Cytoarchitectural and metabolic adaptations in muscles with mitochondrial and cytosolic creatine kinase deficiencies

We have blocked creatine kinase (CK) mediated phosphocreatine (PCr) <==> ATP transphosphorylation in mitochondria and cytosol of skeletal muscle by knocking out the genes for the mitochondrial (ScCKmit) and the cytosolic (M-CK) CK isoforms in mice. Animals which carry single or double mutations, if kept and tested under standard laboratory conditions, have surprisingly mild changes in muscle physiology. Strenuous ex vivo conditions were necessary to reveal that MM-CK absence in single and double mutants leads to a partial loss of tetanic force output. Single ScCKmit deficiency has no noticeable effects but in combination the mutations cause slowing of the relaxation rate. Importantly, our studies revealed that there is metabolic and cytoarchitectural adaptation to CK defects in energy metabolism. The effects involve mutation type-dependent alterations in the levels of AMP, IMP, glycogen and phosphomonoesters, changes in activity of metabolic enzymes like AMP-deaminase, alterations in mitochondrial volume and contractile protein (MHC isoform) profiles, and a hyperproliferation of the terminal cysternae of the SR (in tubular aggregates). This suggests that there is a compensatory resiliency of loss-of-function and redirection of flux distributions in the metabolic network for cellular energy in our mutants.     

Synthesis and differential properties of creatine analogues as inhibitors for human creatine kinase isoenzymes

Fourteen new creatine analogues, all with a guanidine function and either a polar or an apolar group instead of the creatine carboxylic function, were tested as potential inhibitors for human creatine kinase by kinetic analysis of their effects on the reaction rate. Only compounds bearing an apolar aromatic moiety, which was spaced from the guanidine function by at least two bonds, proved to have a significant inhibitory activity and showed a mixed-type inhibition similar to that of creatine. Among these compounds 2,6-dichlorobenzylguanidine (Ki = 5.6 mM and 39.8 mM for muscle-type and brain-type creatine kinases, respectively) and 3-(2,6-dichlorophenyl)propylguanidine (Ki = 15 mM and 4.5 mM) were the more potent inhibitors and showed a significant isoenzyme selectivity between muscle- and brain-type creatine kinases. Our results are in agreement with recent data that suggest the location of a hydrophobic pocket near the guanidine-binding domain of the enzyme. The observed selectivity in isoenzyme inhibition may be useful to study structural differences in catalytic centers.     

Purification and characterization of human sarcomeric mitochondrial creatine kinase

PURPOSE: A dose-escalation study of irinotecan hydrochloride (CPT-11) combined with fixed-dose cisplatin was conducted to determine the maximum-tolerated dose (MTD), dose-limiting toxicities, and objective response rate in patients with advanced gastric cancer. PATIENTS AND METHODS: Twenty-four patients with or without prior chemotherapy were enrolled. All patients were assessable for toxicities and response. On day 1, CPT-11 was administered as a 90-minute intravenous (I.V.) infusion, which was followed 2 hours later by a 120-minute I.V. infusion of cisplatin 80 mg/m2. CPT-11 alone at the same dose was administered again on day 15. The treatment was repeated every 4 weeks until disease progression was observed. The initial dose of CPT-11 was 60 mg/m2, and was escalated in increments of 10 mg/m2 until severe or life-threatening toxicity was observed. RESULTS: The MTD of this combination was CPT-11 80 mg/m2. At this dose level, 16.7% of patients (two of 12) had leukopenia of less than 1,000/microL, 66.7% (eight of 12) had neutropenia of less than 500/microL, and 16.7% (two of 12) had severe diarrhea of grade 4 during the first course. The dose-limiting toxicity was neutropenia. Ten patients achieved a partial response (PR), and the overall response rate was 41.7% among 24 patients (95% confidence interval, 21.9% to 61.4%). CONCLUSION: The recommended dose and schedule is CPT-11 70 mg/m2 on days 1 and 15 and cisplatin 80 mg/m2 on day 1 every 4 weeks. This combination of CPT-11 and cisplatin, considered to be active against advanced gastric cancer with acceptable toxicity, should be further assessed in a phase II study.     

