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Brain energy deficit has been a suggested cause of Huntington disease (HD), but ATP depletion has not reliably been shown in preclinical models, possibly because of the immediate post-mortem changes in cellular energy metabolism. To examine a potential role of a low energy state in HD, we measured, for the first time in a neurodegenerative model, brain levels of high energy phosphates using microwave fixation, which instantaneously inactivates brain enzymatic activities and preserves in vivo levels of analytes. We studied HD transgenic R6/2 mice at ages 4, 8, and 12 weeks. We found significantly increased creatine and phosphocreatine, present as early as 4 weeks for phosphocreatine, preceding motor system deficits and decreased ATP levels in striatum, hippocampus, and frontal cortex of R6/2 mice. ATP and phosphocreatine concentrations were inversely correlated with the number of CAG repeats. Conversely, in mice injected with 3-nitroproprionic acid, an acute model of brain energy deficit, both ATP and phosphocreatine were significantly reduced. Increased creatine and phosphocreatine in R6/2 mice was associated with decreased guanidinoacetate N-methyltransferase and creatine kinase, both at the protein and RNA levels, and increased phosphorylated AMP-dependent protein kinase (pAMPK) over AMPK ratio. In addition, in 4-month-old knock-in Hdh(Q111/+) mice, the earliest metabolic alterations consisted of increased phosphocreatine in the frontal cortex and increased the pAMPK/AMPK ratio. Altogether, this study provides the first direct evidence of chronic alteration in homeostasis of high energy phosphates in HD models in the earliest stages of the disease, indicating possible reduced utilization of the brain phosphocreatine pool.

Amyloid peptide (Aβ) aggregation in the brain is a characteristic feature of Alzheimer disease (AD). Previously, we reported the discovery of focally elevated creatine deposits in brain tissue from TgCRND8 mice, which express double mutant (K670N/M671L and V717F) amyloid protein precursor. In this study, frozen hippocampal tissue sections from 5-, 8-, 11-, 14-, and 17-month old TgCRND8 and littermate control mice were examined with Fourier transform infrared microspectroscopy to explore the distribution of lipid, creatine, and dense core plaque deposits. Lipid distribution throughout the hippocampus was similar in transgenic (Tg) and non-Tg littermates at all ages. Dense core plaques were always found to lie within a thin (30–50 μm) lipid envelope, confirmed by imaging through serial sections. Creatine deposits were found in all TgCRND8 mice; the extent of deposition increased with age. Minor creatine deposits appeared in the oldest littermate controls. Distribution in the serial sections showed moderate correlation between layers, slightly disturbed by the freeze/thaw process. Creatine deposits in Tg mice were not specifically co-localized with plaques or lipid halos. The dimension of the lipid envelope is comparable with that of the diffuse halo of nonaggregated amyloid, implying a dynamic association in vivo, postulated to have a significant role in the evolving neurotoxicity.

As recently demonstrated by our group (da-Silva, W. S., Gomez-Puyou, A., Gomez-Puyou, M. T., Moreno-Sanchez, R., De Felice, F. G., de Meis, L., Oliveira, M. F., and Galina, A. (2004) J. Biol. Chem. 279, 39846–39855) mitochondrial hexokinase activity (mt-HK) plays a preventive antioxidant role because of steady-state ADP re-cycling through the inner mitochondrial membrane in rat brain. In the present work we show that ADP re-cycling accomplished by the mitochondrial creatine kinase (mt-CK) regulates reactive oxygen species (ROS) generation, particularly in high glucose concentrations. Activation of mt-CK by creatine (Cr) and ATP or ADP, induced a state 3-like respiration in isolated brain mitochondria and prevention of H2O2 production obeyed the steady-state kinetics of the enzyme to phosphorylate Cr. The extension of the preventive antioxidant role of mt-CK depended on the phosphocreatine (PCr)/Cr ratio. Rat liver mitochondria, which lack mt-CK activity, only reduced state 4-induced H2O2 generation when 1 order of magnitude more exogenous CK activity was added to the medium. Simulation of hyperglycemic conditions, by the inclusion of glucose 6-phosphate in mitochondria performing 2-deoxyglucose phosphorylation via mt-HK, induced H2O2 production in a Cr-sensitive manner. Simulation of hyperglycemia in embryonic rat brain cortical neurons increased both ΔΨm and ROS production and both parameters were decreased by the previous inclusion of Cr. Taken together, the results presented here indicate that mitochondrial kinase activity performed a key role as a preventive antioxidant against oxidative stress, reducing mitochondrial ROS generation through an ADP-recycling mechanism.

The creatine/phosphocreatine system, regulated by creatine kinase, plays an important role in maintaining energy balance in the brain. Energy metabolism and the function of creatine kinase are known to be affected in Alzheimer diseased brain and in cells exposed to the beta-amyloid peptide. We used infrared microspectroscopy to examine hippocampal, cortical, and caudal tissue from 21-89-week-old transgenic mice expressing doubly mutant (K670N/M671L and V717F) amyloid precursor protein and displaying robust pathology from an early age. Microcrystalline deposits of creatine, suggestive of perturbed energetic status, were detected by infrared microspectroscopy in all animals with advanced plaque pathology. Relatively large creatine deposits were also found in hippocampal sections from post-mortem Alzheimer diseased human brain, compared with hippocampus from non-demented brain. We therefore speculate that this molecule is a marker of the disease process.

The neuronal ceroid lipofuscinoses (NCLs) constitute a range of progressive neurological disorders primarily affecting children. Although six of the causative genes have been characterized, the underlying disease pathogenesis for this family of disorders is unknown. Using a metabolomics approach based on high resolution 1H NMR spectroscopy of the cortex, cerebellum, and remaining regions of the brain in conjunction with statistical pattern recognition, we report metabolic deficits associated with juvenile NCL in a Cln3 knock-out mouse model. Tissue from Cln3 null mutant mice aged 1–6 months was characterized by an increased glutamate concentration and a decrease in γ-amino butyric acid (GABA) concentration in aqueous extracts from the three regions of the brain. These changes are consistent with the reported altered expression of genes involved in glutamate metabolism in older mice and imply a change in neurotransmitter cycling between glutamate/glutamine and the production of GABA. Further variations in myo-inositol, creatine, and N-acetyl-aspartate were also identified. These metabolic changes were distinct from the normal aging/developmental process. Together, these changes represent the first documented pre-symptomatic symptoms of the Cln3 mouse at 1 month of age and demonstrate the versatility of 1H NMR spectroscopy as a tool for phenotyping mouse models of disease.

