Phosphorylation Of Creatine Research Articles

Decreased mitochondrial creatine kinase 2 impairs skeletal muscle mitochondrial function independently of insulin in type 2 diabetes.

Increased plasma creatine concentrations are associated with the risk of type 2 diabetes, but whether this alteration is associated with or causal for impairments in metabolism remains unexplored. Because skeletal muscle is the main disposal site of both creatine and glucose, we investigated the role of intramuscular creatine metabolism in the pathophysiology of insulin resistance in type 2 diabetes. In men with type 2 diabetes, plasma creatine concentrations were increased, and intramuscular phosphocreatine content was reduced. These alterations were coupled to reduced expression of sarcomeric mitochondrial creatine kinase 2 (CKMT2). In C57BL/6 mice fed a high-fat diet, neither supplementation with creatine for 2 weeks nor treatment with the creatine analog β-GPA for 1 week induced changes in glucose tolerance, suggesting that increased circulating creatine was associated with insulin resistance rather than causing it. In C2C12 myotubes, silencing Ckmt2 using small interfering RNA reduced mitochondrial respiration, membrane potential, and glucose oxidation. Electroporation-mediated overexpression of Ckmt2 in skeletal muscle of high-fat diet-fed male mice increased mitochondrial respiration, independent of creatine availability. Given that overexpression of Ckmt2 improved mitochondrial function, we explored whether exercise regulates CKMT2 expression. Analysis of public data revealed that CKMT2 content was up-regulated by exercise training in both humans and mice. We reveal a previously underappreciated role of CKMT2 in mitochondrial homeostasis beyond its function for creatine phosphorylation, independent of insulin action. Collectively, our data provide functional evidence for how CKMT2 mediates mitochondrial dysfunction associated with type 2 diabetes.

Measurement of Futile Creatine Cycling Using Respirometry.

Thermogenic adipose tissue plays a vital function in regulating whole-body energy expenditure and nutrient homeostasis due to its capacity to dissipate chemical energy as heat, in a process called non-shivering thermogenesis. A reduction of creatine levels in adipocytes impairs thermogenic capacity and promotes diet-induced obesityKazak et al, Cell 163, 643-55, 2015; Kazak et al, Cell Metab 26, 660-671.e3, 2017; Kazak et al, Nat Metab 1, 360-370, 2019). Mechanistically, thermogenic respiration can be promoted by the liberation of an excess quantity of ADP that is dependent on addition of creatine. A model of a two-enzyme system, which we term the Futile Creatine Cycle, has been posited to support this thermogenic action of creatine. Futile creatine cycling can be monitored in purified mitochondrial preparations wherein creatine-dependent liberation of ADP is monitored through the measurement of oxygen consumption under ADP-limiting conditions. The current model proposes that, in thermogenic fat cells, mitochondria-targeted creatine kinase B (CKB) uses mitochondrial-derived ATP to phosphorylate creatine (Rahbani JF, Nature 590, 480-485, 2021). The creatine kinase reaction generates phosphocreatine and ADP, and ADP stimulates respiration. Next, a pool of mitochondrial phosphocreatine is directly hydrolyzed by a phosphatase, to regenerate creatine. The liberated creatine can then engage mitochondrial CKB to trigger another round of this cycle to support ADP-dependent respiration. In this model, the coordinated action of creatine phosphorylation and phosphocreatine hydrolysis triggers a futile cycle that produces a molar excess of mitochondrial ADP to promote thermogenic respiration (Rahbani JF, Nature 590, 480-485, 2021; Kazak and Cohen, Nat Rev Endocrinol 16, 421-436, 2020). Here, we provide a detailed method to perform respiratory measurements on isolated mitochondria and calculate the stoichiometry of creatine-dependent ADP liberation. This method provides a direct measure of the futile creatine cycle.

HIF-Dependent CKB Expression Promotes Breast Cancer Metastasis, Whereas Cyclocreatine Therapy Impairs Cellular Invasion and Improves Chemotherapy Efficacy.