Maintained coupling of oxidative phosphorylation to creatine kinase activity in sarcomeric mitochondrial creatine kinase-deficient mice

The importance of mitochondrial creatine kinase (mi-CK) in oxidative muscle was tested by studying the functional properties of in situ mitochondria in saponin-skinned muscle fibres from sarcomeric mi-CK-deficient (mutant) mice. Biochemical analyses showed that the lack of mi-CK in mutant muscle was associated with a decrease in specific activity of MM-CK in mutant ventricle, and increase in mutant soleus (oxidative) muscle. Lactate dehydrogenase activity and isoenzyme analysis showed an increased glycolytic metabolism in mutant soleus. No change was observed in ventricular muscle. In control animals, the apparent K(m) of mitochondrial respiration for ADP in ventricle and soleus (232 +/- 36 and 381 +/- 63 microM, respectively) was significantly reduced in the presence of creatine (52 +/- 8 and 45 +/- 12 microM, respectively). There was no change in the K(m) in oxidative fibres from mutant mice (258 +/- 27 and 399 +/- 66 microM, respectively) compared with control, though surprisingly, it was also significantly decreased in the presence of creatine (144 +/- 8 and 150 +/- 27 microM, respectively) despite the absence of mi-CK. It is proposed that in mutant (and perhaps normal) oxidative tissue, cytosolic MM-CK can relocate to the outer mitochondrial membrane, where it is coupled to oxidative phosphorylation by close proximity to porin, and the adenine nucleotide translocase. Such an effect can preserve the functioning of the CK shuttle and the energetic properties of mi-CK deficient tissue.     

Expression of brain-type creatine kinase and ubiquitous mitochondrial creatine kinase in the fetal rat brain: evidence for a nuclear energy shuttle

To test the hypothesis that embryonic brain cells utilize a creatine phosphate energy shuttle, we examined the pattern of creatine kinase (CK) isoform expression and localization in the fetal rat brain. Moderate levels of CK activity are present at embryonic day 14 (7 U/mg protein) and decrease slightly until 3 days postpartum followed by a rapid, fourfold up-regulation to adult levels by 1 month (18 U/mg protein). In parallel with changes in enzyme activity, there is a biphasic and coordinate pattern of expression of brain-type CK (BCK) and ubiquitous mitochondrial CK (uMtCK) determined by nondenaturing electrophoresis and immunoblot analysis. The localization of CK isoforms was examined by immunocytochemistry, and, during the fetal period, BCK and uMtCK immunoreactivity was detected throughout the central and peripheral nervous system, especially in neuroepithelial regions of the cerebral vesicles and spinal cord. In large cells within the olfactory neuroepithelium and ventral spinal cord, differential compartmentation of CK isoforms was evident, with BCK localized primarily in cell nuclei, whereas uMtCK immunoreactivity was present in the cell body (but not within nuclei). In olfactory bulb neuroepithelium, both isoforms were expressed in the middle zone of the germinal layer associated with DNA synthesis. In embryonic skeletal and cardiac muscle, which also express BCK, the same compartmentation of BCK was seen, with BCK localized primarily in the cell nucleus of cardiac and skeletal myoblasts. These results demonstrate a coordinate pattern of expression and compartmentation of BCK and uMtCK isoforms in the fetal brain that, in some cells, provides the anatomic basis for a nuclear energy shuttle.     

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.     

Combined myofibrillar and mitochondrial creatine kinase deficiency impairs mouse diaphragm isotonic function

Creatine kinase (CK) is an enzyme central to cellular high-energy phosphate metabolism in muscle. To characterize the physiological role of CK in respiratory muscle during dynamic contractions, we compared the force-velocity relationships, power, and work output characteristics of the diaphragm (Dia) from mice with combined myofibrillar and sarcomeric mitochondrial CK deficiency (CK[-/-]) with CK-sufficient controls (Ctl). Maximum velocity of shortening was significantly lower in CK[-/-] Dia (14.1 +/- 0.9 Lo/s, where Lo is optimal fiber length) compared with Ctl Dia (17.5 +/- 1.1 Lo/s) (P < 0.01). Maximum power was obtained at 0.4-0.5 tetanic force in both groups; absolute maximum power (2,293 +/- 138 W/m2) and work (201 +/- 9 J/m2) were lower in CK[-/-] Dia compared with Ctl Dia (2,744 +/- 146 W/m2 and 284 +/- 26 J/m2, respectively) (P < 0.05). The ability of CK[-/-] Dia to sustain shortening during repetitive isotonic activation (75 Hz, 330-ms duration repeated each second at 0.4 tetanic force load) was markedly impaired, with CK[-/-] Dia power and work declining to zero by 37 +/- 4 s, compared with 61 +/- 5 s in Ctl Dia. We conclude that combined myofibrillar and sarcomeric mitochondrial CK deficiency profoundly impairs Dia power and work output, underscoring the functional importance of CK during dynamic contractions in skeletal muscle.     

