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Objective: Hypertension is a major risk factor for cardiovascular disease, kidney disease, and premature death. Increased levels of creatine kinase are associated with development of hypertension. However, it is unknown if creatine, a substrate of CK, is associated with the development of hypertension. We therefore, aimed to investigate the association between plasma creatine concentration and incident hypertension. Methods: We measured fasting plasma creatine concentrations by nuclear magnetic resonance spectroscopy in participants of the population-based PREVEND study. The study outcome was incident hypertension, defined as either a SBP of at least 140mmHg, a DBP of at least 90mmHg, or the new usage of antihypertensive drugs. Participants with hypertension at baseline were excluded. Results: We included 3135 participants (46% men) aged 49=10years. Mean plasma creatine concentrations were 36.2=17.5mmol/l, with higher concentrations in women than in men (42.2=17.6 versus 29.2=17.6mmol/l; P, Journal of Hypertension, 40 (2)

There is great need for the identification of new, potentially modifiable risk factors for the poor health-related quality of life (HRQoL) and of the excess risk of mortality in dialysis-dependent chronic kidney disease patients. Creatine is an essential contributor to cellular energy homeostasis, yet, on a daily basis, 1.6–1.7% of the total creatine pool is non-enzymatically degraded to creatinine and subsequently lost via urinary excretion, thereby necessitating a continuous supply of new creatine in order to remain in steady-state. Because of an insufficient ability to synthesize creatine, unopposed losses to the dialysis fluid, and insufficient intake due to dietary recommendations that are increasingly steered towards more plant-based diets, hemodialysis patients are prone to creatine deficiency, and may benefit from creatine supplementation. To avoid problems with compliance and fluid balance, and, furthermore, to prevent intradialytic losses of creatine to the dialysate, we aim to investigate the potential of intradialytic creatine supplementation in improving outcomes. Given the known physiological effects of creatine, intradialytic creatine supplementation may help to maintain creatine homeostasis among dialysis-dependent chronic kidney disease patients, and consequently improve muscle status, nutritional status, neurocognitive status, HRQoL. Additionally, we describe the rationale and design for a block-randomized, double-blind, placebo-controlled pilot study. The aim of the pilot study is to explore the creatine uptake in the circulation and tissues following different creatine supplementation dosages., Nutrients, 13 (8), ISSN:2072-6643

Muscle wasting, low protein intake, hypoalbuminemia, low body mass, and chronic fatigue are prevalent in hemodialysis patients. Impaired creatine status may be an often overlooked, potential contributor to these symptoms. However, little is known about creatine homeostasis in hemodialysis patients. We aimed to elucidate creatine homeostasis in hemodialysis patients by assessing intradialytic plasma changes as well as intra- and interdialytic losses of arginine, guanidinoacetate, creatine and creatinine. Additionally, we investigated associations of plasma creatine concentrations with low muscle mass, low protein intake, hypoalbuminemia, low body mass index, and chronic fatigue. Arginine, guanidinoacetate, creatine and creatinine were measured in plasma, dialysate, and urinary samples of 59 hemodialysis patients. Mean age was 65 ± 15 years and 63% were male. During hemodialysis, plasma concentrations of arginine (77 ± 22 to 60 ± 19 μmol/L), guanidinoacetate (1.8 ± 0.6 to 1.0 ± 0.3 μmol/L), creatine (26 [16–41] to 21 [15–30] μmol/L) and creatinine (689 ± 207 to 257 ± 92 μmol/L) decreased (all P < 0.001). During a hemodialysis session, patients lost 1939 ± 871 μmol arginine, 37 ± 20 μmol guanidinoacetate, 719 [399–1070] μmol creatine and 15.5 ± 8.4 mmol creatinine. In sex-adjusted models, lower plasma creatine was associated with a higher odds of low muscle mass (OR per halving: 2.00 [1.05–4.14]; P = 0.04), low protein intake (OR: 2.13 [1.17–4.27]; P = 0.02), hypoalbuminemia (OR: 3.13 [1.46–8.02]; P = 0.008) and severe fatigue (OR: 3.20 [1.52–8.05]; P = 0.006). After adjustment for potential confounders, these associations remained materially unchanged. Creatine is iatrogenically removed during hemodialysis and lower plasma creatine concentrations were associated with higher odds of low muscle mass, low protein intake, hypoalbuminemia, and severe fatigue, indicating a potential role for creatine supplementation., Journal of Translational Medicine, 19 (1), ISSN:1479-5876

Background Type 2 diabetes is associated with both impaired insulin action at target tissues and impaired insulin secretion in pancreatic beta cells. Mitochondrial dysfunction may play a role in both insulin resistance and impaired insulin secretion. Plasma creatine has been proposed as a potential marker for mitochondrial dysfunction. We aimed to investigate the association between plasma creatine and incident type 2 diabetes. Methods We measured fasting plasma creatine concentrations by nuclear magnetic resonance spectroscopy in participants of the general population‐based PREVEND study. The study outcome was incident type 2 diabetes, defined as a fasting plasma glucose ≥7.0 mmol/L (126 mg/dl); a random sample plasma glucose ≥11.1 mmol/L (200 mg/dl); self‐report of a physician diagnosis or the use of glucose‐lowering medications based on a central pharmacy registration. Associations of plasma creatine with type 2 diabetes were quantified using Cox proportional hazards models and were adjusted for potential confounders. Results We included 4735 participants aged 52 ± 11 years, of whom 49% were male. Mean plasma creatine concentrations were 36.7 ± 17.6 µmol/L, with lower concentrations in males than in females (30.4 ± 15.1 µmol/L vs. 42.7 ± 17.7 µmol/L; p for difference, Clinical Endocrinology, 94 (4), ISSN:1365-2265, ISSN:0300-0664