Simple SummaryTargeting dysregulated cellular metabolism is a promising avenue to treat metastatic disease. The aim of our study was to identify genes downstream of the hypoxia-inducible factor (HIF)-1 transcription factor that are amenable to therapeutic intervention to treat metastatic breast cancer (MBC). We identified creatine kinase, brain isoform (CKB) as an HIF-dependent gene that strongly promotes invasion and metastasis in estrogen-receptor (ER)-negative breast cancer models. Deletion of Ckb also repressed glycolysis and mitochondrial respiration, leading to a reduction in intracellular ATP. Either the deletion of Ckb or inhibition of creatine kinase (CK) activity using the creatine analog cyclocreatine (cCr) repressed cell invasion, the formation of invadopodia and lung metastasis. In addition, when paired with paclitaxel or doxorubicin, cCr enhanced growth inhibition in an additive or synergistic manner. cCr may be an effective anti-metastatic agent in ER-negative, HIF-1α-positive breast cancers, targeting both cellular metabolism and motility, particularly when paired with conventional cytotoxic agents.The oxygen-responsive hypoxia inducible factor (HIF)-1 promotes several steps of the metastatic cascade. A hypoxic gene signature is enriched in triple-negative breast cancers (TNBCs) and is correlated with poor patient survival. Inhibiting the HIF transcription factors with small molecules is challenging; therefore, we sought to identify genes downstream of HIF-1 that could be targeted to block invasion and metastasis. Creatine kinase brain isoform (CKB) was identified as a highly differentially expressed gene in a screen of HIF-1 wild type and knockout mammary tumor cells derived from a transgenic model of metastatic breast cancer. CKB is a cytosolic enzyme that reversibly catalyzes the phosphorylation of creatine, generating phosphocreatine (PCr) in the forward reaction, and regenerating ATP in the reverse reaction. Creatine kinase activity is inhibited by the creatine analog cyclocreatine (cCr). Loss- and gain-of-function genetic approaches were used in combination with cCr therapy to define the contribution of CKB expression or creatine kinase activity to cell proliferation, migration, invasion, and metastasis in ER-negative breast cancers. CKB was necessary for cell invasion in vitro and strongly promoted tumor growth and lung metastasis in vivo. Similarly, cyclocreatine therapy repressed cell migration, cell invasion, the formation of invadopodia and lung metastasis. Moreover, in common TNBC cell line models, the addition of cCr to conventional cytotoxic chemotherapy agents was either additive or synergistic to repress tumor cell growth.

Creatine Supplementation Exhibits Sex Differences in White Adipose Tissue and Increases Mitochondrial Markers in Female Rats

Activation of white adipose tissue (WAT) thermogenesis, known as WAT browning, has emerged as an attractive approach to treat obesity and obesity‐related diseases. Traditionally BAT or browning WAT has been defined by the expression of uncoupling protein 1 (UCP1), a mitochondrial protein that uncouples respiration from ATP production. However, recent work has highlighted creatine metabolism or futile cycling as a potential thermogenic pathway. Creatine‐driven futile cycling is a catabolic process which results in ATP oxidation by mitochondrial creatine kinase, resulting in the phosphorylation of creatine and increases in ADP concentrations that drive thermogenic respiration. The purpose of the present study was two‐fold: 1) to determine the effects of creatine supplementation on markers of WAT browning, and 2) to determine if creatine supplementation exhibits sex differences. Thirty‐two Sprague‐Dawley rats (16 male, 16 female) were randomly assigned to one of four experimental groups: control, 2.5g/L, 5g/L and 10g/L of creatine monohydrate (CM) and 1% sucrose via drinking water. Rats had ad libitum access to their drinking water and a standard chow diet (AIN‐93G) throughout the study. Following the 8‐week intervention, brown interscapular adipose (BAT) and white subcutaneous inguinal (iWAT) fat depots were collected. Western blotting analysis demonstrated no significant changes in BAT samples for both males and females. Males had lower cytochrome C than females with 10g/L of creatine monohydrate (p<0.05). In iWAT, mitochondrial markers (COXIV, PDH, citrate synthase, and cytochrome C) and GAMT exhibit no changes in males, however, a decrease in UCP‐1 at 5g/L compared to control was observed (p<0.05). Females exhibit a lower control PDH content than males (p<0.01) in iWAT, accompanied by an increase in PDH levels at all doses of CM compared to control (p<0.05). Furthermore, in females COXIV and cytochrome C presented significant increases with 5g/L and 10g/L, respectively (p<0.05). Both males and females did not present with body weight differences despite dose differences. This study presents novel work demonstrating that female WAT exhibits less mitochondrial markers than males under control conditions, however it demonstrates the potential for CM supplementation to increase female adipose thermogenicity to a similar level observed in males.Support or Funding InformationSupported by NSERC, Canada