Twenty-two amino acid residues from transmembrane domain 3 of the creatine transporter were replaced, one at a time, with cysteine. The background for mutagenesis was a C144S mutant retaining ∼75% of wild-type transport activity but resistant to methanethiosulfonate (MTS) reagents. Each substitution mutant was tested for creatine transport activity and sensitivity to the following MTS reagents: 2-aminoethyl methanethiosulfonate (MTSEA), 2-(trimethylammonium) ethyl methanethiosulfonate (MTSET), and 2-sulfonatoethyl methanethiosulfonate (MTSES). Two mutants (G134C and Y148C) were inactive, but most mutants showed significant levels of creatine transport. Treatment with MTSEA inhibited the activity of the W154C, Y147C, and I140C mutants. Creatine partially protected I140C from inactivation, and this residue, like Cys-144 in the wild-type CreaT, is predicted to be close to a creatine binding site. MTSEA inactivation of Y147C was dependent on Na+ and Cl- suggesting that solvent accessibility was ion-dependent. Helical wheel and helical net projections indicate that the three MTSEA-sensitive mutants (W154C, Y147C, and I140C) and two inactive mutants (V151C and Y148C) are aligned on a face of an α-helix, suggesting that they form part of a substrate pathway. The W154C mutant, located near the external face of the membrane, was accessible to the larger MTS reagents, whereas those implicated in creatine binding were only accessible to the smaller MTSEA. Consideration of our data, together with a study on the serotonin transporter (Chen, J. G., Sachpatzidis, A., and Rudnick, G. (1997) J. Biol. Chem. 272, 28321–28327), suggests that involvement of residues from transmembrane domain 3 is a common feature of the substrate pathway of Na+- and Cl--dependent neurotransmitter transporters.

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.

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.

Parallel activation of heart mitochondria NADH and ATP production by Ca2+ has been shown to involve the Ca2+-sensitive dehydrogenases and the F0F1-ATPase. In the current study we hypothesize that the response time of Ca2+-activated ATP production is rapid enough to support step changes in myocardial workload (∼100 ms). To test this hypothesis, the rapid kinetics of Ca2+ activation ofmV˙ O2, [NADH], and light scattering were evaluated in isolated porcine heart mitochondria at 37 °C using a variety of optical techniques. The addition of Ca2+ was associated with an initial response time (IRT) ofmV˙ O2 that was dose-dependent with a minimum IRT of 0.27 ± 0.02 s (n = 41) at 535 nm Ca2+. The IRTs for NADH fluorescence and light scattering in response to Ca2+ additions were similar tomV˙ O2. The Ca2+ IRT formV˙ O2 was significantly shorter than 1.6 mm ADP (2.36 ± 0.47 s; p ≤ 0.001, n = 13), 2.2 mm Pi (2.32 ± 0.29, p ≤ 0.001, n = 13), or 10 mm creatine (15.6.±1.18 s, p ≤ 0.001, n = 18) under similar experimental conditions. Calcium effects were inhibited with 8 μm ruthenium red (2.4 ± 0.31 s;p ≤ 0.001, n = 16) and reversed with EGTA (1.6 ± 0.44; p ≤ 0.01, n= 6). Estimates of Ca2+ uptake into mitochondria using optical Ca2+ indicators trapped in the matrix revealed a sufficiently rapid uptake to cause the metabolic effects observed. These data are consistent with the notion that extramitochondrial Ca2+ can modify ATP production, via an increase in matrix Ca2+ content, rapidly enough to support cardiac work transitions in vivo.

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.

Functional properties of in situ mitochondria and of mitochondrial creatine kinase were studied in saponin-skinned fibers taken from normal and M-creatine kinase-deficient mice. In control animals, apparent Km values of mitochondrial respiration for ADP in cardiac (ventricular) and slow-twitch (soleus) muscles (137 +/- 16 microM and 209 +/- 10 microM, respectively) were manyfold higher than that in fast-twitch (gastrocnemius) muscle (7.5 +/- 0.5 microM). Creatine substantially decreased the Km values only in cardiac and slow-twitch muscles (73 +/- 11 microM and 131 +/- 21 microM, respectively). As compared to control, in situ mitochondria in transgenic ventricular and slow-twitch muscles showed two times lower Km values for ADP, and the presence of creatine only slightly decreased the Km values. In mutant fast-twitch muscle, a decrease rather than increase in mitochondrial sensitivity to ADP occurred, but creatine still had no effect. Furthermore, in these muscles, relatively low oxidative capacity was considerably elevated. It is suggested that in the mutant mice, impairment of energy transport function in ventricular and slow-twitch muscles is compensated by a facilitation of adenine nucleotide transportation between mitochondria and cellular ATPases; in fast-twitch muscle, mainly energy buffering function is depressed, and that is overcome by an increase in energy-producing potential.

The hypothesis tested was whether creatine kinase (CK) equilibrates with its substrates and products in the cytosol as if in solution. We used the creatine analogs cyclocreatine (cCr) or β-guanidopropionate (βGPA) to test if mass action ratios (Γ) for CK in muscle could be predicted from combined equilibrium constants (Kcomb) measured in solutions mimicking the intracellular environment. Mice were fed cCr or βGPA and their muscles assayed for substrates and products of the CK reaction by 31P NMR spectroscopy and high performance liquid chromatography. After three weeks of feeding, Γ was indistinguishable from Kcomb in cCr-treated muscles demonstrating both PCr/Cr and phospho-analog/analog must have equilibrated with a constant and uniform cellular ATP/ADP ratio. In βGPA-treated muscles, Γ was smaller than Kcomb due to a higher content of muscle βGPA. Feeding βGPA for 9-12 weeks resulted in a closer agreement between Kcomb and Γ, suggesting ATP/ADP ratios are not uniform within the muscle perhaps due to transient metabolic stress in some cells. From this analysis it follows that calculation of free ADP from the CK equilibrium for a heterogeneous population of cells with respect to total Cr and ATP content is correct only if chemical potentials of these cells are uniform.