The CK/PCr-system, with creatine (Cr) as an energy precursor, plays a crucial role in cellular physiology. In the kidney, as in other organs and cells with high and fluctuating energy requirements, energy-charged phospho-creatine (PCr) acts as an immediate high-energy source and energy buffer, and as an intracellular energy transport vehicle. A maximally filled total Cr (Cr plus PCr) pool is a prerequisite for optimal functioning of the body and its organs, and health. Skeletal- and cardiac muscles of dialysis patients with chronic kidney disease (CKD) are depleted of Cr in parallel with the duration of dialysis. The accompanying accumulation of cellular damage seen in CKD patients lead to a deterioration of musculo-skeletal and neurological functioning and poor quality of life (QOL). Therefore, to counteract Cr depletion, it is proposed to supplement CKD patients with Cr. The anticipated benefits include previously documented improvements in the musculo-skeletal system, brain and peripheral nervous system, as well as improvements in the common comorbidities of CKD patients (see below). Thus, with a relatively simple, safe and inexpensive Cr supplementation marked improvements in quality of life (QOL) and life span are likely reached. To avoid Cr and fluid overload by oral Cr administration, we propose intradialytic Cr supplementation, whereby a relatively small amount of Cr is added to the large volume of dialysis solution to a final concentration of 1–10 mM. From there, Cr enters the patient’s circulation by back diffusion during dialysis. Because of the high affinity of the Cr transporter (CRT) for Cr affinity for Cr (Vmax of CRT for Cr = 20–40 μM Cr), Cr is actively transported from the blood stream into the target cells and organs, including skeletal and cardiac muscle, brain, proximal tubules of kidney epithelial cells, neurons, and leukocytes and erythrocytes, which all express CRT and depend on the CK/PCr system. By this intradialytic strategy, only as much Cr is taken up by the body as is needed to fill the tissue Cr pools and no excess Cr has to be excreted, as is the case with oral Cr. Because aqueous solutions of Cr are not very stable, Cr must be added immediately before dialysis either as solid Cr powder or from a frozen Cr stock solution to the dialysate, or alternatively, Cr could become an additional component of a novel dry dialysate mixture in a cartridge device., Medical Hypotheses, 99, ISSN:0306-9877, ISSN:1532-2777

International audience; In this review we analyze the recent important and remarkable advancements in studies of compartmentation of adenine nucleotides in muscle cells due to their binding to macromolecular complexes and cellular structures, which results in non-equilibrium steady state of the creatine kinase reaction. We discuss the problems of measuring the energy fluxes between different cellular compartments and their simulation by using different computer models. Energy flux determinations by (18)O transfer method have shown that in heart about 80% of energy is carried out of mitochondrial intermembrane space into cytoplasm by phosphocreatine fluxes generated by mitochondrial creatine kinase from adenosine triphosphate (ATP), produced by ATP Synthasome. We have applied the mathematical model of compartmentalized energy transfer for analysis of experimental data on the dependence of oxygen consumption rate on heart workload in isolated working heart reported by Williamson et al. The analysis of these data show that even at the maximal workloads and respiration rates, equal to 174 μmol O(2) per min per g dry weight, phosphocreatine flux, and not ATP, carries about 80-85% percent of energy needed out of mitochondria into the cytosol. We analyze also the reasons of failures of several computer models published in the literature to correctly describe the experimental data.

The creatine/creatine kinase (CK) system plays a key role in cellular energy buffering and transport. In vertebrates, CK has four isoforms expressed in a tissue-specific manner. In the process of creatine biosynthesis several other important metabolites are formed. The anticancer effect of creatine had been reported in the past, and recent literature has reported low creatine content in several types of malignant cells. Furthermore, creatine can protect cardiac mitochondria from the deleterious effects of some anticancer compounds. Previous work from our laboratory showed progressive decrease of phosphocreatine, creatine and CK upon transformation of skeletal muscle into sarcoma. It was convincingly demonstrated that prominent expression of creatine-synthesizing enzymes L-arginine: glycine amidinotransferase and N-guanidinoacetate methyltransferase occurs in sarcoma, Ehrlich ascites carcinoma and sarcoma 180 cells; whereas, both these enzymes are virtually undetectable in skeletal muscle. Creatine transporter also remained unaltered in malignant cells. The anticancer effect of methylglyoxal had been known for a long time. The present work shows that this anticancer effect of methylglyoxal is significantly augmented in presence of creatine. On creatine supplementation the effect of methylglyoxal plus ascorbic acid was further augmented and there was no visible sign of tumor. Moreover, creatine and CK, which were very low in sarcoma tissue, were significantly elevated with the concomitant regression of tumor.

The creatine/creatine kinase system decreases drastically in sarcoma. In the present study, an investigation of catalytic activities, western blot and mRNA expression unambiguously demonstrates the prominent expression of the creatine-synthesizing enzymes l-arginine:glycine amidinotransferase and N-guanidinoacetate methyltransferase in sarcoma, Ehrlich ascites carcinoma and Sarcoma 180 cells, whereas both enzymes were virtually undetectable in normal muscle. Compared to that of normal animals, these enzymes remained unaffected in the kidney or liver of sarcoma-bearing mice. High activity and expression of mitochondrial arginase II in sarcoma indicated increased ornithine formation. Slightly or moderately higher levels of ornithine, guanidinoacetate and creatinine were observed in sarcoma compared to muscle. Despite the intrinsically low level of creatine in Ehrlich ascites carcinoma and Sarcoma 180 cells, these cells could significantly take up and release creatine, suggesting a functional creatine transport, as verified by measuring mRNA levels of creatine transporter. Transcript levels of arginase II, ornithine-decarboxylase, S-adenosyl-homocysteine hydrolase and methionine-synthase were significantly upregulated in sarcoma and in Ehrlich ascites carcinoma and Sarcoma 180 cells. Overall, the enzymes related to creatine and arginine/methionine metabolism were found to be significantly upregulated in malignant cells. However, the low levels of creatine kinase in the same malignant cells do not appear to be sufficient for the building up of an effective creatine/phosphocreatine pool. Instead of supporting creatine biosynthesis, l-arginine:glycine amidinotransferase and N-guanidinoacetate methyltransferase appear to be geared to support cancer cell metabolism in the direction of polyamine and methionine synthesis because both these compounds are in high demand in proliferating cancer cells.