Interactions of mitochondrial ATP synthesis and the creatine kinase equilibrium in skeletal muscle

This study aimed to evaluate the effect of FR128998, (1s,6s)-1-benzyl-10-(3-pyridyl-methyl)-7-thia-10-azaspiro [5,6]-dodecan-11-one 7,7-dioxide hydrochloride, a novel platelet activating factor (PAF) receptor antagonist, on endotoxin lipopolysaccharide-induced disseminated intravascular coagulation in rats. Experimental disseminated intravascular coagulation was induced by an infusion of lipopolysaccharide at 0.25 mg/kg/h for 4 h. Simultaneous infusion of FR128998 (0.25 and 1.0 mg/kg/h) with lipopolysaccharide dose dependently inhibited thrombocytopenia, but not leukopenia. The changes in coagulation parameters of disseminated intravascular coagulation, i.e., prolongation of activated partial thromboplastin time and elevated levels of fibrinogen/fibrin degradation products, were also prevented by the treatment with FR128998. In addition, FR128998 attenuated the increase in serum tumor necrosis factor (TNF) which appeared during the initial stage of disseminated intravascular coagulation. FR128998 (10 microM) also inhibited the TNF production by peripheral blood leukocytes or alveolar macrophages stimulated by lipopolysaccharide in vitro. Furthermore, TNF production induced by PAF itself in vitro was also inhibited in the presence of FR128998. These data indicate that PAF plays a pivotal role in the development of disseminated intravascular coagulation via TNF production.     

The protection of creatine kinase MM sub-bands by EDTA during storage

We examined the effect of a 5 mmol/l concentration of EDTA on the stabilization of the five serum creatine kinase MM isoenzymes, resolved by thin-layer isoelectric focusing. In patient sera, total CK and CK-MB activities were stable during storage of the samples for two months at 4 degrees C even in the absence of EDTA. However, EDTA stabilized the labile MM and MM1 sub-bands, which are the first to appear in the blood after the release from the damaged tissue and its addition to blood samples intended for determining the MM sub-band pattern is recommended. The stabilizing effect of EDTA was emphasized at higher temperatures. EDTA protected the CK-MM pattern in myocardium extracts made in normal serum and incubated at 37 degrees C during 40 h, but was unnecessary when myocardium was homogenized in heat-inactivated serum. It is thought that EDTA could act by inhibiting a heat-labile component of human serum.     

Cellular relocalisation of protein kinase C-theta caused by staurosporine and some of its analogues

The microbial product staurosporine is a protein kinase C (PKC) inhibitor with some phorbol ester-agonistic properties. It is known to cause the translocation of the PKC isoenzymes epsilon and delta from the cellular cytosol to the membrane and nucleus. We tested the hypothesis that it also affects the cellular localisation of the novel PKC isoenzyme theta, and that staurosporine analogues, some of which are currently under clinical evaluation as potential anticancer drugs, have a similar effect. Their ability to alter PKC-theta distribution was studied in human-derived A549 lung carcinoma cells. Western blot analysis and confocal microscopy after indirect immunofluorescence staining showed that staurosporine (100 nM), like the phorbol ester 12-O-tetradecanoylphorhol-13-acetate (25 nM) caused the translocation of PKC-theta from the cytosol to the membrane and the nucleus. The bisindolylmaleimide GF 109203X mimicked staurosporine, but had a weaker effect. Ro 31-8220 and UCN-01 decreased cytosolic PKC-theta only at 1 microM. CGP 41251 had no effect on PKC-theta in either experimental design. The results show that some, but not all, staurosporine analogues share the partial phorbol ester-agonistic PKC-translocatory activity of the parent molecule.     