The discovery of oxidative phosphorylation: a conceptual off-shoot from the study of glycolysis

The origins of oxidative phosphorylation, initially known as aerobic phosphorylation, grew out of three research areas of muscle metabolism, creatine phosphorylation, aerobic metabolism of lactic acid in muscle, and studies on the nature and role of adenosine triphosphate (ATP). Much of this work centred round the laboratory of Otto Meyerhof, and most of those contributing to the study of aerobic phosphorylation were influenced by that laboratory: particularly Lipmann and also Ochoa. The work of Engelhardt on ATP levels in blood also appears to have been influenced by the studies of Meyerhof’s laboratory. However, with the work of Kalckar, influenced by Lipmann, biochemists began to realise the potential importance of the process. This was confirmed and extended by Belitzer and Ochoa and the theoretical contribution of Lipmann. Thus it is not easy to identify a single point that marked the initiation of studies in this field. The early work was based on the use of tissue homogenates and cells but ultimately this approach limited research possibilities. The development of techniques based on mitochondria opened up new possibilities and brought this first phase of the study of oxidative phorphorylation to a close.

Creatine Kinase Elevation Following Eccentric Exercise in Females as a Marker for Training Stress

Creatine kinase is an enzyme used by various tissue types to produce the energy substrate phosphocreatine (PCr). Cytosolic creatine kinase (CKc) is found within skeletal, cardiac as well as brain tissue and is responsible for catalyzing the phosphorylation of creatine to PCr when muscles are at rest. Observed CKc elevation following eccentric loading has previously been reported as an indicator for muscle adaptation or damage. PURPOSE: To determine the degree and duration of CKc elevation post-eccentric loading in females as a marker for training stress. METHODS: Baseline CKc levels were measured in four female subjects (22+2 yrs) prior to eccentric loading. Approximately 30 minutes of eccentric gymnastics strengthening activities were performed by all subjects. CKc was measured pre-exercise, 24 and 48 hours post-exercise (Reflotron®). RESULTS: Although a one-way Repeated Measures ANOVA was not significant (p>0.05), baseline CKc increased 23.96%, 130.97%, 442.53% and 133.66% at 24 hours. The insignificant difference is likely due to the high degree of CKc variability seen between subjects. All CKc values did follow the elevation trend expected at 24 hours post-exercise. CONCLUSION: Measures of CKc alone appear too variable to reply upon for a sole marker of training stress. When coupled with other parameters to account for variability between individuals, such as cortisol and/or a perceived effort or muscle soreness scale, CKc could provide a relevant measurement to help assess training stress.

Regulation of Creatine Kinase Activity by Phosphorylation of Serine‐199 by AMP‐Activated Kinase

Creatine kinase (CK) catalyzes the reversible phosphorylation of creatine by MgATP to form phosphocreatine and MgADP. An accessible reservoir of high energy phosphate in the form of phosphocreatine is useful in excitable tissues (i.e. brain, heart, or muscle) which require large energy fluxes. Thus, CK is important to energy homeostasis in cells. Previous work has suggested that AMP‐activated kinase may regulate the activity of CK by phosphorylation, presumably at a serine residue (Ponticos, et al. EMBO J. 17:1688.). To confirm this hypothesis, a set of serines in CK were changed to glutamate by site‐directed mutagenesis to assess if any of these residues were possible phosphorylation sites. Notably, the S199E variant of CK showed a 200‐fold reduction in the catalytic rate constant (phosphocreatine formation) compared to wild‐type suggesting that phosphorylation of S199 is likely involved in regulation of CK. The possible means by which phosphorylation alters the active site will be discussed. These results point to a higher degree of cellular control of ATP concentration by regulating the activity of CK. Such a regulatory mechanism may have implications on better understanding energy flux within a cell.