In vertebrates, phosphocreatine and ATP are continuously interconverted by the reversible reaction of creatine kinase in accordance with cellular energy needs. Sarcoma tissue and its normal counterpart, creatine-rich skeletal muscle, are good source materials to study the status of creatine and creatine kinase with the progression of malignancy. We experimentally induced sarcoma in mouse leg muscle by injecting either 3-methylcholanthrene or live sarcoma 180 cells into one hind leg. Creatine, phosphocreatine and creatine kinase isoform levels decreased as malignancy progressed and reached very low levels in the final stage of sarcoma development; all these parameters remained unaltered in the unaffected contralateral leg muscle of the same animal. Creatine and creatine kinase levels were also reduced significantly in frank malignant portions of human sarcoma and gastric and colonic adenocarcinoma compared with the distal nonmalignant portions of the same samples. In mice, immunoblotting with antibodies against cytosolic muscle-type creatine kinase and sarcomeric mitochondrial creatine kinase showed that both of these isoforms decreased as malignancy progressed. Expressions of mRNA of muscle-type creatine kinase and sarcomeric mitochondrial creatine kinase were also severely downregulated. In human sarcoma these two isoforms were undetectable also. In human gastric and colonic adenocarcinoma, brain-type creatine kinase was found to be downregulated, whereas ubiquitous mitochondrial creatine kinase was upregulated. These significantly decreased levels of creatine and creatine kinase isoforms in sarcoma suggest that: (a) the genuine muscle phenotype is lost during sarcoma progression, and (b) these parameters may be used as diagnostic marker and prognostic indicator of malignancy in this tissue.

Heterotrimeric AMP-activated protein kinase (AMPK) is crucial for energy homeostasis of eukaryotic cells and organisms. Here we report on (i) bacterial expression of untagged mammalian AMPK isoform combinations, all containing gamma(1), (ii) an automated four-dimensional purification protocol, and (iii) biophysical characterization of AMPK heterotrimers by small angle x-ray scattering in solution (SAXS), transmission and scanning transmission electron microscopy (TEM, STEM), and mass spectrometry (MS). AMPK in solution at low concentrations (~1 mg/ml) largely consisted of individual heterotrimers in TEM analysis, revealed a precise 1:1:1 stoichiometry of the three subunits in MS, and behaved as an ideal solution in SAXS. At higher AMPK concentrations, SAXS revealed concentration-dependent, reversible dimerization of AMPK heterotrimers and formation of higher oligomers, also confirmed by STEM mass measurements. Single particle reconstruction and averaging by SAXS and TEM, respectively, revealed similar elongated, flat AMPK particles with protrusions and an indentation. In the lower AMPK concentration range, addition of AMP resulted in a significant decrease of the radius of gyration by approximately 5% in SAXS, which indicates a conformational switch in AMPK induced by ligand binding. We propose a structural model involving a ligand-induced relative movement of the kinase domain resulting in a more compact heterotrimer and a conformational change in the kinase domain that protects AMPK from dephosphorylation of Thr(172), thus positively affecting AMPK activity.

Problems of quantitative investigation of intracellular diffusion and compartmentation of metabolites are analyzed. Principal controversies in recently published analyses of these problems for the living cells are discussed. It is shown that the formal theoretical analysis of diffusion of metabolites based on Fick's equation and using fixed diffusion coefficients for diluted homogenous aqueous solutions, but applied for biological systems in vivo without any comparison with experimental results, may lead to misleading conclusions, which are contradictory to most biological observations. However, if the same theoretical methods are used for analysis of actual experimental data, the apparent diffusion constants obtained are orders of magnitude lower than those in diluted aqueous solutions. Thus, it can be concluded that local restrictions of diffusion of metabolites in a cell are a system-level properties caused by complex structural organization of the cells, macromolecular crowding, cytoskeletal networks and organization of metabolic pathways into multienzyme complexes and metabolons. This results in microcompartmentation of metabolites, their channeling between enzymes and in modular organization of cellular metabolic networks. The perspectives of further studies of these complex intracellular interactions in the framework of Systems Biology are discussed.

Problems of quantitative investigation of intracellular diffusion and compartmentation of metabolites are analyzed. Principal controversies in recently published analyses of these problems for the living cells are discussed. It is shown that the formal theoretical analysis of diffusion of metabolites based on Fick’s equation and using fixed diffusion coefficients for diluted homogenous aqueous solutions, but applied for biological systems in vivo without any comparison with experimental results, may lead to misleading conclusions, which are contradictory to most biological observations. However, if the same theoretical methods are used for analysis of actual experimental data, the apparent diffusion constants obtained are orders of magnitude lower than those in diluted aqueous solutions. Thus, it can be concluded that local restrictions of diffusion of metabolites in a cell are a system-level properties caused by complex structural organization of the cells, macromolecular crowding, cytoskeletal networks and organization of metabolic pathways into multienzyme complexes and metabolons. This results in microcompartmentation of metabolites, their channeling between enzymes and in modular organization of cellular metabolic networks. The perspectives of further studies of these complex intracellular interactions in the framework of Systems Biology are discussed.

Hyperammonemic disorders in pediatric patients lead to poorly understood irreversible effects on the developing brain that may be life-threatening. We showed previously that some of these NH4+-induced irreversible effects might be due to impairment of axonal growth that can be protected under ammonium exposure by creatine co-treatment. The aim of the present work was thus to analyse how the genes of arginine:glycine amidinotransferase (AGAT) and guanidinoacetate methyltransferase (GAMT), allowing creatine synthesis, as well as of the creatine transporter SLC6A8, allowing creatine uptake into cells, are regulated in rat brain cells under NH4+ exposure. Reaggregated brain cell three-dimensional cultures exposed to NH4Cl were used as an experimental model of hyperammonemia in the developing central nervous system (CNS). We show here that NH4+ exposure differentially alters AGAT, GAMT and SLC6A8 regulation, in terms of both gene expression and protein activity, in a cell type-specific manner. In particular, we demonstrate that NH4+ exposure decreases both creatine and its synthesis intermediate, guanidinoacetate, in brain cells, probably through the inhibition of AGAT enzymatic activity. Our work also suggests that oligodendrocytes are major actors in the brain in terms of creatine synthesis, trafficking and uptake, which might be affected by hyperammonemia. Finally, we show that NH4+ exposure induces SLC6A8 in astrocytes. This suggests that hyperammonemia increases blood-brain barrier permeability for creatine. This is normally limited due to the absence of SLC6A8 from the astrocyte feet lining microcapillary endothelial cells, and thus creatine supplementation may protect the developing CNS of hyperammonemic patients.

