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).

Octamer formation and coupling of cardiac sarcomeric mitochondrial creatine kinase are mediated by charged N-terminal residues.

Mitochondrial creatine kinases form octameric structures composed of four active and stable dimers. Octamer formation has been postulated to occur via interaction of the charged amino acids in the N-terminal peptide of the mature enzyme. We altered codons for charged amino acids in the N-terminal region of mature sarcomeric mitochondrial creatine kinase (sMtCK) to those encoding neutral amino acids. Transfection of normal sMtCK cDNA or those with the mutations R42G, E43G/H45G, and K46G into rat neonatal cardiomyocytes resulted in enzymatically active sMtCK expression in all. After hypoosmotic treatment of isolated mitochondria, mitochondrial inner membrane-associated and soluble sMtCK from the intermembranous space were measured. The R42G and E43G/H45G double mutation caused destabilization of the octameric structure of sMtCK and a profound reduction in binding of sMtCK to the inner mitochondrial membrane. The other mutant sMtCK proteins had modest reductions in binding. Creatine-stimulated respiration was markedly reduced in mitochondria isolated from cells transfected with the R42G mutant cDNA as compared with those transfected with normal sMtCK cDNA. We conclude that neutralization of charges in N-terminal peptide resulted in destabilization of octamer structure of sMtCK. Thus, charged amino acids at the N-terminal moiety of mature sMtCK are essential for octamer formation, binding of sMtCK with inner mitochondrial membrane, and coupling of sMtCK to oxidative phosphorylation.

Cardiac phosphocreatine deficiency induced by GPA during postnatal development in rat.

The effect of chronic administration of beta-guanidinopropionic acid (GPA) on the protein profiling, energy metabolism and right ventricular (RV) function was studied in the rat heart during the weaning and adolescence period. GPA was given in tap water (1-1.5%) using pair drink controls. The feeding of animals with GPA solution for a six week period resulted in elevation of heart to body weight ratio due to body growth retardation. GPA accumulated in the myocardium up to 67.37 +/- 5.3 mumoles.g dry weight and the tissue content of total creatine, phosphocreatine and ATP was significantly decreased to 15%, 9% and 65% of control values respectively. Total activity of creatine kinase (CK) was not changed, but the proportion of mitochondrial (Mi) CK isoenzyme was decreased; the percentage of MB isoenzyme of CK was significantly higher. GPA treatment resulted in an elevation of the content of cardiac collagenous proteins and decrease of non-collagenous proteins in the heart; in parallel, a decrease of the collagen I to collagen III ratio was detected. The function of the RV was assessed using an isolated perfused heart with RV performing pressure-volume work. As compared to pair-drink controls, RV function was significantly impaired the GPA group: at any given right atrial filling pressure, the RV systolic pressure and the rate of pressure development were decreased by almost a factor of two. Elevation of the RV diastolic pressure with increasing pulmonary artery diastolic pressure was also significantly steeper in the GPA group which also showed decrease of cardiac output, especially at high outflow resistance. It may be assumed that chronic administration of GPA deeply influenced metabolic parameters, protein profiles and contractile function of the developing heart. On the other hand, concentrations of glucose, total lipids and triglycerides in blood plasma were not affected. All these data confirm the concept that the CK system is of central importance both for heart function and for the regulation of normal growth of cardiac myocytes.

Control of cellular respiration in vivo by mitochondrial outer membrane and by creatine kinase. A new speculative hypothesis: possible involvement of mitochondrial-cytoskeleton interactions.

The current problems of regulation of myocardial energy metabolism and oxidative phosphorylation in vivo are considered. With this purpose, retarded diffusion of ADP in cardiomyocytes was studied by analysis of elevated apparent Km for this substrate in regulation of respiration of saponin-skinned cardiac fibers, as compared to isolated mitochondria. Recently published data showing the importance of the outer mitochondrial membrane were compared with new experimental results on the proteolysis of skinned fibers and tissue homogenates. In both cases 10 min incubation and 0.125 mg/ml of trypsin resulted in a decrease of apparent Km for ADP from 297 +/- 35 and 228 +/- 16 to 109 +/- 2 and 36 +/- 16, respectively. Thus, the permeability of the outer mitochondrial membrane for ADP may be controlled by some unknown cytoplasmic protein(s), probably related to the cytoskeleton, which are separated from mitochondria during their isolation. The extent of expression of this protein(s) depends on the energy state and type of muscle. Activation of mitochondrial creatine kinase reaction coupled to oxidative phosphorylation overcomes the diffusion difficulties of ADP by amplifying the stimulatory effect of ADP on respiration. It is concluded that both cytoplasmic and mitochondrial creatine kinases, adenylate kinase and cytoplasmic factor controlling outer membrane permeability may participate in metabolic feedback regulation of respiration in muscle cells.