An interaction of mitochondrial creatine kinase with purified outer mitochondrial porin (voltage-dependent anion channel) was shown by co-sedimentation assays as well as by gel permeation chromatography. Porin formed high M(r) complexes with wild-type mitochondrial creatine kinase as well as with an N-terminal deletion mutant, lacking the first five N-terminal amino acids. The complexes were identified by creatine kinase activity in parallel with immunoblotting using specific antibodies against the two proteins. In addition, porin induced octamerization of the N-terminal creatine kinase mutant, which under the same conditions without porin, did not polymerize but remained more than 90% dimeric. Furthermore, binding of mitochondrial creatine kinase to porin affected the conductance of porin when reconstituted in "black membranes." At 10 mV the pore in the complex adopted a low conductance (1.5-2 nanosiemens) state, compared to the high conductance state (3-4 nanosiemens) of the free incorporated pores. The former state of the pore is known to be cationically selective. Thus, besides a specific structural interaction, a defined physiological function is assumed of the mitochondrial creatine kinase-porin complexes that are discussed here.

L-Arginine-glycine amidinotransferase (transamidinase) is the first and rate-limiting step in creatine biosynthesis. Rats fed a creatine-supplemented diet or hypophysectomized rats have only 20% of the kidney transamidinase activity as intact rats fed a creatine-free diet. A cDNA clone corresponding to transamidinase was isolated by immunoscreening of a lambda gt11 expression library prepared from rat kidney mRNA. The transamidinase cDNA had an open reading frame containing the known sequence of the amino-terminal peptide of transamidinase. Based on the cDNA sequence, transamidinase is synthesized as a precursor with an amino-terminal extension of 50 amino acids, consistent with its mitochondrial localization. Comparison of the transamidinase sequence with the protein data base identified only a single, related protein. Remarkably, this protein, which has a 37% amino acid identity with transamidinase, is also an amidinotransferase, catalyzing streptomycin biosynthesis in Streptomyces griseus. Transamidinase cDNA was used to investigate the regulation of mRNA levels by creatine and growth hormone. Hypophysectomized rats were fed a creatine-free or a creatine-supplemented diet and maintained with and without injections of growth hormone. An excellent correlation was found between changes in transamidinase activity and mRNA levels in response to creatine and growth hormone. Thus, the regulation of transamidinase by creatine and growth hormone is at a pretranslational level. In addition, the two effectors do not act independently but interact at a pretranslational level to control transamidinase gene expression.

A cDNA has been cloned from rabbit brain that is a new member of the emerging family of Na(+)-dependent plasma membrane transporters for several neurotransmitters and structurally related compounds. Functional expression of this cDNA in COS-7 cells identifies its product as a Na(+)- and Cl(-)-dependent creatine transporter with a Km of approximately 35 microM. Its creatine transporter activity is efficiently antagonized by 3-guanidinopropionate, a well characterized alternative substrate of creatine transport in several tissues, and by 4-guanidinobutyrate. More distant structural analogues of creatine are much less efficient or inactive as antagonists, indicating a high substrate specificity of the transporter. Northern blot hybridization detects the expression of its mRNA in most tissues tested, most prominently in kidney, heart, and muscle, but not in liver and intestine. A full-length cDNA clone was also isolated from a muscle cDNA library and found to contain the same coding sequence. Substrate affinity and specificity of the cloned transporter are very similar to the endogenous creatine transporter of the COS-7 cells and to the creatine transport activities of several cell types described in the literature. Moreover, its mRNA is most abundant in the tissues known to possess high creatine uptake capacity.

The effect of temperature on the apparent equilibrium constant of creatine kinase (ATP:creatine N-phosphotransferase (EC 2.7.3.2)) was determined. At equilibrium the apparent K' for the biochemical reaction was defined as [formula: see text] The symbol sigma denotes the sum of all the ionic and metal complex species of the reactant components in M. The K' at pH 7.0, 1.0 mM free Mg2+, and ionic strength of 0.25 M at experimental conditions was 177 +/- 7.0, 217 +/- 11, 255 +/- 10, and 307 +/- 13 (n = 8) at 38, 25, 15, and 5 degrees C, respectively. The standard apparent enthalpy or heat of the reaction at the specified conditions (delta H' degree) was calculated from a van't Hoff plot of log10K' versus 1/T, and found to be -11.93 kJ mol-1 (-2852 cal mol-1) in the direction of ATP formation. The corresponding standard apparent entropy of the reaction (delta S' degree) was +4.70 J K-1 mol-1. The linear function (r2 = 0.99) between log10 K' and 1/K demonstrates that both delta H' degree and delta S' degree are independent of temperature for the creatine kinase reaction, and that delta Cp' degree, the standard apparent heat capacity of products minus reactants in their standard states, is negligible between 5 and 38 degrees C. We further show from our data that the sign and magnitude of the standard apparent Gibbs energy (delta G' degree) of the creatine kinase reaction was comprised mostly of the enthalpy of the reaction, with 11% coming from the entropy T delta S' degree term. The thermodynamic quantities for the following two reference reactions of creatine kinase were also determined. [formula: see text] The delta H degree for Reaction 2 was -16.73 kJ mol-1 (-3998 cal mol-1) and for Reaction 3 was -23.23 kJ mol-1 (-5552 cal mol-1) over the temperature range 5-38 degrees C. The corresponding delta S degree values for the reactions were +110.43 and +83.49 J K-1 mol-1, respectively. Using the delta H' degree of -11.93 kJ mol-1, and one K' value at one temperature, a second K' at a second temperature can be calculated, thus permitting bioenergetic investigations of organs and tissues using the creatine kinase equilibria over the entire physiological temperature range.

The targeting of creatine kinase isoenzymes to specific sites within muscle cells provides a system for the regeneration of ATP in situ from ADP and creatine phosphate. We have recently reported the colocalization of brain-type (B) creatine kinase and the nonsarcomeric mitochondrial creatine kinase isoenzymes in the thick ascending limb of the loop of Henle in the rat kidney, suggesting that creatine kinase may regenerate ATP for sodium transport (Friedman, D.L., and Perryman, M.B. (1991) J. Biol. Chem. 266, 22404-22410). In order to test the hypothesis regarding the association of B creatine kinase with sodium transport, we examined the creatine kinase enzymes in the rectal (salt-secreting) gland of the dogfish shark which contains high levels of the Na+/K(+)-ATPase. The creatine kinase isoform composition was determined by non-denaturing electrophoresis, immunoblotting, protein purification, and amino acid sequence analysis. The results demonstrate both B creatine kinase and mitochondrial creatine kinase proteins are present in the rectal gland, an isoform composition which is the same as in the mammalian kidney. By using a combination of chromatographic techniques, shark B creatine kinase was purified to homogeneity and partial sequence data was obtained from two cyanogen bromide peptide fragments. One of these fragments contains the active site and is identical at all sequenced residues with the corresponding region from the echinoderm sperm flagellar creatine kinase, and is 96% homologous with both chicken and rat B creatine kinase subunits. The other fragment corresponds to a region near the N-terminal of mammalian creatine kinases and is 89% homologous with B creatine kinase from chicken. The localization of these isoforms was examined by immunocytochemistry using subunit specific antisera. Mitochondrial creatine kinase and B creatine kinase immunoreactivity are detected in all tubules, and is restricted to the basal region of the cells, which is the site of the Na+/K(+)-ATPase. The conservation of creatine kinase isoform expression in excretory tissue, and the localization of creatine kinase immunoreactivity in the basal region of the tubule cells, demonstrate that subcellular compartmentation of B creatine kinase may underly the functional coupling of creatine kinase activity with sodium transport.