Preclinical and clinical studies suggest that striatal transplantation of neural stem cells (NSCs) and neural progenitor cells (NPCs) may be an appealing and valuable system for treating Huntington's disease. Nevertheless, for a neural replacement to become an effective translational treatment for Huntington's disease, a certain number of difficulties must be addressed, including how to improve the integration of transplanted cell grafts with the host tissue, to elevate the survival rates of transplanted cells, and to ensure their directed differentiation into specific neuronal phenotypes. Research focusing on the translational applications of creatine (Cr) supplementation in NSC and NPC cell replacement therapies continues to offer promising results, pointing to Cr as a factor with the potential to improve cell graft survivability and encourage differentiation toward GABAergic phenotypes in models of striatal transplantation. Here, we evaluate research examining the outcomes of Cr supplementation and how the timing of supplementation regimes may affect their efficacy. The recent studies indicate that Cr's effects vary according to the developmental stage of the cells being treated, noting the dynamic differences in creatine kinase expression over the developmental stages of differentiating NPCs. This research continues to move Cr supplementation closer to the widespread clinical application and suggests such techniques warrant further examination.

International audience; Doxorubicin (DXR) belongs to the most efficient anticancer drugs. However, its use is limited by a risk of cardiotoxicity, which is not completely understood. Recently, we have shown that DXR impairs essential properties of purified mitochondrial creatine kinase (MtCK), with cardiac isoenzyme (sMtCK) being particularly sensitive. In this study we assessed the effects of DXR on respiration of isolated structurally and functionally intact heart mitochondria, containing sMtCK, in the presence and absence of externally added creatine (Cr), and compared these effects with the response of brain mitochondria expressing uMtCK, the ubiquitous, non-muscle MtCK isoenzyme. DXR impaired respiration of isolated heart mitochondria already after short-term exposure (minutes), affecting both ADP- and Cr-stimulated respiration. During a first short time span (minutes to 1 h), detachment of MtCK from membranes occurred, while a decrease of MtCK activity related to oxidative damage was only observed after longer exposure (several hours). The early inhibition of Cr-stimulated respiration, in addition to impairment of components of the respiratory chain involves a partial disturbance of functional coupling between MtCK and ANT, likely due to interaction of DXR with cardiolipin leading to competitive inhibition of MtCK/membrane binding. The relevance of these findings for the regulation of mitochondrial energy production in the heart, as well as the obvious differences of DXR action in the heart as compared to brain tissue, is discussed.

Journal of muscle research and cell motility, 36 (4), ISSN:0142-4319, ISSN:1573-2657

This review re-evaluates regulatory aspects of substrate supply in heart. In aerobic heart, the preferred substrates are always free fatty acids, and workload-induced increase in their oxidation is observed at unchanged global levels of ATP, phosphocreatine and AMP. Here, we evaluate the mechanisms of regulation of substrate supply for mitochondrial respiration in muscle cells, and show that a system approach is useful also for revealing mechanisms of feedback signalling within the network of substrate oxidation and particularly for explaining the role of malonyl-CoA in regulation of fatty acid oxidation in cardiac muscle. This approach shows that a key regulator of fatty acid oxidation is the energy demand. Alterations in malonyl-CoA would not be the reason for, but rather the consequence of, the increased fatty acid oxidation at elevated workloads, when the level of acetyl-CoA decreases due to shifts in the kinetics of the Krebs cycle. This would make malonyl-CoA a feedback regulator that allows acyl-CoA entry into mitochondrial matrix space only when it is needed. Regulation of malonyl-CoA levels by AMPK does not seem to work as a master on–off switch, but rather as a modulator of fatty acid import.

International audience; Doxorubicin and other anthracyclines are among the most potent chemotherapeutic drugs for the treatment of acute leukaemia, lymphomas and different types of solid tumours such as breast, liver and lung cancers. Their clinical use is, however, limited by the risk of severe cardiotoxicity, which can lead to irreversible congestive heart failure. There is increasing evidence that essential components of myocardial energy metabolism are among the highly sensitive and early targets of doxorubicin-induced damage. Here we review doxorubicin-induced detrimental changes in cardiac energetics, with an emphasis on the emerging importance of defects in energy-transferring and -signalling systems, like creatine kinase and AMP-activated protein kinase.

The fundamental principle of cardiac behaviour is described by the Frank-Starling law relating force of contraction during systole with end-diastolic volume. While both work and respiration rates increase linearly with imposed load, the basis of mechano-energetic coupling in heart muscle has remained a long-standing enigma. Here, we highlight advances made in understanding of complex cellular and molecular mechanisms that orchestrate coupling of mitochondrial oxidative phosphorylation with ATP utilization for muscle contraction. Cardiac system bioenergetics critically depends on an interrelated metabolic infrastructure regulating mitochondrial respiration and energy fluxes throughout cellular compartments. The data reviewed indicate the significance of two interrelated systems regulating mitochondrial respiration and energy fluxes in cells: (1) the creatine kinase, adenylate kinase and glycolytic pathways that communicate flux changes generated by cellular ATPases within structurally organized enzymatic modules and networks; and (2) a secondary system based on mitochondrial participation in cellular calcium cycle, which adjusts substrate oxidation and energy-transducing processes to meet increasing cellular energy demands. By conveying energetic signals to metabolic sensors, coupled phosphotransfer reactions provide a high-fidelity regulation of the excitation–contraction cycle. Such integration of energetics with calcium signalling systems provides the basis for ‘metabolic pacing’, synchronizing the cellular electrical and mechanical activities with energy supply processes.

International audience; Mitochondrial creatine kinase (MtCK), together with cytosolic creatine kinase isoenzymes and the highly diffusible CK reaction product, phosphocreatine, provide a temporal and spatial energy buffer to maintain cellular energy homeostasis. Mitochondrial proteolipid complexes containing MtCK form microcompartments that are involved in channeling energy in form of phosphocreatine rather than ATP into the cytosol. Under situations of compromised cellular energy state, which are often linked to ischemia, oxidative stress and calcium overload, two characteristics of mitochondrial creatine kinase are particularly relevant: its exquisite susceptibility to oxidative modifications and the compensatory up-regulation of its gene expression, in some cases leading to accumulation of crystalline MtCK inclusion bodies in mitochondria that are the clinical hallmarks for mitochondrial cytopathies. Both of these events may either impair or reinforce, respectively, the functions of mitochondrial MtCK complexes in cellular energy supply and protection of mitochondria form the so-called permeability transition leading to apoptosis or necrosis.