Myocardial adaptation to long-term action of substances associated with decreased intensity of cardiac function.

The effect of prolonged treatment of rats with 6-n-propyl-2-thiouracil (PTU), verapamil, or propranolol on cardiac pump function and the properties of myofibrils and mitochondria was studied. After 6-8 weeks of treatment, the heart rate and maximal cardiac output of the isolated heart of rats treated with verapamil or propranolol were higher than those in the control group. The PTU treatment was followed by lower heart rate and maximal work. Calcium sensitivity (pCa50 value) of skinned ventricular fibers was higher in all experimental groups compared to the control by 0.07-0.15 units. Myofibrillar Mg2+, Ca(2+)-ATPase activity measured in isolated Triton-skinned cardiomyocytes was considerably lower after PTU treatment than that in respective controls (0.128 +/- 0.013 vs 0.178 +/- 0.010 mumol Pi/min/mg protein). In contrast, long-term treatment with verapamil or propranolol was accompanied by increased activity to 0.223 +/- 0.018 and 0.254 +/- 0.015 mumol Pi/min/mg protein, respectively. Neither the basal mitochondrial respiration rate of saponin-skinned cardiac fibers nor its enhancement after addition of low ADP concentration or creatine was significantly altered in any experimental group. Also no difference between control and experimental groups was observed in the total activity of creatine kinase or relative percentage of its isoenzymes extracted from cardiac tissue. Thus the changes in cardiac pump function after prolonged treatment with agents decreasing cardiac function may be attributed to concomitant alterations of myofibrils while mitochondria remain relatively intact.

Metabolic compartmentation and substrate channelling in muscle cells. Role of coupled creatine kinases in in vivo regulation of cellular respiration--a synthesis.

The published experimental data and existing concepts of cellular regulation of respiration are analyzed. Conventional, simplified considerations of regulatory mechanism by cytoplasmic ADP according to Michaelis-Menten kinetics or by derived parameters such as phosphate potential etc. do not explain relationships between oxygen consumption, workload and metabolic state of the cell. On the other hand, there are abundant data in literature showing microheterogeneity of cytoplasmic space in muscle cells, in particular with respect to ATP (and ADP) due to the structural organization of cell interior, existence of multienzyme complexes and structured water phase. Also very recent experimental data show that the intracellular diffusion of ADP is retarded in cardiomyocytes because of very low permeability of the mitochondrial outer membrane for adenine nucleotides in vivo. Most probably, permeability of the outer mitochondrial membrane porin channels is controlled in the cells in vivo by some intracellular factors which may be connected to cytoskeleton and lost during mitochondrial isolation. All these numerous data show convincingly that cellular metabolism cannot be understood if cell interior is considered as homogenous solution, and it is necessary to use the theories of organized metabolic systems and substrate-product channelling in multienzyme systems to understand metabolic regulation of respiration. One of these systems is the creatine kinase system, which channels high energy phosphates from mitochondria to sites of energy utilization. It is proposed that in muscle cells feed-back signal between contraction and mitochondrial respiration may be conducted by metabolic wave (propagation of oscillations of local concentration of ADP and creatine) through cytoplasmic equilibrium creatine and adenylate kinases and is amplified by coupled creatine kinase reaction in mitochondria. Mitochondrial creatine kinase has experimentally been shown to be a powerful amplifier of regulatory action of weak ADP fluxes due to its coupling to adenine nucleotide translocase. This phenomenon is also carefully analyzed.

Influence of the mitochondrial outer membrane and the binding of creatine kinase to the mitochondrial inner membrane on the compartmentation of adenine nucleotides in the intermembrane space of rat heart mitochondria.