A quantitative study of bioenergetics in skeletal muscle lacking carbonic anhydrase III using 31 P magnetic resonance spectroscopy

Oxidative slow skeletal muscle contains carbonic anhydrase III in high concentration, but its primary function remains unknown. To determine whether its lack handicaps energy metabolism and/or acid elimination, we measured the intracellular pH and energy phosphates by (31)P magnetic resonance spectroscopy in hind limb muscles of wild-type and CA III knockout mice during and after ischemia and intense exercise (electrical stimulation). Thirty minutes of ischemia caused phosphocreatine (PCr) to fall and P(i) to rise while pH and ATP remained constant in both strains of mice. PCr and P(i) kinetics during ischemia and recovery were not significantly different between the two genotypes. From this we conclude that under neutral pH conditions resting muscle anaerobic metabolism, the rate of the creatine kinase reaction, intracellular buffering of protons, and phosphorylation of creatine by mitochondrial oxygen metabolism are not influenced by the lack of CA III. Two minutes of intense stimulation of the mouse gastrocnemius caused PCr, ATP, and pH to fall and ADP and P(i) to rise, and these changes, with the exception of ATP, were all significantly larger in the CA III knockouts. The rate of return of pH and ADP to control values was the same in wild-type and mutant mice, but in the mutants PCr and P(i) recovery were delayed in the first minute after stimulation. Because the tension decrease during fatigue is known to be the same in the two genotypes, we conclude that a lack of CA III impairs mitochondrial ATP synthesis.

Origin and properties of cytoplasmic and mitochondrial isoforms of taurocyamine kinase

Taurocyamine kinase (TK) is a member of the highly conserved family of phosphagen kinases that includes creatine kinase (CK) and arginine kinase. TK is found only in certain marine annelids. In this study we used PCR to amplify two cDNAs coding for TKs from the polychaete Arenicola brasiliensis, cloned these cDNAs into the pMAL plasmid and expressed the TKs as fusion proteins with the maltose-binding protein. These are the first TK cDNA and deduced amino acid sequences to be reported. One of the two cDNA-derived amino acid sequences of TKs shows a high amino acid identity to lombricine kinase, another phosphagen kinase unique to annelids, and appears to be a cytoplasmic isoform. The other sequence appears to be a mitochondrial isoform; it has a long N-terminal extension that was judged to be a mitochondrial targeting peptide by several on-line programs and shows a higher similarity in amino acid sequence to mitochondrial creatine kinases from both vertebrates and invertebrates. The recombinant cytoplasmic TK showed activity for the substrates taurocyamine and lombricine (9% of that of taurocyamine). However, the mitochondrial TK showed activity for taurocyamine, lombricine (30% of that of taurocyamine) and glycocyamine (7% of that of taurocyamine). Neither TK catalyzed the phosphorylation of creatine. Comparison of the deduced amino acid sequences of mitochondrial CK and TK indicated that several key residues required for CK activity are lacking in the mitochondrial TK sequence. Homology models for both cytoplasmic and mitochondrial TK, constructed using CK templates, provided some insight into the structural correlation of differences in substrate specificity between the two TKs. A phylogenetic analysis using amino acid sequences from a broad spectrum of phosphagen kinases showed that annelid-specific phosphagen kinases (lombricine kinase, glycocyamine kinase and cytoplasmic and mitochondrial TKs) are grouped in one cluster, and form a sister-group with CK sequences from vertebrate and invertebrate groups. It appears that the annelid-specific phosphagen kinases, including cytoplasmic and mitochondrial TKs, evolved from a CK-like ancestor(s) early in the divergence of the protostome metazoans. Furthermore, our results suggest that the cytoplasmic and mitochondrial isoforms of TK evolved independently.

Isoleucine 69 and Valine 325 Form a Specificity Pocket in Human Muscle Creatine Kinase

Creatine kinase (CK) catalyzes the reversible phosphorylation of creatine by ATP. From a structural perspective, the enzyme utilizes two flexible loop regions to sequester and position the substrates for catalysis. There has been debate over the specific roles of the flexible loops in substrate specificity and catalysis in CK and other related phosphagen kinases. In CK, two hydrophobic loop residues, I69 and V325, make contacts with the N-methyl group of creatine. In this study, we report the alteration of the substrate specificity of CK through the mutagenesis of V325. The V325 to glutamate mutation results in a more than 100-fold preference for glycocyamine, while mutation of V325 to alanine results in a slight preference of the enzyme for cyclocreatine (1-carboxymethyl-2-iminoimidazolidine). This study enhances our understanding of how the active sites of phosphagen kinases have evolved to recognize their respective substrates and catalyze their reactions.