The CRT (creatine transporter) is a member of the Na+- and Cl−-dependent neurotransmitter transporter family and is responsible for the import of creatine into cells, and thus is important for cellular energy metabolism. We established for CRT an expression system in HEK-293 cells that allowed biochemical, immunological and functional analysis of CRT wild-type and glycosylation-deficient mutants. Analysis of HA (haemagglutinin)-tagged CRT-NN (wild-type rat CRT with an HA-tag at the C-terminus) revealed several monomeric immunoreactive species with apparent molecular masses of 58, 48 and 43 kDa. The 58 kDa species was shown to be plasma-membrane-resident by EndoHf (endoglycosidase Hf) and PNGase F (peptide N-glycosidase F) treatments and represents fully glycosylated CRT, whereas the 48 kDa and 43 kDa species were glycosylation intermediates and non-glycosylated CRT respectively. Glycosylation-deficient mutants (Asn192Asp, Asn197Asp and Asn192Asp/Asn197Asp) showed altered electrophoretic mobility, indicating that CRT is indeed N-glycosylated. In addition, a prominent CRT band in the range of 75–91 kDa was also detected. Pharmacological inhibition of N-linked glycosylation by tunicamycin in CRT-NN-expressing cells gave a similar reduction in molecular mass, corroborating the finding that Asn192 and Asn197 are major N-glycosylation sites in CRT. Although the apparent Km was not significantly affected in glycosylation-deficient mutants compared with CRT-NN, we measured reduced Vmax values for all mutants (21–28% residual activity), and 51% residual activity after enzymatic deglycosylation of surface proteins in intact CRT-NN cells by PNGase F. Moreover, immunocytochemical analysis of CRT-NN- and CRT-DD-expressing cells (where CRT-DD represents a non-glycosylated double mutant of CRT, i.e. Asn192Asp/Asn197Asp) showed a lower abundance of CRT-DD in the plasma membrane. Taken together, our results suggest that plasma-membrane CRT is glycosylated and has an apparent monomer molecular mass of 58 kDa. Furthermore, N-linked glycosylation is neither exclusively important for the function of CRT nor for surface trafficking, but affects both processes. These findings may have relevance for closely related neurotransmitter transporter family members.

International audience; Parkinson's disease is a disabling neurodegenerative disorder of unknown etiology characterized by a predominant and progressive loss of dopaminergic neurons in the substantia nigra. Recent findings suggest that impaired energy metabolism plays an important role in the pathogenesis of this disorder. The endogenously occurring guanidino compound creatine is a substrate for mitochondrial and cytosolic creatine kinases. Creatine supplementation improves the function of the creatine kinase/phosphocreatine system by increasing cellular creatine and phosphocreatine levels and the rate of ATP resynthesis. In addition, mitochondrial creatine kinase together with high cytoplasmic creatine levels inhibit mitochondrial permeability transition, a major step in early apoptosis. In the present study, we analyzed the effects of externally added creatine on the survival and morphology of dopaminergic neurons and also addressed its neuroprotective properties in primary cultures of E14 rat ventral mesencephalon. Chronic administration of creatine [5 mM] for 7 days significantly increased survival (by 1.32-fold) and soma size (by 1.12-fold) of dopaminergic neurons, while having no effect on other investigated morphological parameters. Most importantly, concurrent creatine exerted significant neuroprotection for dopaminergic neurons against neurotoxic insults induced by serum and glucose deprivation (P < 0.01), 1-methyl-4-phenyl pyridinium ion (MPP+) [15 microM] and 6-hydroxydopamine (6-OHDA) [90 microM] exposure (P < 0.01). In addition, creatine treatment significantly protected dopaminergic cells facing MPP+-induced deterioration of neuronal morphology including overall process length/neuron (by 60%), number of branching points/neuron (by 80%) and area of influence per individual neuron (by 60%). Less pronounced effects on overall process length/neuron and number of branching points/neuron were also found after 6-OHDA exposure (P < 0.05) and serum/glucose deprivation (P < 0.05). In conclusion, our findings identify creatine as a rather potent natural survival- and neuroprotective factor for developing nigral dopaminergic neurons, which is of relevance for therapeutic approaches in Parkinson's disease and for the improvement of cell replacement strategies.

International audience; Creatine (Cr) plays a key role in cellular energy metabolism and is found at high concentrations in metabolically active cells such as skeletal muscle and neurons. These, and a variety of other cells, take up Cr from the extra cellular fluid by a high affinity Na(+)/Cl(-)-dependent creatine transporter (CrT). Mutations in the crt gene, found in several patients, lead to severe retardation of speech and mental development, accompanied by the absence of Cr in the brain. In order to characterize CrT protein(s) on a biochemical level, antibodies were raised against synthetic peptides derived from the N- and C-terminal cDNA sequences of the putative CrT-1 protein. In total homogenates of various tissues, both antibodies, directed against these different epitopes, recognize the same two major polypetides on Western blots with apparent Mr of 70 and 55 kDa. The C-terminal CrT antibody (alpha-CrTCOOH) immunologically reacts with proteins located at the inner membrane of mitochondria as determined by immuno-electron microscopy, as well as by subfractionation of mitochondria. Cr-uptake experiments with isolated mitochondria showed these organelles were able to transport Cr via a sulfhydryl-reagent-sensitive transporter that could be blocked by anti-CrT antibodies when the outer mitochondrial membrane was permeabilized. We concluded that mitochondria are able to specifically take-up Cr from the cytosol, via a low-affinity CrT, and that the above polypeptides would likely represent mitochondrial CrT(s). However, by mass spectrometry techniques, the immunologically reactive proteins, detected by our anti-CrT antibodies, were identified as E2 components of the alpha-keto acid dehydrogenase multi enzyme complexes, namely pyruvate dehydrogenase (PDH), branched chain keto acid dehydrogenase (BC-KADH) and alpha-ketoglutarate dehydrogenase (alpha-KGDH). The E2 components of PDH are membrane associated, whilst it would be expected that a mitochondrial CrT would be a transmembrane protein. Results of phase partitioning by Triton X-114, as well as washing of mitochondrial membranes at basic pH, support that these immunologically cross-reactive proteins are, as expected for E2 components, membrane associated rather than transmembrane. On the other hand, the fact that mitochondrial Cr uptake into intact mitoplast could be blocked by our alpha-CrTCOOH antibodies, indicate that our antisera contain antibodies reactive to proteins involved in mitochondrial transport of Cr. The presence of specific antibodies against CrT is supported by results from plasma membrane vesicles isolated from human and rat skeletal muscle, where both 55 and 70 kDa polypeptides disappeared and a single polypeptide with an apparent electrophoretic mobility of approximately 60 kDa was enriched. This latter is most likely representing the genuine plasma membrane CrT. Due to the fact that all anti-CrT antibodies that were independently prepared by several laboratories seem to cross-react with non-CrT polypeptides, specifically with E2 components of mitochondrial dehydrogenases, further research is required to characterise on a biochemical/biophysical level the CrT polypeptides, e.g. to determine whether the approximately 60 kDa polypeptide is indeed a bona-fide CrT and to identify the mitochondrial transporter that is able to facilitate Cr-uptake into these organelles. Therefore, the anti-CrT antibodies available so far should only be used with these precautions in mind. This holds especially true for quantitation of CrT polypeptides by Western blots, e.g. when trying to answer whether CrT's are up- or down-regulated by certain experimental interventions or under pathological conditions. In conclusion, we still hold to the scheme that besides the high-affinity and high-efficiency plasmalemma CrT there exists an additional low affinity high Km Cr uptake mechanism in mitochondria. However, the exact biochemical nature of this mitochondrial creatine transport, still remains elusive. Finally, similar to the creatine kinase (CK) isoenzymes, which are specifically located at different cellular compartments, also the substrates of CK are compartmentalized in cytosolic and mitochondrial pools. This is in line with 14C-Cr-isotope tracer studies and a number of [31P]-NMR magnetization transfer studies, as well as with recent [1H]-NMR spectroscopy data.