The influence of the mitochondrial outer membrane and that of the binding of creatine kinase to the mitochondrial inner membrane on the compartmentation of adenine nucleotides in the intermembrane space of rat heart mitochondria were investigated under conditions of maximal rates of mitochondrial creatine kinase. To this end, experiments were performed in reconstituted systems consisting of functionally intact rat heart mitochondria or mitoplasts and pyruvate kinase, both competing at ADP formed by mitochondrial creatine kinase in the presence of high concentrations of adenine nucleotides. Results showed that removal of the mitochondrial outer membrane diminished the compartmentation effects, supporting the relevance of the mitochondrial outer membrane to compartmentation effects in the intermembrane space. Compartmentation effects were clearly seen even in KCl-containing media which release the creatine kinase from the inner membrane, indicating that the localization of the ADP-regenerating enzyme in the mitochondrial intermembrane space within the outer membrane is a prerequisite for dynamic compartmentation of adenine nucleotides. Under these conditions, the binding of creatine kinase to the inner membrane is of minor importance. Lowering the ATP concentrations diminished the extent of AdN compartmentation due to decreased creatine kinase rates. Under these conditions of low AdN fluxes, the release of creatine kinase from the inner membrane entailed another decrease in the compartmentation.

[Cellular mechanisms of impaired cardiac energetics in patients with dilated cardiomyopathy: decrease in mitochondrial respiration and creatine kinase expression]. / Kletochnye mekhanizmy narusheniia énergetiki serdtsa u bol'nykh s dilatatsionnoi kardiomiopatiei: umen'shenie mitokhondrial'nogo dykhaniia i ékspressii kreatinkinazy.

The mitochondrial functional characteristics were assessed in the biopsy specimens from patients with various Functional Classes dilated cardiomyopathy (DCMP). The assessment was made by using endomyocardial biopsy specimens weighing 2-4 mg which had been taken from 39 patients aged 19-64 years during coronary ventriculography and cardiac transplantation. The status of mitochondria and the efficiency of mitochondrial creatine kinase functioning were evaluated by recording the respiration of saponin-skinned muscular fibers. The maximum mitochondrial respiration rate calculated on a dry weight basis was not substantially different in all functional classes of DCMP, while the acceptor control index (Vmax/V0) and the level of creatine-activated respiration decreased with an increase in the functional class of DCMP. The findings show a good positive correlation between ejection fraction and creatine-stimulated respiration values and a linear negative correlation between this parameter and end-diastolic pressures. Thus, the respiratory parameters of mitochondria in the endomyocardial biopsy specimens may be used to assess the severity of cardias lesions.

The creatine kinase system and cardiomyopathy.

Changes in the creatine kinase system, cellular energetics, regulation of respiration and alterations in parameters of contractility in experimental animals (myopathic hamsters), and in patients with dilated cardiomyopathy were studied. 31P-NMR methods were used to show that cardiomyopathic hearts are characterized by decreased work index, lower tissue ATP, phosphocreatine, and total creatine contents and diminished creatine kinase activity and energy fluxes. In isolated mitochondria, only the creatine kinase activity was decreased. Both in cardiomyopathic hamsters and human hearts a share of mitochondrial creatine kinase in the total tissue enzyme activity was decreased from 33% to 18% and that of BB elevated from 5% in control to 20%, at an unchanged relative level of MM. In saponins-skinned cardiac fibers on cardiomyocytes creatine (Cr, 25 mM) decreased Km for ADP in regulation of respiration from 133 +/- 20 to 20 +/- 4 microM due to activation of coupled mitochondrial creatine kinase-oxidative phosphorylation reactions in control hamster hearts. In the case of cardiomyopathy it decreased Km for ADP only to 81 +/- 13 microM. In endocardial biopsy samples from the hearts of patients with dilated cardiomyopathy taken during angiography, creatine stimulated respiration was decreased by 36% of control value, which correlated well with increase of end-diastolic pressure and fall in ejection fraction. Thus, changes in mitochondrial creatine kinase expression diminished the efficiency of cellular regulation of respiration in cardiomyopathic hearts that may have functional consequences for hemodynamics or may be adaptive alterations in response to decreased contractility.

Intracellular alterations of the creatine kinase isoforms in brains of schizophrenic patients.