Functional aspects of the X-ray structure of mitochondrial creatine kinase: a molecular physiology approach

Mitochondrial creatine kinase (Mi-CK) is a central enzyme in energy metabolism of tissues with high and fluctuating energy requirements. In this review, recent progress in the functional and structural characterization of Mi-CK is summarized with special emphasis on the solved X-ray structure of chicken Mib-CK octamer (Fritz-Wolf et al., Nature 381, 341-345, 1996). The new results are discussed in a historical context and related to the characteristics of CK isoforms as known from a large number of biophysical and biochemical studies. Finally, two hypothetical functional aspects of the Mi-CK structure are proposed: (i) putative membrane binding motifs at the top and bottom faces of the octamer and (ii) a possible functional role of the central 20 A channel.     

Mercury contamination during pH measurement and its effect on creatine kinase activity

Inhibition of activity of the enzyme creatine kinase occurs when small volumes of assay mixtures have their pH measured using a combination pH electrode prior to addition of the enzyme. This inhibition can be attributed to diffusion of ionic mercury from the calomel reference electrode through the saturated potassium chloride salt bridge and the ground glass-liquid junction to the test solution. The concentration of mercury accumulating in a solution by this process can be sufficient to affect the activity of an enzyme, and EDTA cannot be used successfully to scavenge mercury ion and avoid the inhibition.     

Mutation of cis-proline 207 in mitochondrial creatine kinase to alanine leads to increased acid stability

SETTING: Hamad General Hospital, the tertiary health centre for Doha, Qatar. OBJECTIVE: The purpose of the study was to define and correlate the role of radiology with clinical and pathological findings in abdominal tuberculosis. MATERIAL AND METHODS: A total of 59 patients (47 males and 12 females) diagnosed bacteriologically and/ or histologically for abdominal tuberculosis were radiologically assessed. Evaluation was based on the analysis of plain abdominal radiographs, gastro-intestinal contrast studies (barium meal follow through and barium enema), ultrasonography and computed tomography. RESULTS: Plain abdominal radiographs performed in 38 patients were positive in 19 cases (50%). Gastrointestinal contrast examinations were positive in 27 out of 34 cases (80%). Ultrasound examinations were abnormal in 25 out of 31 cases (81%), while computed tomography, performed in 24 patients, revealed abnormal findings in 19 cases (80%). Combined radiographic and imaging procedures revealed peritoneal involvement (ascites) in 16 patients (27%), bowel involvement in 36 (61%), mass lesion in 11 (19%), lymphadenopathy in 13 (22%) and organ involvement in 13 (22%). CONCLUSION: There was no single radiological method that provided all necessary information suggestive of abdominal tuberculosis. Although unequivocal diagnosis of abdominal tuberculosis can only be made by culture and histological findings, combined computed tomography and ultrasound findings were the most important imaging tools in the diagnostic process for abdominal tuberculosis, while contrast studies helped to assess the extent of bowel disease, hence influencing decisions concerning surgery.     

Dextran strongly increases the Michaelis constants of oxidative phosphorylation and of mitochondrial creatine kinase in heart mitochondria

A minimal model of glycogen metabolism in muscle tissue is analyzed in accordance with metabolic control analysis. The model contains two branch points. Rather than contributing to complexity of the analysis, this branching allows expression of the control coefficients in a simplified form. Glucose 6-phosphate is the metabolite at the first branch point, and the analysis is simplified further by the fact that glucose 6-phosphate is the substrate for enzymes which catalyze near-equilibrium reactions. Control of the concentration of glucose 6-phosphate is of interest because of its pivotal location in the metabolic system, but also because it interacts with an allosteric site on glycogen synthase to stimulate glycogen synthase activity. It is shown that the control which the transporter and enzymes involved in glycogen synthesis exert on glycolytic flux is proportional to the control which these components exert on glucose 6-phosphate concentration. Thus, glycolysis plays a major role in control of glucose 6-phosphate concentration. It is concluded that control of glycogen synthesis is not a rigid parameter of any component of this metabolic system. Rather the distribution of control is flexible and shifts from one portion of the system to another in response to shifts in the physiological state. An important element in determining the distribution of control of glycogen synthesis is the change in the sensitivity of the allosteric site of glycogen synthase to glucose 6-phosphate which is brought about by conversion of glycogen synthase to the dephosphorylated, glucose 6-phosphate-independent, state.     

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.