Methylglyoxal (MG) (pyruvaldehyde) is a reactive carbonyl compound produced in glycolysis. MG can form covalent adducts on proteins resulting in advanced glycation end products that may alter protein function. Here we report that MG covalently modifies the mitochondrial permeability transition pore (PTP), a high conductance channel involved in the signal transduction of cell death processes. Incubation of isolated mitochondria with MG for a short period of time (5 min), followed by removal of excess free MG, prevented both ganglioside GD3- and Ca2+-induced PTP opening and the ensuing membrane depolarization, swelling, and cytochrome c release. Under these conditions MG did not significantly interfere with mitochondrial substrate transport, respiration, or oxidative phosphorylation. The suppression of permeability transition was reversible following extended incubation in MG-free medium. Of the 29 physiological carbonyl and dicarbonyl compounds tested only MG and its analogue glyoxal were able to specifically alter the behavior of the PTP. Using a set of arginine-containing peptides, we found that the major MG-derived arginine adduct formed, following a short time exposure to MG, was the 5-hydro-5-methylimidazol-4-one derivative. These findings demonstrate that MG rapidly modifies the PTP covalently and stabilizes the PTP in the closed conformation. This is probably due to the formation of an imidazolone adduct on an arginine residue involved in the control of PTP conformation (Linder, M. D., Morkunaite-Haimi, S., Kinnunen, P. J. K., Bernardi, P., and Eriksson, O. (2002) J. Biol. Chem. 277, 937–942). We deduce that the permeability transition constitutes a potentially important physiological target of MG.

Mitochondria from transgenic mice, expressing enzymatically active mitochondrial creatine kinase in liver, were analyzed for opening of the permeability transition pore in the absence and presence of creatine kinase substrates but with no external adenine nucleotides added. In mitochondria from these transgenic mice, cyclosporin A-inhibited pore opening was delayed by creatine or cyclocreatine but not by β-guanidinopropionic acid. This observation correlated with the ability of these substrates to stimulate state 3 respiration in the presence of extramitochondrial ATP. The dependence of transition pore opening on calcium and magnesium concentration was studied in the presence and absence of creatine. If mitochondrial creatine kinase activity decreased (i.e. by omitting magnesium from the medium), protection of permeability transition pore opening by creatine or cyclocreatine was no longer seen. Likewise, when creatine kinase was added externally to liver mitochondria from wild-type mice that do not express mitochondrial creatine kinase in liver, no protective effect on pore opening by creatine and its analog was observed. All these findings indicate that mitochondrial creatine kinase activity located within the intermembrane and intercristae space, in conjunction with its tight functional coupling to oxidative phosphorylation, via the adenine nucleotide translocase, can modulate mitochondrial permeability transition in the presence of creatine. These results are of relevance for the design of creatine analogs for cell protection as potential adjuvant therapeutic tools against neurodegenerative diseases.

One of the most important duties of a cell is energy homoeostasis. Several kinases, including AMP-activated protein kinase (AMPK), creatine kinase and adenylate kinase, are involved in the immediate response to stress, resulting in energy depletion. Here, we present our view of events preceding the downstream processes mediated by AMPK and leading to reduced energy expenditure and increased energy production. Unfortunately, AMPK is very poorly defined at the molecular level. Thus a procedure for production of AMPK in milligram amounts is presented which will greatly facilitate the functional and structural characterization of this protein kinase.

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.

Acid stress in Escherichia coli involves a complex resource- and energy-consuming response mechanism. By overexpression of arginine kinase from Limulus polyphemus in E. coli, we improved the recovery from a transient pH stress. While wild type E. coli resumed growth after a transient pH reduction to pH 3 for 1 h with a rate that was 25% lower than before the stress, the arginine kinase expressing strain continued to grow as rapidly as before. This effect is presumably caused by the physiological function of arginine kinase as a short term energy buffer in the form of phosphoarginine, but a pH-buffering effect cannot be excluded.

International audience; To meet high cellular demands, the energy metabolism of cardiac muscles is organized by precise and coordinated functioning of intracellular energetic units (ICEUs). ICEUs represent structural and functional modules integrating multiple fluxes at sites of ATP generation in mitochondria and ATP utilization by myofibrillar, sarcoplasmic reticulum and sarcolemma ion‐pump ATPases. The role of ICEUs is to enhance the efficiency of vectorial intracellular energy transfer and fine tuning of oxidative ATP synthesis maintaining stable metabolite levels to adjust to intracellular energy needs through the dynamic system of compartmentalized phosphoryl transfer networks. One of the key elements in regulation of energy flux distribution and feedback communication is the selective permeability of mitochondrial outer membrane (MOM) which represents a bottleneck in adenine nucleotide and other energy metabolite transfer and microcompartmentalization. Based on the experimental and theoretical (mathematical modelling) arguments, we describe regulation of mitochondrial ATP synthesis within ICEUs allowing heart workload to be linearly correlated with oxygen consumption ensuring conditions of metabolic stability, signal communication and synchronization. Particular attention was paid to the structure–function relationship in the development of ICEU, and the role of mitochondria interaction with cytoskeletal proteins, like tubulin, in the regulation of MOM permeability in response to energy metabolic signals providing regulation of mitochondrial respiration. Emphasis was given to the importance of creatine metabolism for the cardiac energy homoeostasis.