Postmortem brain tissues of schizophrenic patients were found to contain 5-10 times less water-soluble creatine kinase (BB CK) and 1.5-3 times less mitochondrial creatine kinase as compared to control. The major part of BB CK in schizophrenic brain tissues, contrary to control, was found to be insoluble in water (particulate form of BB CK) and could be extracted from brain tissue with strong denaturating agents. The particulate form of BB CK did not have any enzymatic activity but activity was found after the solubilization of this isoenzyme. The observed BB CK translocation into the particulate inactive form and the decrease of mitochondrial CK content to schizophrenic brains may reflect changes in the synthesis and the utilization of creatine phosphate.

Phosphocreatine pathway for energy transport: ADP diffusion and cardiomyopathy.

Chemically skinned (by treatment with saponin, 40 micrograms/ml) isolated cardiomyocytes were used to study the intracellular diffusion of ADP and creatine (Cr). Stimulation of respiration was studied in these cardiomyocytes without intact sarcolemma and in isolated heart mitochondrial by addition of ADP and Cr in the presence of 0.2 mM ATP (via mitochondrial creatine kinase reaction: Cr + MgATP = MgADP + PCr). The Michaelis constant (Km) for Cr was similar in both cases, 5.67 +/- 0.11 (SD) mM in skinned myocytes and 6.9 +/- 0.2 mM in mitochondria, showing that there is no significant restriction to the diffusion of this substrate. However, the apparent Km for external ADP increased from 17.6 +/- 1.0 microM for mitochondria to 250 +/- 38 microM for skinned cardiomyocytes, showing decreased diffusivity of ADP as a result of binding to cellular structures. In the presence of 25 mM Cr, the Km for ADP for myocytes decreased to 35.6 +/- 5.6 microM due to the coupling of the creatine kinase and oxidative phosphorylation reactions. Provision of substrate for the creatine kinase reaction amplified the weak ADP signal in the regulation of respiration. The activity of the mitochondrial creatine kinase was decreased by a factor of two in cardiomyopathic hamsters and human hearts and was associated with a twofold decrease in creatine-stimulated respiration. These data show a potentially key role of mitochondrial creatine kinase in the regulation of cellular respiration and the possible importance of changes in its activity for the functional disturbances of the cardiomyopathic heart.

Alterations in the creatine kinase system in the myocardium of cardiomyopathic hamsters.

Immunochemical and biochemical methods were used to assess quantitatively the changes in the heart creatine kinase system in the myopathic Syrian hamsters, line CHF I46. Cardiomyopathy in I75-200 day old animals was characterized by decreased content of mitochondria and lower total creatine kinase activity. In isolated mitochondria only the creatine kinase activity was decreased while cytochromes aa3 content and respiration rate were unchanged. The share of mitochondrial creatine kinase in the total tissue enzyme activity was decreased from 33% to I8% and that of BB form was elevated from 5% in control to 20%, at unchanged relative level of MM. Immunoassay showed decreased amount of the mitochondrial creatine kinase in the tissue and its decreased ratio to cytochromes aa3. The results show altered expression of creatine kinase isoenzymes in cardiomyopathy.

Heart mitochondrial creatine kinase revisited: the outer mitochondrial membrane is not important for coupling of phosphocreatine production to oxidative phosphorylation.

The state of mitochondrial creatine kinase (CKmi-mi) in intact dog heart mitochondria and mitoplasts and the mechanism of its functional coupling with the oxidative phosphorylation system have been reinvestigated under different osmotic conditions and ionic compositions of the medium. It has been established that in a medium which mimics the cardiac cell cytoplasma, dissociation of CKmi-mi from the membrane of mitoplasts increases when the mitoplasts are swollen due to hypoosmotic treatment. It was shown by EPR that hypoosmotic treatment results in the enhancement of the mobility of phospholipids in the membrane bilayer. It has been also shown that when CKmi-mi is detached from the inner membrane in intact mitochondria in isotonic KCl solution, the effects of the coupling between CKmi-mi and oxidative phosphorylation via ATP/ADP translocase disappear in spite of the presence of CKmi-mi in the intermembrane space and intactness of the outer mitochondrial membrane. Therefore, this coupling cannot be explained by the "compartmented coupling" mechanism or "dynamic adenine nucleotide compartmentation" in the intermembrane space due to diffusion limitation for adenine nucleotides through the outer mitochondrial membrane, as has been supposed by several authors (F.N. Gellerich et al. (1987) Biochim. Biophys. Acta 890, 117-126; S.P.J. Brooks and C.H. Suelter (1987) Arch. Biochem. Biophys. 253, 122-132). The data obtained show that the displacement of the enzyme from the membrane results in significantly increased sensitivity of the coupled processes of aerobic phosphocreatine synthesis to inhibition by the product, phosphocreatine. Thus, all results show that under physiological osmotic and ionic conditions CKmi-mi remains firmly attached to the inner mitochondrial membrane and effectively coupled with ATP/ADP translocase due to intimate dynamic interaction between those proteins.