Creatine supplementation affects sprint endurance in juvenile rainbow trout

Fingerling rainbow trout were supplemented with equal amounts of creatine (Cr) by two routes: dietary (12.5 mg Cr per g food); or intraperitoneal injection (0.5 mg Cr per g fish). Endurance in a fixed velocity sprint test (at a speed of 7 BL s −1), and resting levels of white muscle metabolites (total creatine [a measure of free creatine plus phosphocreatine (PCr), ATP, lactate and glycogen] were assessed following 7 days of supplementation and compared to controls. None of the treatments had a significant effect on growth, muscle total creatine, percent phosphorylation of creatine, ATP or lactate. However, resting muscle glycogen was elevated in creatine-supplemented fish. Higher muscle glycogen corresponded to significantly greater endurance in creatine-supplemented fish. Although fish do not actively transport additional creatine into the muscle, a mechanism whereby circulating creatine acts to enhance muscle glycogen is present. These results suggest that the improved endurance may be due to an insulin-dependent mechanism (similar to that elucidated in mammalian studies) that allows fish to supercompensate muscle glycogen stores, thus extending endurance through enhanced glycolytic flux.

An unusually low pK(a) for Cys282 in the active site of human muscle creatine kinase.

All phosphagen kinases contain a conserved cysteine residue which has been shown by crystallographic studies, on both creatine kinase and arginine kinase, to be located in the active site. There are conflicting reports as to whether this cysteine is essential for catalysis. In this study we have used site-directed mutagenesis to replace Cys282 of human muscle creatine kinase with serine and methionine. In addition, we have replaced Cys282, conserved across all creatine kinases, with alanine. No activity was found with the C282M mutant. The C282S mutant showed significant, albeit greatly reduced, activity in both the forward (creatine phosphorylation) and reverse (MgADP phosphorylation) reactions. The K(m) for creatine was increased approximately 10-fold, but the K(m) for phosphocreatine was relatively unaffected. The V and V/K pH-profiles for the wild-type enzyme were similar to those reported for rabbit muscle creatine kinase, the most widely studied creatine kinase isozyme. However, the V/K(creatine) profile for the C282S mutant was missing a pK of 5.4. This suggests that Cys282 exists as the thiolate anion, and is necessary for the optimal binding of creatine. The low pK of Cys282 was also determined spectrophotometrically and found to be 5.6 +/- 0.1. The S284A mutant was found to have reduced catalytic activity, as well as a 15-fold increase in K(m) for creatine. The pK(a) of Cys282 in this mutant was found to be 6.7 +/- 0.1, indicating that H-bonding to Ser284 is an important, but not the sole, factor contributing to the unusually low pK(a) of Cys282.

Interactions between bioenergetics and mitochondrial biogenesis

We studied the interaction between energy metabolism and mitochondrial biogenesis during myogenesis in C2C12 myoblasts. Metabolic rate was nearly constant throughout differentiation, although there was a shift in the relative importance of glycolytic and oxidative metabolism, accompanied by increases in pyruvate dehydrogenase activation state and total activity. These changes in mitochondrial bioenergetic parameters observed during differentiation occurred in the absence of a hypermetabolic stress. A chronic (3 day) energetic stress was imposed on differentiated myotubes using sodium azide to inhibit oxidative metabolism. When used at low concentrations, azide inhibited more than 70% of cytochrome oxidase (COX) activity without changes in bioenergetics (either lactate production or creatine phosphorylation) or mRNA for mitochondrial enzymes. Higher azide concentrations resulted in changes in bioenergetic parameters and increases in steady state COX II mRNA levels. Azide did not affect mtDNA copy number or mRNA levels for other mitochondrial transcripts, suggesting azide affects stability, rather than synthesis, of COX II mRNA. These results indicate that changes in bioenergetics can alter mitochondrial genetic regulation, but that mitochondrial biogenesis accompanying differentiation occurs in the absence of hypermetabolic challenge.

The active site histidines of creatine kinase. A critical role of His 61 situated on a flexible loop.