In spite of all the fascinating properties of oral creatine supplementation, the mechanism(s) mediating its intestinal absorption has(have) not been investigated. The purpose of this study was to characterize intestinal creatine transport. [(14)C] creatine uptake was measured in chicken enterocytes and rat ileum, and expression of the creatine transporter CRT was examined in human, rat and chicken small intestine by reverse transcription-polymerase chain reaction, Northern blot, in situ hybridization, immunoblotting and immunohistochemistry. Results show that enterocytes accumulate creatine against its concentration gradient. This accumulation was electrogenic, Na(+)- and Cl(-)-dependent, with a probable stoichiometry of 2 Na(+): 1 Cl(-): 1 creatine, and inhibited by ouabain and iodoacetic acid. The kinetic study revealed a K(m) for creatine of 29 microM. [(14)C] creatine uptake was efficiently antagonized by non-labelled creatine, guanidinopropionic acid and cyclocreatine. More distant structural analogues of creatine, such as GABA, choline, glycine, beta-alanine, taurine and betaine, had no effect on intestinal creatine uptake, indicating a high substrate specificity of the creatine transporter. Consistent with these functional data, messenger RNA for CRT was detected only in the cells lining the intestinal villus. The sequences of partial clones, and of the full-length cDNA clone, isolated from human and rat small intestine were identical to previously cloned CRT cDNAs. Immunological analysis revealed that CRT protein was mainly associated with the apical membrane of the enterocytes. This study reports for the first time that mammalian and avian enterocytes express CRT along the villus, where it mediates high-affinity, Na(+)- and Cl(-)-dependent, apical creatine uptake.

Immunoblotting of isolated mitochondria from rat heart, liver, kidney, and brain with antibodies made against N- and C-terminal peptide sequences of the creatine transporter, together with in situ immunofluorescence staining and immunogold electron microscopy of adult rat myocardium, revealed two highly related polypeptides with molecular masses of ∼70 and ∼55 kDa in mitochondria. These polypeptides were localized by immunoblotting of inner and outer mitochondrial membrane fractions, as well as by immunogold labeling in the mitochondrial inner membrane. In addition, a novel creatine uptake via a mitochondrial creatine transport activity was demonstrated by [14C]creatine uptake studies with isolated mitochondria from rat liver, heart, and kidney showing a saturable low affinity creatine transporter, which was largely inhibited in a concentration-dependent manner by the sulfhydryl-modifying reagent NEM, as well as by the addition of the above anti-creatine transporter antibodies to partially permeabilized mitochondria. Mitochondrial creatine transport was to a significant part dependent on the energetic state of mitochondria and was inhibited by arginine, and to some extent also by lysine, but not by other creatine analogues and related compounds. The existence of an active creatine uptake mechanism in mitochondria indicates that not only creatine kinase isoenzymes, but also creatine transporters and thus a certain proportion of the creatine kinase substrates, might be subcellularly compartmentalized. Our data suggest that mitochondria, shown here to possess creatine transport activity, may harbor such a creatine/phosphocreatine pool.

Study design: To evaluate a potential protective effect of increased creatine levels in spinal cord injury (SCI) in an animal model. Objectives: Acute SCI initiates a series of cellular and molecular events in the injured tissue leading to further damage in the surrounding area. This secondary damage is partly due to ischemia and a fatal intracellular loss of energy. Phospho-creatine in conjunction with the creatine kinase isoenzyme system acts as a potent intracellular energy buffer. Oral creatine supplementation has been shown to elevate the phospho-creatine content in brain and muscle tissue, leading to neuroprotective effects and increased muscle performance. Setting: Zurich, Switzerland. Methods: Twenty adult rats were fed for 4 weeks with or without creatine supplemented nutrition before undergoing a moderate spinal cord contusion. Results: Following an initial complete hindlimb paralysis, rats of both groups substantially recovered within 1 week. However, creatine fed animals scored 2.8 points better than the controls in the BBB open field locomotor score (11.9 and 9.1 points respectively after 1 week; P=0.035, and 13 points compared to 11.4 after 2 weeks). The histological examination 2 weeks after SCI revealed that in all rats a cavity had developed which was comparable in size between the groups. In creatine fed rats, however, a significantly smaller amount of scar tissue surrounding the cavity was found. Conclusions: Thus creatine treatment seems to reduce the spread of secondary injury. Our results favour a pretreatment of patients with creatine for neuroprotection in cases of elective intramedullary spinal surgery. Further studies are needed to evaluate the benefit of immediate creatine administration in case of acute spinal cord or brain injury.

The mdx mouse serves as animal model for Duchenne muscular dystrophy. Energy status in muscles of mdx mice is impaired and we have demonstrated recently that the energy precursor creatine exerts beneficial effects on mdx skeletal muscle cells in culture. Here we show that feeding a creatine-enriched diet to new-born mdx mice strongly reduced the first wave of muscle necrosis four weeks after birth. Necrosis of the fast-twitch muscle extensor digitorum longus was inhibited by 63+/-14% (P

We hypothesized that creatine (Cr) supplementation would preserve energy metabolism and thus ameliorate the energy failure and the extent of brain edema seen after severe but transient cerebral hypoxia-ischemia (HI) in the neonatal rat model. Six-day-old (P6) rats received subcutaneous Cr monohydrate injections for 3 consecutive days (3 g/kg body weight/day), followed by 31P-magnetic resonance spectroscopy (MRS) at P9. In a second group, P4 rats received the same Cr dose as above for 3 days prior to unilateral common carotid artery ligation followed 1 h later by 100 min of hypoxia (8% O2) at P7. Rats were maintained at 37°C rectal temperature until magnetic resonance imaging was performed 24 h after HI. Cr supplementation for 3 days significantly increased the energy potential, i.e. the ratio of phosphocreatine to β-nucleotide triphosphate (PCr/βNTP) and PCr/inorganic phosphate (PCr/Pi) as measured by 31P-MRS. Rats with hemispheric cerebral hypoxic-ischemic insult that had received Cr showed a significant reduction (25%) of the volume of edemic brain tissue compared with controls as calculated from diffusion-weighted images (DWI). Thus, prophylactic Cr supplementation demonstrated a significant neuroprotective effect 24 h after transient cerebral HI. We hypothesize that neuroprotection is probably due to the availability of a larger metabolic substrate pool leading to a reduction of the secondary energy failure because DWI has been reported to correlate with the PCr/Pi ratio in the acute phase of injury. Additional protection by Cr may be related to prevention of calcium overload, prevention of mitochondrial permeability transition pore opening and direct antioxidant effects.