Specific inhibition of ATP-ADP translocase in cardiac mitoplasts by antibodies against mitochondrial creatine kinase.

Mitochondrial creatine kinase was purified from rat hearts and used to produce antibodies in chicken and rabbits. Antibodies were purified to a high degree of homogeneity by an affinity chromatography method. Chicken antibodies against mitochondrial creatine kinase inhibited this enzyme in rat-heart mitochondrial inner membrane and matrix preparation, and simultaneously blocked oxidative phosphorylation. Under these conditions respiratory chain activities remained unchanged, but adenine nucleotide translocase was inhibited. Removal of mitochondrial creatine kinase from the membrane by pretreatment with 0.15 M KCl and 20 mM ADP completely abolished the effect of antibodies against mitochondrial creatine kinase on oxidative phosphorylation. Noninhibitory antibodies from rabbit with high affinity to rat mitochondrial creatine kinase inhibited neither creatine kinase activity nor oxidative phosphorylation. These data show close and specific spatial arrangement of mitochondrial creatine kinase and adenine nucleotide translocase in mitochondria. It is supposed that there is a fixed orientation of these proteins in the cardiolipin domain in the membrane and that their interaction may occur by a frequent collision due to their lateral movement.

Heart mitochondria in physiological salt solution: not ionic strength but salt composition is important for association of creatine kinase with the inner membrane surface.

In physiological salt solution (PSS) which mimicks the cardiac cells cytoplasm and contains 120 mM K-MES, 10 mM NaCl, 20 mM imidazole, pH 7.2, 20 mM taurine, 15 mM creatine, 15 mM Na2phosphocreatine, 5 mM Na2ATP, 8 mM MgCl2, 5 mM K2HPO4, 3 mM glutamate, 3 mM malate, 0.5 mM dithiothreitol and 10 mg/ml of bovine serum albumine both isolated mitochondria and intracellular structures in skinned fibers stay intact. In PSS mitochondrial creatine kinase remains firmly attached to the inner membrane surface. CKmi-mi is extracted from cardiac mitoplasts in 0.125 M KCl solution, but addition of 10 mM sodium borate to this KCl solution completely inhibits dissociation of CKmi-mi. Therefore, not ionic strength but ion composition is important for association of CKmi-mi with mitochondrial membrane. Functional and structural studies using antibodies against CKmi-mi showed that in PSS CKmi-mi is bound to the inner mitochondrial membrane in spatially close relationship to adenine nucleotide translocase (ANT). Thus, under physiological conditions CKmi-mi is structurally and functionally coupled to ANT in cardiac mitochondria and functions to catalyze almost complete utilization of mitochondrial ATP for aerobic phosphocreatine synthesis.

Some properties of cardiac troponin T structure.

Troponin T is eluted in multiple peaks when the whole bovine cardiac troponin complex is subjected to DEAE-cellulose chromatography in the presence of 8 M-urea. The heterogeneity observed is due to the presence of two forms of troponin T, differing in their Mr values, amino acid content, degree of phosphorylation and aggregation. Cardiac troponin T contains up to 0.8 mol of phosphate/mol of protein. Rabbit skeletal-muscle troponin T kinase phosphorylates the single site located in the N-terminal pentapeptide of cardiac troponin T. The composition of this peptide, (Ser,Asx,Glx,Glx)Val, is similar to that of skeletal-muscle troponin T. The single thiol group of cardiac troponin T is located some 50-70 residues from the N-terminus. The C-terminal sequence of cardiac troponin T is Trp-Lys, i.e. as is the case of skeletal-muscle troponin T.