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.     

Separation of mitochondrial creatine kinase (MiMi-CK) from cytosolic creatine kinase isoenzymes by Cibachrome-Blue affinity chromatography

The mitochondrial isoenzyme of creatine kinase (MiMi-CK) was separated by affinity chromatography on Cibachrome-Blue-Sepharose (Sepharose-Blue, Pharmacia). While the soluble CK isoforms (BB-CK and MM-CK) were specifically eluted by raising the pH of the column buffer from pH 6.0 to pH 8.0, MiMi-CK remained bound under these conditions but was specifically eluted by subsequent addition of ADP to the pH 8.0 buffer. This one-step method allows a fast and efficient separation of MiMi-CK from MM-and BB-CK isoenzymes and at the same time an enrichment of MiMi-CK by about 50-fold. Since MiMi-CK can be assayed separately after isolation by affinity chromatography on Sepharose-Blue, this method may be of clinical importance.     

Differential heat sensitivity of human creatine kinase isoenzymes.

Creatine kinase isoenzymes (MM, MB and BB) were isolated from human tissue by ion-exchange chromatography. The B subunit was found to be more heat sensitive than the M subunit. BB and MB isoenzymes respond similarly to heat inactivation. Our results are in contrast with the body temperature inactivation of the brain isoenzyme reported by Lindsey and Diamond.     

Aggregation and folding of recombinant human creatine kinase

The processes of aggregation and refolding of recombinant human creatine kinase (rHCK) were studied. Most of the rHCK expressed in E. coli was present in the insoluble traction and it could be solubilized in 6 M urea solution. Unfolding of rHCK in 6 M urea showed biphasic kinetic courses (kappa1 = 6.5 x 10(-3) s(-1); kappa2 = 0.54 x 10(-3) s(-1)) as observed by maximum fluorescence wavelength change. During refolding of the rHCK dissolved in urea, significant aggregation was noticed following first-order kinetics. Aggregation rate constants were influenced by the concentration of NaCl, which increased the difference in transition-free energy (deltadeltaG), showing that stabilization of folding intermediates by NaCl could efficiently reduce the formation of insoluble aggregates. Formations of aggregate were also reduced by adjusting temperature, pH, and concentration of rHCK. Refolding of rHCK under the optimized condition which prevented the aggregation also showed multi-kinetic phases (kappa1 = 3.0 x 10(-3) s(-1); kappa2 = 0.64 x 10(-3) s(-1)). Under optimized conditions applied in this study, rHCK could correctly refold retrieving the high specific enzymatic activity.     

Molecular heterogeneity of creatine kinase isoenzymes

The [32P]phosphoamino acids in proteins of first trimester and term-cultured human placentas have been separated and their relative amounts were measured. A significant phosphorylation of tyrosine residues could be detected in the cultured placental tissue at different stages of gestation. The phosphotyrosine accounts for 2-4% of the total acid-stable phosphate in the phosphoamino acids after partial acid hydrolysis. The difference in the extent of [32P]tyrosine in various placentas seems to be a function of biological variation of the individual placentas, rather than a function of placental age and stage of gestation. In contrast, a significant difference in the phosphorylation ratio of serine and threonine could be measured between first trimester and term placentas. As more evidence is accumulating that protein phosphorylation of tyrosine is involved in the processes of cellular growth and proliferation, our findings of the relatively high tyrosine phosphorylation in human placenta strongly suggest that this type of protein phosphorylation may play an important role in the placental growth and development. Furthermore, these findings may correlate with the existence of the endogenous RNA virus-like particles found in normal human placenta.     

Aggregation of creatine kinase during refolding and chaperonin-mediated folding of creatine kinase

The course of refolding and reactivation of urea-denatured creatine kinase (ATP; creatine N-phosphotransferase, EC 2.7.3.2) has been studied in the absence and presence of molecular chaperonin GroEL. The enzyme was denatured in Tris--HCl buffer containing 6 M urea for 1 h. In the refolding studies, the denatured enzyme was diluted 60-fold into the same buffer containing GroEL or not for activity, turbidity, fluorescence measurements and polyacrylamide gel electrophoresis. The results show that the reactivation process is dependent of creatine kinase concentration in the concentration range 2.5--4 microM. The levels of activity recovery decrease with increasing enzyme concentration because of the formation of wrong aggregates. The molecular chaperonin GroEL can bind the refolding intermediate of creatine kinase and thus prevent the formation of wrong aggregates. This intermediate is an inactive dimeric form that is in a conformation resembling the 'molten globule' state.     