Epoxycreatine (N-(2,3-epoxypropyl)-N-amidinoglycine) is an affinity label of creatine kinase that irreversibly and completely inactivates the enzyme (Marletta, M. A., and Kenyon, G. L. (1979) J. Biol. Chem. 254, 1879-1886). To identify active site residues of rabbit muscle creatine kinase, the site of modification of it by epoxycreatine has been determined. Separation by high performance liquid chromatography of a tryptic digest of [14C]epoxycreatine-modified creatine kinase yielded two radiolabeled peptides. The larger of these consisted of amino acids Ala-266 through Arg-291 and was labeled with epoxycreatine at Cys-282. Attempts to purify completely the other labeled peptide were not successful; however, it was possible to obtain, by tandem mass spectrometry, a collision-induced dissociation spectrum of it from a mixture of several peptides. This peptide was a fragment (amino acids Val-279 through Arg-291) of the previously identified peptide and was also labeled at Cys-282. Model studies with cysteine and epoxycreatine have demonstrated that opening of the oxirane ring occurs by attack of the cysteine thiolate at the terminal carbon of the epoxide. These results are consistent with previous studies on the base lability of the label; however, a carboxyl group in the active site is not labeled, as had been previously suggested. These results provide evidence that Cys-282 is located in or near the creatine-binding site and will also be important in identifying and delineating the boundaries of the active site of creatine kinase.

The isolated perfused rat pancreas was used to test the hypothesis that total cellular ATP or the ratio of ATP/free ADP plays the primary role in coupling intermediary metabolism to the biophysical events that are the basis of glucose-stimulated insulin release. The pancreas was preperfused for 20 min with 4.0 mM of a physiological mixture of 20 amino acids plus 4.2 mM glucose, and insulin release was then stimulated for 150 s by suddenly increasing the glucose to 8.3 mM. The pancreas was sampled at 24, 48, 72, and 150 s after the switch. The content of total ATP, ADP, AMP, Pi, phosphocreatine, and creatine were measured in beta-cell enriched cores of pancreatic islets microdissected from freeze-dried pancreas cryostat sections. Metabolites were measured by quantitative histochemical enzymatic cycling techniques. Modeling studies were carried out to assess the impact of biochemical analytical results on the membrane potential of the beta-cells. The level of free ADP was calculated using the creatine kinase equilibrium reaction and an intracellular pH of 7.2. First phase insulin release was stimulated at least 10-fold with the maximum reached 45 s after adding high glucose. The biochemical analytical data demonstrate that the total cellular level of the putative coupling factor ATP and of the ratios ATP/free ADP and ATP/free ADP x Pi are not significantly influenced by a glucose level change that causes a more than 10-fold surge of insulin release. The strength and limitations of the present experimental strategy and the implications of the results for our understanding of metabolic coupling in glucose-stimulated insulin release are discussed.

Creatine kinase enzymes are present in tissues such as muscle and brain to interconvert creatine phosphate and ADP, thus providing a system to interconnect energy production and utilization (Bessman, S. P., and Carpenter, C. L. (1985) Annu. Rev. Biochem. 54, 831-862). Creatine kinase isoenzymes in kidney have received little attention since kidney contains relatively low creatine kinase activity compared with muscle and brain and because there is disagreement regarding the identity of the specific isoforms expressed in kidney. Using a combination of chromatographic and immunological techniques, we have identified two isoforms of creatine kinase in rat kidney supernatants, B creatine kinase, and the non-sarcomeric form of the mitochondrial creatine kinase, which represent 82 and 15%, respectively, of the total creatine kinase activity in this tissue. The identity of the non-muscle form of the mitochondrial creatine kinase was confirmed by N-terminal sequence analysis and compared with recently published cDNA sequences (Haas, R. C., and Strauss, A. W. (1990) J. Biol. Chem. 265, 6921-6927). We prepared multiple antisera specific for each isoform using synthetic peptide immunogens based upon nonhomologous regions from the primary sequence of each creatine kinase isoform. Immunocytochemical results demonstrate that both creatine kinase isoforms are colocalized in the inner stripe of the outer medulla in tubules of the distal nephron. A similar distribution of creatine kinase isoforms was obtained when different layers of the renal cortex and medulla were examined for creatine kinase activity and isozyme content using nondenaturing electrophoresis. In general, the distribution of creatine kinase enzymes in kidney corresponds to the regions of greatest ATP utilization, oxygen consumption, and sodium transport. These results suggest a role for creatine kinase enzymes in the coupling of ion transport and oxidative phosphorylation in the distal nephron of the mammalian kidney.

To further evaluate the bioenergetic role of phosphocreatine, we assessed several parameters in normal and depleted rat hearts. Rats were fed (8 weeks) a diet containing either 1% beta-guanidinoproprionic acid or 2% beta-guanidinobutyric acid (beta-GBA), resulting in an 80% phosphocreatine depletion compared to controls. Left ventricular pressure-volume curves were obtained to determine contractile function. At any volume, the developed pressure in depleted hearts was lower than in controls. At the plateau, the rate-pressure product was between 37-45% lower: 34,000 (beta-GBA), 30,174 (beta-guanidinoproprionic acid) versus 54,400 (control). 31P NMR spectroscopy on beta-GBA-treated hearts obtained the [ATP] and [phosphocreatine], which with saturation transfer estimated the rates of creatine kinase and ATP production. In depleted hearts, the rate constant for ATP synthesis from phosphocreatine was increased 33%. However, the flux was 72% lower. ATP production from ADP and Pi were similar under normal conditions, in spite of higher rates of oxygen consumption in the depleted hearts. The addition of 50 mM creatine to control perfusate had no effect on function or high energy phosphates. In contrast, a 28% increase in function and a 52% increase in [phosphocreatine] was seen in beta-GBA hearts. There was a marked increase in free [ADP] in beta-GBA hearts, resulting in a lower estimated ATP phosphorylation potential. Overall, the results suggest that phosphocreatine may play an important function by optimizing the thermodynamics of cardiac high energy phosphate utilization.