A histidine residue with a pKa of 7 has been inferred to act as a general acid-base catalyst for the reaction of creatine kinase (CK), catalyzing the reversible phosphorylation of creatine by ATP. The chicken sarcomeric muscle mitochondrial isoenzyme Mib-CK contains several histidine residues that are conserved throughout the family of creatine kinases. By X-ray crystal structure analysis, three of them (His 61, His 92, and His 186) were recently shown to be located close to the active site of the enzyme. These residues were exchanged against alanine or aspartate by in vitro mutagenesis, and the six mutant proteins were expressed in E. coli and purified. Structural integrity of the mutant proteins was checked by small-angle X-ray scattering. Kinetic analysis showed the mutant His 61 Asp to be completely inactive in the direction of ATP consumption while exhibiting a residual activity of 1.7% of the wild-type (wt) activity in the reverse direction. The respective His to Ala mutant of residue 61 showed approximately 1% wt activity in the forward and 10% wt activity in the reverse reaction. All other mutants showed near wt activities. Changes in the kinetic parameters K(m) or Vmax, as well as a significant loss of synergism in substrate binding, could be observed with all active mutants. These effects were most pronounced for the binding of creatine and phosphocreatine, whereas ATP or ADP binding were less severely affected. Based on our results, we assume that His 92 and His 186 are involved in the binding of creatine and ATP in the active site, whereas His 61 is of importance for the catalytic reaction but does not serve as an acid-base catalyst in the transphosphorylation of creatine and ATP. In addition, our data support the idea that the flexible loop bearing His 61 is able to move towards the active site and to participate in catalysis.

31P NMR investigation of energy metabolism in perifused MMQ cells.

The MMQ cell line is a unique prolactin-secreting rat pituitary cell line. MMQ cells entrapped in agarose gel threads are metabolically active, as determined by the uptake and phosphorylation of creatine and the maintenance of high energy phosphates for over 15 h. Forskolin activates the catalytic subunit of adenylyl cyclase and, in MMQ cells, elevates the level of cAMP and stimulates prolactin secretion. 31P NMR spectroscopy was used to investigate the energy metabolism of the MMQ cells during stimulation by forskolin. The ability to measure small changes in the energy status of these cells was enhanced by increasing the PCr levels in the cells. Administration of forskolin to the perifused MMQ cells resulted in acute, reversible, and dose-dependent changes in the 31P NMR spectra of the cells within 12 to 24 min of the beginning of forskolin exposure. Several lines of evidence indicate that the changes observed in the MMQ cells are the composite result of the interaction of forskolin with adenylyl cyclase and the plasma membrane glucose transporter. Also, preincubation of the MMQ cells with the dopamine agonist, bromocriptine, attenuates the forskolin-stimulated decrease in the PCr resonance by approximately 50%. This attenuation indicates that the forskolin-stimulated changes in energy metabolism are probably related to the prolactin secretion process.

ATP cofactor regeneration via the glycolytic pathway

In this communication the glycolytic pathway is shown to be a useful means of regenerating ATP in situ. The system has the rather attractive advantage that the two phosphoryl donor substrates, glucose and inorganic phosphate, are readily available at minimal cost. In addition, the free energy change for the ATP regeneration reaction is much more negative than other previously utilized ATP regeneration systems. Hence, the phosphorylation of compounds having high free energies of hydrolysis is thermodynamically more favorable using the glycolytic pathway as a means of ATP regeneration. The utility of the method is demonstrated by coupling the creatine kinase-catalyzed phosphorylation of creatine to the glycolytic conversion of glucose to lactic acid.

Correlations between cardiac protein synthesis rates, intracellular pH and the concentrations of creatine metabolites

We have examined in detail the correlations between protein synthesis rates, intracellular pH (pHi) and the concentrations of creatine metabolites in the rat heart perfused anterogradely in vitro. Using perfusion buffers ranging from pH 7.2 to 8.2 at 37 degrees C, we were able to manipulate pHi from between 7.24 to 7.66, i.e. from the slightly acidotic to the alkalinotic as compared with the physiological values of pHi (about pH 7.29). The dependence of pHi on extracellular pH (pHo) was linear, with the value of delta pHi/delta pHo being 0.4-0.5. Protein synthesis rates were significantly stimulated when pHi was increased above its physiological value, and they were strongly correlated with pHi. They were also strongly correlated with phosphocreatine concentrations (and with creatine concentrations and phosphocreatine/creatine concentration ratios). Adenine nucleotide (ATP, ADP and AMP) concentrations and the ATP/ADP concentration ratio were not systematically altered by manipulating pHi, and protein synthesis rates showed only a relatively weak dependence on these variables. Since creatine kinase catalyses a reaction that is close to equilibrium in the perfused heart, and since phosphorylation of creatine involves release of a proton, we argue that the changes in phosphocreatine and creatine concentrations are manifestations of alterations in pHi. In this regard, we show that [log[( phosphocreatine]/[creatine]) + log [( ADP]/[ATP])] [the value of which gives [pHi--log (mass action ratio)]] is positively correlated with pHi, although the slope of the line is 0.7, as opposed to the ideal value of unity. We discuss three hypotheses to account for our observations: (i) protein synthesis rates are influenced directly by pHi, (ii) pHi affects the concentrations of creatine metabolites, which in turn affect protein synthesis rates, and (iii) pHi affects the value of an unidentified co-variable, which in turn affects protein synthesis.

