplasmic and mitochondrial creatine kinase oligomeric states

Adsorption to the air/water interface of isoenzymes of creatine kinase was investigated using surface pressure-area isotherms and Brewster angle microscopy (BAM) observations. Octameric mitochondrial creatine kinase (mtCK) exhibits a significant affinity for the air/water interface. Whatever the mode of formation of the interfacial film, i.e., injection of the protein in the subphase or spreading onto the buffer surface, the final arrangement and conformation adopted by mtCK molecules lead to a similar result. In contrast, the dimeric isoenzymes mtCK and cytosolic MMCK do not induce any surface pressure variation. However, when the subphase contains 0.3M NaCl, both isoenzymes adsorb to the interface. When treated with 0.8 or 3M GdnHCl, muscle creatine kinase (MMCK) becomes surface active and occupies a greater surface than mtCK. This result contrasts with previous observations, often derived from monomeric proteins, that their surface activity is increased upon unfolding. It underlines the possible influence exerted by the protein oligomeric state on its interfacial activity. At a subphase pH of 8.8, which corresponds to the pI of octameric mtCK, the profiles of the isotherms obtained with dimeric and octameric states and the resistance to compression of the protein monolayers are significantly affected when compared to those recorded at pH 7.4. These data suggest that the octamer is more hydrophobic than the dimer and may contribute to explaining why octamers bind to the inner mitochondrial membrane while dimers do not.     

Muscle creatine kinase isoenzyme expression in adult human brain.

Previous studies have suggested that MM creatine kinase is a muscle-specific protein and is not present in adult brain tissue. We have isolated a protein from human brain with an apparent molecular weight of 43,000 as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis which is identical to the muscle M creatine kinase isoenzyme subunit at all 30 sequenced amino acid residues and possesses creatine kinase enzymatic activity following nondenaturing agarose-gel electrophoresis. Immunohistochemistry localizes M creatine kinase to discrete areas of adult human brain. Northern blot analysis of both total and poly(A)-selected RNA isolated from brain did not detect M creatine kinase mRNA. However, polymerase chain reaction amplification of cDNA synthesized from human placenta, heart, and brain mRNA detected M creatine kinase message in both heart and brain but not placenta which contains no detectable M creatine kinase protein. N1E115 and NS20Y, mouse neuroblastoma cell lines which have been used as models of neural cell differentiation, were found also to express MM creatine kinase. Moreover, a transiently transfected reporter gene with 4,800 base pairs of M creatine kinase upstream region fused to chloramphenicol acetyltransferase was expressed during differentiation of these neural cell lines. In summary, MM creatine kinase is present in human brain and we suggest the M creatine kinase upstream region is sufficient to modulate M creatine kinase expression in certain neuronal cells and may be regulated independently from other muscle genes.     

Resonance energy transfer between the active sites of myocardial-type creatine kinase (isozyme MB)

The single reactive sulfhydryl group, located in the active site of each subunit of dimeric creatine kinase from rabbit muscle (isozyme MM), was selectively labeled with 3-(4-maleimidylphenyl)-7-(diethylamino)-4-methylcoumarin (CPM). Isozyme BB, purified to homogeneity from rabbit brain, was conjugated with the sulfhydryl-specific reagent 5'-(iodoacetamido)fluorescein (5'-IAF). Spectral analyses demonstrated that 1.8 mol of CPM and 1.9 mol of 5'-IAF had reacted per mol of protein. Labeled isozymes were combined, denatured in 8 M urea, and renatured by dialysis, producing the parent labeled homodimers and forming the heterolabeled hybrid dimer, creatine kinase MB. Similar hybridizations were performed to prepare singly labeled hybrids, starting with labeled and unlabeled homodimers. The hybrid isozymes were isolated by ion-exchange chromatography, and spectral analyses of singly labeled heterodimers revealed overlap between the absorption spectrum of MB labeled with acetamidofluorescein on the B subunit and the corrected fluorescence emission spectrum of MB labeled with CPM on the M subunit. Analyses included evaluation of the quantum yield of the CPM-labeled hybrid, estimation of the range of the orientation factor K2 from fluorescence polarization and anisotropy studies, and determination of J, the spectral overlap integral for the fluorescence donor (CPM-labeled MB) and acceptor (acetamidofluorescein-labeled MB). Results of these experiments permitted an estimation of R0, the distance between the donor and the acceptor at which energy transfer is 50% efficient. Comparison of the relative fluorescence of the donor in the presence (heterolabeled hybrid) and absence (hybrid conjugated with CPM on the M subunit) of the acceptor or determination of the normalized sensitization of the acceptor fluorescence led to an evaluation of the transfer efficiency and the actual transfer distance of between 27 and 52 A.(ABSTRACT TRUNCATED AT 250 WORDS)     