Serum creatine kinase isoenzymes in progressive muscular dystrophy

Creatine kinase isoenzymes in sera and muscle biopsies obtained from 50 controls, 72 patients with progressive muscular dystrophy (PMD), 68 patients with other neuromuscular disorders, 17 carriers of Duchenne-type PMD and 15 patients with myocardial infarction were studied. MB isoenzyme was detected in the sera of 58 patients with PMD and 56 out of 61 muscle biopsies. The MB activity varied between 4 and 400 IU/1 or 3.4--22% of total activity. The MB activity was demonstrated in a considerably smaller number of cases with polymyositis, dystrophic myotonia and Kugelberg-Welander disease. The MB isoenzyme in sera of PMD persisted for many years. It is admitted that the MB isoenzyme in the serum of patients with PMD originates chiefly from skeletal muscle.     

Functional aspects of creatine kinase isoenzymes in endothelial cells

To characterize the isoenzyme distribution of creatine kinase (CK) in endothelial cells (ECs) and its functional role during substrate depletion, ECs from aorta (AECs) and microvasculature (MVECs) of pig and rat were studied. In addition, high- energy phosphates were continuously monitored by (31)P NMR spectroscopy in pig AECs attached to microcarrier beads. CK activity per milligram of protein in rat AECs and MVECs (0.08 +/- 0.01 and 0.15 +/- 0.08 U/mg, respectively) was <3% of that of cardiomyocytes (6.46 +/- 1.02 U/mg). Rat and pig AECs and MVECs displayed cytosolic BB-CK, but no MM-CK. Gel electrophoresis of mitochondrial fractions of rat and pig ECs indicated the presence of mitochondrial Mi-CK, mostly in dimeric form. The presence of Mi(a)-CK was demonstrated by indirect immunofluorescence staining using Mi(a)-CK antibodies. When perifused with creatine-supplemented medium, phosphocreatine (PCr) continuously increased with time (1.2 +/- 0.6 nmol x h(-1) x mg x protein(-1)), indicating creatine uptake and CK activity. Glucose withdrawal from the medium induced a rapid decrease in PCr, which was fully reversible on glucose addition, demonstrating temporal buffering of an energy deficit. Because both cytosolic and mitochondrial CK isoforms are present in ECs, the CK system may also contribute to energy transduction ("shuttle hypothesis").     

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.     

Divergent enzyme kinetics and structural properties of the two human mitochondrial creatine kinase isoenzymes

The mitochondrial isoenzymes of creatine kinase (MtCK), ubiquitous uMtCK and sarcomeric sMtCK, are key enzymes of oxidative cellular energy metabolism and play an important role in human health and disease. Very little is known about uMtCK in general, or about sMtCK of human origin. Here we have heterologously expressed and purified both human MtCK isoenzymes to perform a biochemical, kinetic and structural characterization. Both isoenzymes occurred as octamers, which can dissociate into dimers. Distinct Stokes' radii of uMtCK and sMtCK in solution were indicative for conformational differences between these equally sized proteins. Both human MtCKs formed 2D-crystals on cardiolipin layers, which revealed further subtle differences in octamer structure and stability. Octameric human sMtCK displayed p4 symmetry with lattice parameters of 145 A, indicating a 'flattening' of the octamer on the phospholipid layer. pH optima and enzyme kinetic constants of the two human isoenzymes were significantly different. A pronounced substrate binding synergism (Kd > Km) was observed for all substrates, but was most pronounced in the forward reaction (PCr production) of uMtCK and led to a significantly lower Km for creatine (1.01 mM) and ATP (0.11 mM) as compared to sMtCK (creatine, 7.31 mM; ATP, 0.68 mM).     

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     

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.     