Analyses of isolated intact diaphragm muscle show that at rest only about 30% of the total cellular Pi is metabolically reactive as indicated by 18O incorporation from [18O]water, whereas up to 90% becomes metabolically active incrementally with contractile frequency. Kinetics of [gamma-18O]ATP appearance show that about 90% of the cellular ATP is metabolically active and suggest slowly and rapidly metabolizing compartments of ATP in resting muscle and only rapidly metabolizing compartments in contracting muscle. Rates of [18O]creatine phosphate [( 18O]CrP) appearance are consistent with creatine kinase-catalyzed phosphoryl exchange functioning in an obligatory phosphoryl shuttle system. In noncontracting muscle, ATP turnover rate was 83 nmol.mg protein-1.min-1, and the P/O ratio was determined to be 3.2. ATP utilization increases in direct proportion to contractile frequency with each contracture consuming the equivalent of 0.96 nmol of ATP.mg protein-1 or 2.5-3.5 molecules of ATP/myosin active site. Basal concentrations of nucleotide polyphosphates are not altered when ATP utilization rates increase during contraction. At high contractile frequencies, decreases in CrP concentration occur, but this accounts for less than 4% of total high energy phosphoryls consumed. If metabolic intermediates are free in the aqueous cellular cytosol, each twitch contracture would result in a decrease in ATP concentration of no more than 2% and increases in ADP and AMP concentrations of less than 20 and 7%, respectively. Thus, changes in metabolite concentration must be highly localized or metabolic regulation can be accomplished by a nonallosteric mechanism.

ADP is an important regulator of hepatic metabolism. Despite its importance the level of free ADP in the liver remains controversial. Recently, we engineered transgenic mice which express high levels of creatine kinase in liver. The reaction catalyzed by creatine kinase was assumed to be at equilibrium and used to calculate a free ADP level of 0.059 mumol/g wet weight. In this report we test the equilibrium assumption by studying the free ADP level as a function of enzyme activity or substrate content. Over a 5-fold range of creatine kinase activity, from 150-800 mumol/min/g wet weight, there was no change in the free ADP level. The average value of ADP for these mice was 0.061 +/- 0.016 mumol/g wet weight. Similarly, altering hepatic creatine content from 1.6 to 30 mumol/g wet weight had no effect on the calculated total free ADP level. The average value of ADP for the creatine levels was 0.048 +/- 0.015 mumol/g wet weight. Finally, the free ADP level was calculated using the equilibrium with cyclocreatine rather than creatine as substrate. The equilibrium of the reaction with cyclocreatine lies 30 times more toward phosphorylation than does the equilibrium with creatine. A free ADP level of 0.063 +/- 0.031 mumol/g wet weight was calculated using cyclocreatine. This value is not different from that found with creatine. These results show that the equilibrium assumption used to calculate free ADP levels in transgenic mouse liver is valid, and the presence of creatine kinase does not affect ADP levels.

The oxygen dependence of mitochondrial respiration was investigated using suspensions of mitochondria and quiescent ventricular myocytes isolated from adult rat hearts. A new optical method was used to determine oxygen concentration in the suspending media. The P50 for respiration for coupled mitochondria at a high [ATP]/[ADP].[Pi] ratio and oxidizing glutamate/malate was 0.45 +/- 0.03 microM but was increased to 0.57 +/- 0.02 microM by the addition of succinate to the substrate mixture. This value was decreased to less than 0.06 +/- 0.01 microM when the ATP/ADP.Pi ratio was decreased with the uncoupler, carbonyl cyanide p-trifluoromethoxyphenylhydrazone. The P50 value in resting myocytes was 2.23 +/- 0.13 microM at a Vmax of 13.22 +/- 1.38 nmol of O2/g, dry weight/min. During resting conditions, the creatine phosphate/creatine and ATPfree/ADPfree ratios were high in these cells, 6.81 +/- 1.11 and 1131 +/- 185, respectively. Addition of 1 mM Ca2+ to the suspending media increased the P50 by 50% whereas respiration rose by only 10%. Respiratory rate was increased up to about 10-fold by uncoupling the cells, but the P50 increased by less than 3-fold. When these uncoupled cells were inhibited with Amytal to lower the rate of oxygen consumption to that of resting cells, the P50 fell to 1.25 +/- 0.14 microM. Diffusion models indicate that in resting myocytes, the oxygen concentration difference from sarcolemma to cell core was approximately 1.84 microM with an additional difference of about 0.27 microM attributed to the unstirred layer of media surrounding each cell. The intracellular oxygen diffusivity coefficient in myocytes was calculated to be 0.30 x 10(-5) cm2/s. The results show that the oxygen dependence of respiration is modulated by the cellular metabolic state. At near maximal levels of respiration or on recovery from hypoxic episodes, oxygen diffusion may become an important determinant of the oxygen dependence of myocardial respiration.

Breast muscle of young chicks fed chow diets containing the creatine analog 1-carboxymethyl-2-iminoimidazolidine (cyclocreatine) accumulated up to 40 mumol/g wet weight of the synthetic phosphagen 1-carboxymethyl-2-imino-3-phosphonoimidazolidine (cyclocreatine-P2-). ATP levels were sustained at high values substantially longer in breast muscle of cyclocreatine-fed chicks, compared to control-fed chicks, during total ischemia initiated 2 h after injection of both groups with the beta-adrenergic agonist isoproterenol (5 mg/kg subcutaneous). For example, in chicks fed 0.5% cyclocreatine for 10-19 days ATP levels in isoproterenol-stimulated breast muscles after 1 h of ischemia at 37 degrees C were 6.1 mumol/g, compared to 1.9 mumol/g for the control-fed group, and after 2 h of ischemia were 3.5 mumol/g compared to 0.6 mumol/g for controls. Creatine-P reserves in isoproterenol-stimulated breast muscles of all dietary groups were essentially exhausted within the first hour of ischemia. In contrast, breast muscle of chicks fed either 1 or 0.5% cyclocreatine still contained 28 and 19 mumol/g of cyclocreatine-P, respectively, after 1 h of ischemia; after 2 h of ischemia, the respective cyclocreatine-P values were 20 and 13 mumol/g. Isoproterenol-stimulated chick breast muscle provides the first skeletal muscle model system for studying the molecular mechanisms by which dietary cyclocreatine helps sustain ATP levels during ischemia. Although adaptive factors are also involved, it is suggested that a significant portion of the ATP-sustaining activity of dietary cyclocreatine in ischemic breast muscle can be attributed to the unique thermodynamic properties of the accumulated cyclocreatine-P. These properties enable cyclocreatine-P to continue to thermodynamically buffer the adenylate system and transport high energy phosphate throughout the long muscle fibers at cytosolic pH values and phosphorylation potentials well below the range where the creatine-P system can function effectively. Synergism between glycolysis and this long-acting synthetic phosphagen might well help delay depletion of ATP levels in skeletal muscles during ischemia. Cyclocreatine feeding provides a unique experimental tool for quantitative evaluation of the proposed protective role of ATP against irreversible cellular damage in skeletal and cardiac muscles during ischemic episodes.