Creatine uptake, metabolism, and efflux in human monocytes and macrophages

The uptake of creatine in human monocytes and monocyte-derived macrophages can be divided into two components. One component of uptake is saturable (Km = 30 microM), specific for creatine, requires Na+ in the extracellular medium, and enables cells to take up creatine against a concentration gradient of creatine. The second component of uptake is nonsaturable and accounts for less than 10% of creatine uptake at physiological creatine concentrations (20-60 microM). Both monocytes and macrophages have similar Km values for creatine transport. However, macrophages exhibit a fivefold greater rate of creatine uptake than monocytes (3.6 vs. 0.7 pmol X micrograms protein-1 X h-1), indicating that monocyte differentiation into macrophages is associated with an increase in the number of creatine transporters in the membrane. Monocytes do not contain creatine kinase and do not phosphorylate creatine; monocyte-derived macrophages express creatine kinase and phosphorylate approximately 25% of intracellular creatine. These results and the finding that 2-deoxyglucose inhibits creatine phosphorylation by macrophages without affecting the transport of creatine indicate that phosphorylation of creatine is not required for net creatine accumulation.

Compartmentation of adenine nucleotides and phosphocreatine shuttle in cardiac cells: some new evidence.

The concept of the phosphocreatine shuttle for intracellular energy channelling in muscle cells (1 — 6) assumes compartmentation of adenine nucleotides in mitochondrial and myofibrillar compartments and around membranes. The link between these compartments is considered to be performed by phosphocreatine molecules due to heterogeneous distribution of creatine kinase isoenzymes functioning in mitochondria in direction of creatine phosphorylation and in myofibrils in direction of phosphocreatine dephosphorylation. While different isoenzymes of creatine kinase are well characterized, adenine nucleotide compartmentation still remains to be an unsolved question. In our recent studies described below a competitive enzyme method was used to show the existence of rather closed functional cycles of adenine nucleotides in mitochondria and in myofibrils due to titivation of creatine kinase in those structures. Also, 31P-NMR saturation transfer method was used to show the role of those coupled creatine kinases inside mitochondrial and myofibrillar compartments in the integrated intracellular energy fluxes in isolated perfused rat hearts at different workloads.KeywordsCreatine KinaseOxidative PhosphorylationPyruvate KinaseAdenine NucleotideCreatine Kinase IsoenzymeThese keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

ATP-ADP-dependent phosphorylations of glycolysis metabolites, creatine and glycerol: their compartition and thermodynamic relationship in gastrocnemius muscle cell of exercised guinea pigs.

The concentrations of following metabolites were determined in freeze-clamped gastrocnemius muscle samples: glucose 1-phosphate, glucose 6-phosphate, glucose, fructose 1,6-diphosphate, fructose 6-phosphate, D-glyceraldehyde 3-phosphate. dihydroxyacetone phosphate, phosphoenolpyruvate, pyruvate, glycerol 3-phosphate, glycerol, creatine phosphate, creatine, glycerate 3-phosphate, glycerate 2-phosphate, adenosine monophosphate, adenosine diphosphate, adenosine triphosphate, inorganic phosphate. The results showed that within the limits of experimental error, concentration homeostasis for this metabolites is founded at least in some cases on equilibria between enzymic transformations. Discrepancies between constant mass ratios measured in this study and equilibrium constants allow the free energy variation delta G to keep creatine phosphate at high concentration to be calculated. For the phosphoglycerate mutase system, the equilibrium constant in controls and trained animals is unchanged and corresponds to that in vitro. Training hindered glycolysis and favoured phosphorylation of creatine by glycerol 3-phosphate. Metabolites of the pyruvate kinase and hexokinase system cannot be homogeneously distributed in one space. The creatine kinase system is also separated from the hexokinase und pyruvate kinase system. A compartition of glycolytic process in gastrocnemius muscle seems to be inferred from these results.