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 by31P 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 a-CK was demonstrated by indirect immunofluorescence staining using Mia-CK antibodies. When perifused with creatine-supplemented medium, phosphocreatine (PCr) continuously increased with time (1.2 ± 0.6 nmol · h−1· mg 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”).

The dimeric chicken brain type isoenzyme of creatine kinase (BB-CK) was mutated by a C283S amino acid exchange in the catalytic site to produce a basically inactive dimer (B*B*-CK). The mutated enzyme showed a residual activity of about 4% compared to the wild-type, whereas substrate binding parameters were not altered. The inactivated dimer was hybridized with native dimeric muscle enzyme (MM-CK) to produce a partially inactivated MB*-CK heterodimeric hybrid and also to a his-tagged BB-CK (hBhB-CK) resulting in a partially inactive hBB*-CK homodimer. The generated hybrids were purified by chromatography. The V(max) and substrate binding parameters K(m) and K(d) were determined for both directions of the CK reaction and compared to the parameters of the wild-type enzymes (MM-, BB-, hBhB-, MB-CK). In the direction of ATP synthesis (reverse reaction), the MB*- and hBB*-CK hybrids showed a decrease of V(max) to 34% and 32%, respectively, compared to the unmodified wild-type isoform. The inactivation of a single subunit in MB*-CK led to an increase in the K(d) value resulting in an significant substrate synergism, not seen with the MB-CK wild-type enzyme. In the direction of phosphocreatine synthesis (forward reaction), the modified hybrids showed a decrease of V(max) to 50% of the wild-type enzymes and no significant alterations of the K(m) and K(d) parameters. These results strongly suggest an enzymatic cooperativity of the two subunits in the reverse reaction but independent catalytic function in the forward reaction.

The specific interaction of muscle type creatine-kinase (MM-CK) with the myofibrillar M-line was demonstrated by exchanging endogenous MM-CK with an excess of fluorescently labeled MM-CK in situ, using chemically skinned skeletal muscle fibers and confocal microscopy. No binding of labeled MM-CK was noticed at the I-band of skinned fibers, where the enzyme is additionally located in vivo, as shown earlier by immunofluorescence staining of cryosections of intact muscle. However, when rhodamine-labeled MM-CK was diffused into skinned fibers that had been preincubated with phosphofructokinase (PFK), a glycolytic enzyme known to bind to actin, a striking in vivo-like interaction of Rh-MM-CK with the I-band was found, presumably mediated by binding of Rh-MM-CK to the glycolytic enzyme. Aldolase, another actin-binding glycolytic enzyme was also able to bind Rh-MM-CK to the I-band, but formation of the complex occurred preferably at long sarcomere length (>3.0 μm). Neither pyruvate kinase, although known for its binding to actin, nor phosphoglycerate kinase (PGK), not directly interacting with the I-band itself, did mediate I-band targeting of MM-CK. Anchoring of MM-CK to the I-band via PFK, but not so via aldolase, was strongly pH-dependent and occurred below pH 7.0. Labeling performed at different sarcomere length indicated that the PFK/MM-CK complex bound to thin filaments of the I-band, but not within the actomyosin overlap zones. The physiological consequences of the structural interaction of MM-CK with PFK at the I-band is discussed with respect to functional coupling of MM-CK to glycolysis, metabolic regulation and channeling in multi-enzyme complexes. The in situ binding assay with skinned skeletal muscle fibers described here represents a useful method for further studies of specific protein–protein interactions in a structurally intact contractile system under various precisely controlled conditions.

The efficiency of stimulation of mitochondrial respiration in permeabilized muscle cells by ADP produced at different intracellular sites, e.g. cytosolic or mitochondrial intermembrane space, was evaluated in wild-type and creatine kinase (CK)-deficient mice. To activate respiration by endogenous production of ADP in permeabilized cells, ATP was added either alone or together with creatine. In cardiac fibers, while ATP alone activated respiration to half of the maximal rate, creatine plus ATP increased the respiratory rate up to its maximum. To find out whether the stimulation by creatine is a consequence of extramitochondrial [ADP] increase, or whether it directly correlates with ADP generation by mitochondrial CK in the mitochondrial intermembrane space, an exogenous ADP-trap system was added to rephosphorylate all cytosolic ADP. Under these conditions, creatine plus ATP still increased the respiration rate by 2.5 times, compared with ATP alone, for the same extramitochondrial [ADP] of 14 microM. Moreover, this stimulatory effect of creatine, observed in wild-type cardiac fibers disappeared in mitochondrial CK deficient, but not in cytosolic CK-deficient muscle. It is concluded that respiration rates can be dissociated from cytosolic [ADP], and ADP generated by mitochondrial CK is an important regulator of oxidative phosphorylation.

We have evaluated surface plasmon resonance with avidin-biotin immobilized liposomes tocharacterize membrane binding of ubiquitous mitochondrial creatine kinase (uMtCK). Whilethe sarcomeric sMtCK isoform is well known to bind to negatively charged phospholipids,especially cardiolipin, this report provides the first experimental evidence on the membraneinteraction of an uMtCK isoform. Qualitative measurements showed that liposomes containing16% (w/w) cardiolipin bind octameric as well as dimeric human uMtCK and also cytochromec, but not bovine serum albumin. Quantitative parameters could be derived only for themembrane interaction of octameric human uMtCK using an improved analytical approach.Association and dissociation kinetics of octameric uMtCK fit well to a model for heterogeneousinteraction suggesting two independent binding sites. Rate constants of the two sites differedby one order of magnitude, while their affinity constants were both about 80–100 nM. Thedata obtained demonstrate that surface plasmon resonance with immobilized liposomes is asuitable approach to characterize the binding of peripheral proteins to a lipid bilayer and thatthis method yields consistent quantitative binding parameters.