Hypoxia and anoxia were induced in isolated perfused rat hearts by reduction of perfusate oxygen tension. Ischemia was produced by restricting coronary flow in hearts working against high resistance. Protein degradation was estimated from release of free phenylalanine in the presence of cycloheximide, and from dilution of the specific radioactivity of perfusate phenylalanine. Both anoxia and ischemia inhibited protein degradation as compared to unpaced or paced aerobic control hearts, respectively. The effect of ischemia was greater than that of anoxia. Estimates of net phenylalanine release, in the absence of cycloheximide, suggested that rates of protein synthesis and degradation were more nearly in balance in ischemic, than in anoxic hearts. Although insulin inhibited proteolysis in aerobic tissues, no effect of the hormone was evident in energy-poor hearts. Anoxia and ischemia appeared to affect initial steps in the degradative pathway as evidenced by inhibition of the decrease in activity of Sadenosylmethionine decarboxylase. Inhibition of proteolysis in energy-poor hearts was accompanied by decreased latency of the lysosomal enzymes, P-acetylglucosaminidase and cathepsin D. A progressive reduction in perfusate oxygen tension (hypoxia) indicated that protein degradation was reduced significantly when perfusates were gassed with 30% 02, although levels of nucleotides and creatine phosphate were normal. Anoxia led to depletion of high energy phosphates and more marked inhibition of proteolysis. Lactate and hydrogen ions, metabolites that accumulate in ischemic hearts, inhibited proteolysis in aerobic and anoxic tissues. These results indicate that inhibition of protein degradation may involve both energy-depletion and metabolite accumulation.

Tissues of chicks fed 5% N-methyl-3-guanidinopropionate (N-amidino-N-methyl-beta-alanine) for 12 days accumulated the following amounts of free plus phosphorylated derivatives as mumol/g, wet weight: brain, 5.5; heart, 7.3; leg muscle, 21.0; and breast muscle, 24.4. Since total creatine levels remained nearly the same in brain, N-methyl-3-guanidinopropionate-P provided brain with a supplemental reservoir of high energy phosphate. Tissues of rats fed 2% N-ethylguanidinoacetate (N-amidino-N-ethylglycine) accumulated large amounts of N-ethylguanidinoacetate-P, which has thermodynamic properties similar to creatine-P and is the kinetically most reactive synthetic phosphagen yet described. N-Ethylguanidinoacetate derivatives replaced creatine derivatives mole-for-mole, and the fraction of synthetic to total phosphagen after 19 days was 60% in heart, 54% in slow oxidative muscle, 42% in fast glycolytic muscles, and 22% in brain. N-Ethylguanidinoacetate served as a false end product co-repressor of liver arginine:glycine amidinotransferase levels in both chicks and chick embryos; N-methyl-3-guanidinopropionate and N-propylguanidinoacetate were relatively inactive. Creatinine amidohydrolase reversibly cyclized both N-ethylguanidinoacetate and N-propylguanidinoacetate with even lower Km values than for creatine derivatives, but it did not react significantly with N-methyl-3-guanidinopropionate, 3-guanidinopropionate, or 1-carboxy-methyl-2-imino-imidazolidine (cyclocreatine). Creatine amidinohydrolase also hydrolyzed N-acetimidoylsarcosine, but was relatively unreactive toward N-ethylguanidinoacetate, N-methyl-3-guanidinopropionate, 3-guanidinopropionate, and cyclocreatine. Amidinohydrolase can therefore be used to remove interfering creatine in assays of tissues for coexisting N-ethylguanidinoacetate or N-methyl-3-guanidinopropionate. Assays are now available to follow changes during metabolic stresses of any combination or all of the following phosphagens accumulated by the same tissue: creatine-P, N-ethylguanidinoacetate-P, cyclocreatine-P, N-methyl-3-guanidinopropionate-P, and homocyclocreatine-P.

Creatine kinase from chicken breast muscle can be titrated stoichiometrically with 2 equivalents of 2-chloromercuri-4-nitrophenol, a chromophoric probe specific for thiols, without loss of enzymatic activity (Quiocho, F. A., and Thomson, J. W. (1973) Proc. Nat. Acad. Sci. U. S. A. 70, 2858–2862). The rate of reaction of mercurinitrophenol with creatine kinase was measured using the stopped flow rapid mixing technique and was found to depend on the liganded state of the enzyme. The observed second order rate constant for the unliganded enzyme in N,N-bis(2-hydroxyethyl)glycine (Bicine)-NaOH buffer, at pH 8.0 and 20°, was 4.5 x 106 m-1 s-1. Of the various enzyme-substrate complexes examined, the creatine-enzyme-MgADP-nitrate complex, which is an analog of the transition state complex, showed the most pronounced decrease in reactivity, as much as 200-fold. A 13-fold decrease in the rate was observed for the creatine-enzyme-MgADP complex. Creatine, MgADP, or nitrate added separately decreased the rate by only about 2- to 3-fold. ADP or ATP decreased the rate by 7-fold. These rate differences and those observed for other complexes indicate that creatine kinase undergoes progressively different conformations depending on the complexed state of the enzyme. The rate of reaction of the enzyme isolated from dystrophic muscle was identical with that of the enzyme isolated from normal muscle.