Kinetic, thermodynamic, and developmental consequences of deleting creatine kinase isoenzymes from the heart. Reaction kinetics of the creatine kinase isoenzymes in the intact heart

Creatine kinase (CK) exists as a family of isoenzymes in excitable tissue. We studied isolated perfused hearts from mice lacking genes for either the main muscle isoform of CK (M-CK) or both M-CK and the main mitochondrial isoform (Mt-CK) to determine 1) the biological significance of CK isoenzyme shifts, 2) the necessity of maintaining a high CK reaction rate, and 3) the role of CK isoenzymes in establishing the thermodynamics of ATP hydrolysis. (31)P NMR was used to measure [ATP], [PCr], [P(i)], [ADP], pH, as well as the unidirectional reaction rate of PCr--> [gamma-P]ATP. Developmental changes in the main fetal isoform of CK (BB-CK) were unaffected by loss of other CK isoenzymes. In hearts lacking both M- and Mt-CK, the rate of ATP synthesis from PCr was only 9% of the rate of ATP synthesis from oxidative phosphorylation demonstrating a lack of any high energy phosphate shuttle. We also found that the intrinsic activities of the BB-CK and the MM-CK isoenzymes were equivalent. Finally, combined loss of M- and Mt-CK (but not loss of only M-CK) prevented the amount of free energy released from ATP hydrolysis from increasing when pyruvate was provided as a substrate for oxidative phosphorylation.     

Role of C-terminal sequences in the folding of muscle creatine kinase

Proteinase K selectively nicks the native homodimeric muscle creatine kinase (MM-CK) into two 37.1 kDa N-terminal (K1) and two 5.8 kDa C-terminal (K2) fragments that remain firmly associated in a native-like, although inactive, heterotetrameric structure. This truncated protein has been named (K1K2)(2). To analyze the role of the C-terminal peptide in the protein structure acquisition, we studied in vitro refolding of the guanidinium chloride-denatured (K1K2)(2). Although they never reassociate with K2, in selected conditions the K1 fragments refold slowly to a dimeric state as shown by size exclusion chromatography data. This K1 dimer exhibits a fluorescence emission lambda max of 335 nm, a high degree of tyrosine exposure, strongly binds ANS but not MgADP, a CK substrate, and according to these structural characteristics, could be a dimeric molten globule species. We propose a folding model that takes into account the existence of a new transient intermediate state in the MM-CK refolding process. Besides two monomeric premolten and molten globule kinetic intermediates and the active final dimeric form, an inactive dimer, with partly compacted monomers, must ephemerally exist. Our results strongly suggest that the C-terminal end of the protein accelerates folding and plays a critical role for monomer final packing into a native-like conformation. The data also indicate that MM-CK catalytic efficiency is only acquired after dimerization.     

Effects of osmolytes on human brain-type creatine kinase folding in dilute solutions and crowding systems

The effects of osmolytes on the unfolding and refolding process of recombinant human brain-type creatine kinase (rHBCK) were comparatively, quantitatively studied in dilute solutions and macromolecular crowding systems (simulated by 100g/L polyethylene glycol 2000), respectively. The results showed that the osmolytes, including glycerol, sucrose, dimethylsulfoxide, mannitol, inositol, and xylitol, could both protect the rHBCK from denaturation induced by 0.8M GdnHCl and aid in the refolding of denatured-rHBCK in macromolecular crowding systems. When we examined the effects of sucrose and xylitol on the parameters of residual activity, reaction kinetics and intrinsic fluorescence of rHBCK during unfolding, it was found that the protecting effects of osmolytes in a macromolecular crowding system were more significant compared with those in a dilute solution, which resulted in more residual activities, protected the conformational changes and greatly decreased the rates of both the fast and slow tracks. Regarding the effects of glycerol, sucrose and mannitol on the denatured-rHBCK refolding parameters of refolding yield, reaction kinetics and aggregation, the results indicated that the osmolytes could alleviate the aggregation of rHBCK during refolding in both dilute solutions and macromolecular crowding systems, and the refolding yields and reaction rates under macromolecular crowding environment could be increased by the addition of osmolytes, though higher yields were obtained in the dilute solution. For further insight, osmolyte docking simulations and rHBCK denaturation were conducted successfully and confirmed our experimental results. The predictions based on the docking simulations suggested that the deactivation of guanidine may be blocked by osmolytes because they share common binding sites on rHBCK, and the higher number of interactions with rHBCK by osmolytes than guanidine may be one of the causes of rHBCK refolding. In brief, the additive effects of the exclusive volume effect from the macromolecular crowding system and the osmophobic effects from the osmolytes resulted in better performance of the osmolytes in a macromolecular crowding system, which also led to a better understanding of protein folding in the intracellular environment.     

A brief summary of the history of the detection of creatine kinase isoenzymes

Molecular and Cellular Biochemistry -