Enzyme termini of a phosphocreatine shuttle. Purification and characterization of two creatine kinase isozymes from sea urchin sperm

Two isozymes of creatine kinase have been purified from sperm of the sea urchin, Strongylocentrotus purpuratus. One isozyme was purified from the sperm flagellum, and the other from the head. Both require nonionic detergent for extraction from sperm. The flagellar isozyme is a monomeric species with an Mr of 145,000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and 126,000 from sucrose density gradient and gel filtration analyses. Creatine kinase from sperm heads was localized to the mitochondrion by an antibody raised against mouse muscle creatine kinase. This purified mitochondrial isozyme is multimeric, with an Mr of 47,000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, but 240,000 for the native enzyme. Peptide mapping indicates that the two isozymes are not related. The following kinetic characteristics were observed for the purified flagellar and mitochondrial isozymes, respectively. In the direction of ATP formation, at pH 6.6 and 25 degrees C, specific activities were 235 and 180 units/mg; pH optima were 6.7 and 6.9 and Michaelis constants were 0.13 and 0.055 mM for ADP and 5.8 and 2.7 mM for phosphocreatine. In the direction of phosphocreatine formation, at pH 7.5 and 25 degrees C, specific activities were 29 and 47 units/mg; pH optima were 7.5 and 7.7 and Michaelis constants were 0.89 and 0.31 mM for ATP and 39 and 62 mM for creatine. These unique isozymes constitute the termini of the phosphocreatine shuttle of sea urchin sperm that is responsible for energy transport from the mitochondrion to the distal flagellum (Tombes, R. M., and Shapiro, B. M. (1985) Cell 41, 325-334; Tombes, R. M., Brokaw, C. J., and Shapiro, B. M. (1987) Biophys. J., 52, 75-86).     

Separate nuclear genes encode sarcomere-specific and ubiquitous human mitochondrial creatine kinase isoenzymes

Creatine kinase (EC 2.7.3.2) isoenzymes play a central role in energy transduction. Nuclear genes encode creatine kinase subunits from muscle, brain, and mitochondria (MtCK). We have recently isolated a cDNA clone encoding MtCK from a human placental library which is expressed in many human tissues (Haas, R. C., Korenfeld, C., Zhang, Z., Perryman, B., Roman, D., and Strauss, A. W. (1989) J. Biol. Chem. 264, 2890-2897). With nontranslated and coding region probes, we demonstrated by RNA blot analysis that the MtCK mRNA in sarcomeric muscle is distinct from this placenta-derived, ubiquitous MtCK cDNA. To compare these different mRNAs, a MtCK cDNA clone was isolated from a human heart library and characterized by complete nucleotide sequence analysis. The chemically determined NH2-terminal 26 residues of purified human heart MtCK protein are identical to those predicted from this sarcomeric MtCK cDNA. The human sarcomeric and ubiquitous cDNAs share 73% nucleotide and 80% predicted amino acid sequence identities, but have less than 66% identity with the cytosolic creatine kinases. The sarcomeric MtCK cDNA encodes a 419-amino acid protein which contains a 39-residue transit peptide essential for mitochondrial import. Primer extension analysis predicts a 348-base pair 5'-nontranslated region. RNA blot analysis demonstrates that heart-derived MtCK is sarcomere-specific, but the ubiquitous MtCK mRNA is expressed in most tissues. Thus, separate nuclear genes encode two closely related, tissue-specific isoenzymes of MtCK. Our finding that multiple genes encode different mitochondrial protein isoenzymes is rare.     

Properties of human creatine kinase isoenzymes]

Properties of human creatine kinase isoenzymes (MM, MB and BB) are investigated. The most pronounced differences in properties of these isoenzymes are found under their urea inactivation, heat denaturation and the inhibition by rabbit antisera to isoenzymes. Differences in values of the Mikhaelis constant and substrate and pH dependencies are much less pronounced. The presence of ADP stabilizes creatine kinase isoenzymes under conditions of urea and heat inactivation. Properties of hybrid MB isoenzymes are found to be intermediate with respect to MM and BB isoenzymes. A mode of the interaction of M and B subunits in dimeric molecules of creatine kinase isoenzymes is discussed.     