Ternary complex formation between [3H]Met-tRNAf, [14C]H3-eIF-2, and GTP was measured on nitrocellulose filters. It is shown that [3H]Met-tRNAf and [14C]H3-eIF-2 are present on the filter in equimolar amounts when ATP, creatine phosphate, and creatine kinase are included in the reaction mixture. Under these conditions the factor is 100% active. With small amounts of factor significant losses occur due to adsorption to the wall of the reaction vessels, resulting in seemingly low activities of eIF-2. These losses can be prevented by the presence of "stimulatory" proteins, which enhance the recovery of both [3H]Met-tRNAf and [14C]H3-eIF-2 on the filter but do not alter their ratio.

Creatine kinase (EC 2.7.3.2) of rabbit brain (BB) was purified to hemogeneity as judged by sedimentation velocity analysis in the ultracentrifuge. Nevertheless, the purified enzyme contained as many as seven extraneous protein species when examined by disc electrophoresis in polyacrylamide gel. The purified preparation also showed antigenic heterogeneity when it reacted in agar gel double immunodiffusion analysis with antibodies produced against it in the rooster. At least some of the extraneous proteins appeared to arise from BB during storage. Subsequently BB was purified to hemogeneity by disc electrophoresis in polyacrylamide gel. It was excised from the gel and injected repeatedly with Freund's complete adjuvant into roosters. A single immunoprecipitin line was noted in agar gel double immunodiffusion analysis when partially pure enzyme reacted with the antibodies raised to the electrophoretically pure protein. These antibodies inhibited the enzymatic activity of BB in both soluble and insoluble immune complexes. They did not, however, form immunoprecipitates with or inhibit rabbit muscle (MM) or rabbit hybrid (MB) creatine kinases. The antibodies were therefore specific for BB and did not react with MM or MB.

Two isozymes of creatine kinase have been purified from sperm of the sea urchin, Strongylocentrotus purpuratus. One isozyme was purified from the sperm flagellum, and the other from the head. Both require nonionic detergent for extraction from sperm. The flagellar isozyme is a monomeric species with an Mr of 145,000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and 126,000 from sucrose density gradient and gel filtration analyses. Creatine kinase from sperm heads was localized to the mitochondrion by an antibody raised against mouse muscle creatine kinase. This purified mitochondrial isozyme is multimeric, with an Mr of 47,000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, but 240,000 for the native enzyme. Peptide mapping indicates that the two isozymes are not related. The following kinetic characteristics were observed for the purified flagellar and mitochondrial isozymes, respectively. In the direction of ATP formation, at pH 6.6 and 25 degrees C, specific activities were 235 and 180 units/mg; pH optima were 6.7 and 6.9 and Michaelis constants were 0.13 and 0.055 mM for ADP and 5.8 and 2.7 mM for phosphocreatine. In the direction of phosphocreatine formation, at pH 7.5 and 25 degrees C, specific activities were 29 and 47 units/mg; pH optima were 7.5 and 7.7 and Michaelis constants were 0.89 and 0.31 mM for ATP and 39 and 62 mM for creatine. These unique isozymes constitute the termini of the phosphocreatine shuttle of sea urchin sperm that is responsible for energy transport from the mitochondrion to the distal flagellum (Tombes, R. M., and Shapiro, B. M. (1985) Cell 41, 325-334; Tombes, R. M., Brokaw, C. J., and Shapiro, B. M. (1987) Biophys. J., 52, 75-86).

Mitochondrial creatine kinase was purified from canine myocardium. The preparation exhibited a positively charged isoenzyme free of other creatine kinase isoenzymes and on sodium dodecyl sulfate gel exhibited a single protein band. Amino acid composition showed mitochondrial creatine kinase to be different from that of MM or BB creatine kinase and did not hybridize with the M or B subunits of the cytosolic forms. Antiserum was developed to mitochondrial creatine kinase which did not cross-react with cytosolic creatine kinases. Antiserum to cytosolic creatine kinase exhibited no reaction to mitochondrial creatine kinase. Utilizing the specific antiserum, a radioimmunoassay was developed for the specific detection of mitochondrial creatine kinase. Thus, mitochondrial creatine kinase was purified and shown to be comprised of a unique subunit which is biochemically and immunologically distinct from the cytosolic creatine kinases.

While the equilibrium assumption and the validity of using total measured concentrations for near equilibrium indicator reactions have been widely tested in liver, these have not been systematically evaluated in skeletal muscle. Vascularly isolated dog gracilis muscles were stimulated via the nerve at 4 Hz, and tissue was sampled by quick freezing at rest and after 10, 15, 30, 60, and 180 s of stimulation or after stimulation in the presence of glycolytic blockade by iodoacetate. Phosphocreatine, creatine, and several glycolytic intermediates were measured in tissue extracts. The in vivo mass action ratios for triosephosphate isomerase and aldolase were evaluated relative to substrate concentrations and compared with equilibrium constants determined in vitro. Although there was evidence of substrate binding at low substrate levels for the triosephosphate isomerase reaction, the in vivo mass action ratios for both reactions stabilized at a constant value at moderate substrate levels and in glycolytically blocked muscles. It was concluded that both enzymes are in apparent equilibrium in vivo, but the equilibrium constants are lower than those determined in vitro. The mass action ratios of the combined creatine kinase, lactate dehydrogenase, glyceraldehyde-phosphate dehydrogenase and phosphoglycerate kinase reactions were determined for resting muscles. These reactions are also at equilibrium and the equilibrium constants are consistent with in vitro values.

The presence of mRNA coding for creatine kinase M (Mck) and creatine kinase B (B-CK) in RNA from myogenic and fibrogenic cell cultures, embryonic muscle, and embryonic brain tissue was demonstrated by "in vitro" translation in a heterologous cell-free protein-synthesizing system from rabbit reticulocytes. The products were isolated by sensitive immunochemical methods and their identity with isolated M-CK and B-CK was shown by the following criteria: (a) the in vitro synthesized creatine kinases react with the specific antibody against these antigens; (b) the labeled peptides co-migrate with purified creatine kinase on sodium dodecyl sulfate gels in single bands; (c) the labeled peptides form homo- and heterodimers with isolated enzymatically active creatine kinase, thus behaving like authentic creatine kinases. The assay was shown to be reproducible and gave a linear response with increasing amounts of RNA, allowing relative quantitation of mRNA in polysomal RNA for the creatine kinases M and B. MRNA for M-CK was detected in polusomal RAN and total cellular RNA from myogenic cells. It is also present in polysomal RNA from enbryonic muscle and the fraction binding to oligo(dT)-cellulose. mRNA for B-CK could be found in RNA extracted from young myogenic cultures and the fraction of polysomal embryonic brain RNA binding to oligo(dT)-cellulose.