Mathematical model of compartmentalized energy transfer: Its use for analysis and interpretation of 31P-NMR studies of isolated heart of creatine kinase deficient mice

A mathematical model of the compartmentalized energy transfer in cardiac cells is described and used for interpretation of novel experimental data obtained by using phosphorus NMR for determination of the energy fluxes in the isolated hearts of transgenic mice with knocked out creatine kinase isoenzymes. These experiments were designed to study the meaning and importance of compartmentation of creatine kinase isoenzymes in the cells in vivo. The model was constructed to describe quantitatively the processes of energy production, transfer, utilization, and feedback between these processes. It describes the production of ATP in mitochondrial matrix space by ATP synthase, use of this ATP for phosphocreatine production in the mitochondrial creatine kinase reaction coupled to the adenine nucleotide translocation, diffusional exchange of metabolites in the cytoplasmic space, and use of phosphocreatine for resynthesis of ATP in the myoplasmic creatine kinase reaction. It accounts also for the recently discovered phenomenon of restricted diffusion of adenine nucleotides through mitochondrial outer membrane porin pores (VDAC). Practically all parameters of the model were determined experimentally. The analysis of energy fluxes between different cellular compartments shows that in all cellular compartments of working heart cells the creatine kinase reaction is far from equilibrium in the systolic phase of the contraction cycle and approaches equilibrium only in cytoplasm and only in the end-diastolic phase of the contraction cycle.Experimental determination of the relationship between energy fluxes by a ³¹P-NMR saturation transfer method and workload in isolated and perfused heart of transgenic mice deficient in MM isoenzyme of the creatine kinase, MM -/- showed that in the hearts from wild mice, containing all creatine kinase isoenzymes, the energy fluxes determined increased 3-4 times with elevation of the workload. By contrast, in the hearts in which only the mitochondrial creatine kinase was active, the energy fluxes became practically independent of the workload in spite of the preservation of 26% of normal creatine kinase activity. These results cannot be explained on the basis of the conventional near-equilibrium theory of creatine kinase in the cells, which excludes any difference between creatine kinase isoenzymes. However, these apparently paradoxical experimental results are quantitatively described by a mathematical model of the compartmentalized energy transfer based on the steady state kinetics of coupled creatine kinase reactions, compartmentation of creatine kinase isoenzymes in the cells, and the kinetics of ATP production and utilization reactions. The use of this model shows that: (1) in the wild type heart cells a major part of energy is transported out of mitochondria via phosphocreatine, which is used for complete regeneration of ATP locally in the myofibrils - this is the quantitative estimate for PCr pathway; (2) however, in the absence of MM-creatine kinase in the myofibrils in transgenic mice the contraction results in a very rapid rise of ADP in cytoplasmic space, that reverses the mitochondrial creatine kinase reaction in the direction of ATP production. In this way, because of increasing concentrations of cytoplasmic ADP, mitochondrial creatine kinase is switched off functionally due to the absence of its counterpart in PCr pathway, MM-creatine kinase. This may explain why the creatine kinase flux becomes practically independent from the workload in the hearts of transgenic mouse without MM-CK. Thus, the analysis of the results of studies of hearts of creatine kinase-deficient transgenic mice, based on the use of a mathematical model of compartmentalized energy transfer, show that in the PCr pathway of intracellular energy transport two isoenzymes of creatine kinase always function in a coordinated manner out of equilibrium, in the steady state, and disturbances in functioning of one of them inevitably result in the disturbances of the other component of the PCr pathway. In the latter case, energy is transferred from mitochondria to myofibrils by alternative metabolic pathways, probably involving adenylate kinase or other systems.     

Homogeneous chicken heart mitochondrial creatine kinase purified by dye-ligand and transition-state analog-affinity chromatography

A method for the preparation of homogeneous mitochondrial creatine kinase from chicken heart is presented. The two-column procedure, which can be completed in 2 days, uses Procion red dye and transition-state analog-affinity chromatography. The transition-state analog-affinity chromatographic system utilizes an ADP-hexane-agarose column in conjunction with the transition-state analog complex originally developed by E. J. Milner-White and D. C. Watts (1971, Biochem, J. 122, 727-740) composed of KNO3, MgCl2, creatine, and ADP. The enzyme is a dimer composed of 2 Mr 43,000 subunits. The sequence of the first N-terminal 20 amino acids shows that the enzyme is different from the cytosolic isozymes but similar to human mitochondrial creatine kinase. The enzyme has an extinction coefficient of epsilon 280 nm = 2.22 +/- 0.10 ml X mg-1 X cm-1 and a maximum velocity of 200 IU/ml at pH 7.0. The kinetic constants for the chicken heart mitochondrial isozyme are comparable to values for the canine and human heart isozyme.