Creatine kinase does not catalyze the scrambling of 18O in adenosine 5'-[alpha beta-18O, beta-18O2]triphosphate in the absence of creatine, in the presence of L-arginine or taurocyamine (competitive inhibitors of creatine), or in the presence of poor substrates where single turnover experiments were performed. In order to support this prima facie evidence for an associative mechanism of phosphoryl transfer, an investigation was undertaken of 1-carboxymethyl-2-aminoimidazole, a new substrate analogue of creatine. This analogue has a binding constant for rabbit muscle creatine kinase similar to creatine and 1-carboxymethyl-2-iminoimidazolidine, but the initial rate of phoshorylation by MgATP in the presence of creatine kinase is almost 5 orders of magnitude slower. The phosphorylation product, assigned the structure 1-carboxymethyl-2-imino-3-phospho-4-imidazoline is also a poor substrate for the phosphorylation of MgADP by creatine kinase. These observations can be accounted for by an associative SN2(P) mechanism of phosphoryl transfer and by a microenvironment of the enzyme-bound creatine (or creatine analogue) which lowers the pKa of the guanidino group by several pH units compared with that in aqueous solution.

The tissue contents of the reactants of the myokinase (EC 2.7.4.3) and the combined glyceraldehyde-3-phophate dehydrogenase (EC 1.1.1.29)-3-phosphoglycerate kinase (EC 2.7.2.3) reactions were measured in rapidly inactivated samples of human blood and rat brain, muscle, and liver. The tissue contents of the reactants of the creatine kinase (EC 2.7.3.2) reaction were measured in rat brain and muscle. In vitro the value of the expression: KG+G = [sigma3PG] . [sigmaATP] . [sigmalactate] KLDH = [sigmaHAP]/22] . [sigmaADP][sigmaPi] . [sigmaRUVATE] (1) was found to be 0.725 x 10(7) M-1 at I = 0.25, T = 38 degrees C, and free [Mg2+] = 0.15 mM and the value measured in vivo in red cell was 0.699 x 10(7) M-1. The value of the expression KMYK = ([sigma ATP] [sigma AMP]/[ADP2]) measured under the above conditions and at pH 7.2 was found to be 0.744 while the value found in red cell was 0.784 +/- 0.037. These reactions, therefore, appear to be in a state of near-equilibrium in the red cell and the measured tissue contents of ATP and ADP, which are common reactants in both reactions, approximate closely the activity of these reactants in vivo. In brain and muscle, the value of KG + G/KLDH calculated from the measured tissue contents of the reactants was a factor of 20 or more lower than that expected at equilibrium as was the measured value of the expression: KCK = [sigma ATP] [sigma creatine] divided by [sigma ADP] [sigma creatine-P] [H+] (2) Substitution of calculated free [sigma ADP] values in the expression of KG + G/KLDH gave values of 0.83 +/- 0.19 x 10(7) M-1 for brain and muscle, respectively, which agreed well with the value of 1.65 x 10(7) M-1 measured in vitro at I = 0.25, free [Mg2+] = 1 mM, T = 38 degrees C. This agreement between two highly active enzyme systems in the same compartment is taken as evidence of the existence of near-equilibrium in both these systems and suggests that free cytosolic [sigma ADP] is probably 20-fold lower than measured cell ADP content in mitochondrial-containing tissues.

In order to define further the nature of the apparent close interaction between mitochondrial creatine phosphokinase and oxidative phosphorylation (Erickson-Viitanen, S., Viitanen, P., Geiger, P. J., Yang, W. C. T., and Bessman, S. P. (1982) J. Biol. Chem. 257, 14395-14404), rabbit heart and rat skeletal muscle mitochondria prepared by gentle mechanical homogenization were compared with preparations isolated after tryptic digestion of tissues, a method which has been reported to yield superior mitochondria (Reichert, M., Schaller, H., Kung, W., and Gerber, G. (1978) Acta Biol. Med. Germ. 37, 1167-1176). The ability of de novo synthesized and exported mitochondrial ATP to interact with creatine phosphokinase prior to total mixing of the ATP pool, which we consider to be evidence of compartmentation, could not be demonstrated with mitochondria prepared via the trypsin procedure. Mitochondria from rabbit cardiac muscle treated with digitonin synthesized ATP and creatine phosphate, but failed to show apparent compartmentation of creatine phosphokinase. Km values for ATP were compared for four conditions: 1) respiring, digitonin-treated rabbit heart mitochondria, 2) atractyloside-inhibited, digitonin-treated rabbit heart mitochondria supplied with a pyruvate kinase-phosphoenolpyruvate regenerating system, 3) respiring rabbit heart mitochondria, 4) atractyloside-inhibited rabbit heart mitochondria supplied with an ATP regenerating system. The observed Km values for ATP for conditions 1, 2, and 3 were similar but lower than that for condition 4. These findings suggest that an outer membrane diffusion barrier influences or controls mitochondrial creatine phosphokinase compartmentation.

The first committed reaction in the biosynthesis of creatine is catalyzed by the enzyme L-arginine:glycine amidinotransferase, commonly called transamidinase. Creatine, the end product of the biosynthetic pathway, is known to alter the levels of kidney transamidinase activity. Rats fed a diet containing 0.3% creatine had 26% of the kidney transamidinase activity of the rats fed a creatine-free diet. This reduction in transamidinase activity was correlated with a decrease in transamidinase protein in the creatine-fed rats. The relative synthetic rates and mRNA functional activities of transmidinase were measured in control and creatine-fed rats. The relative synthetic rate of transamidinase in creatine-fed rats was 21% of that found in the control animals. The functional transamidinase mRNA in creatine-fed rats was correspondingly reduced to 37% of the amount in the control animals. Thus, creatine affects transamidinase activity by altering its rate of synthesis at a pretranslational step and represents an example of end-product repression in a higher eukaryote.

Equations have been derived for calculating the concentrations of ATP, ADP, and AMP in equilibrium with phosphocreatine and creatine by use of association constants for the protonated species and Mg2+ complexes of the reactants and the equilibrium constants for the AMP kinase and creatine kinase reactions. The equations have been used to calculate the expected concentrations of free ATP, ADP, and AMP as high energy phosphate is discharged by a representative muscle. Arguments are advanced for the validity of the results as an estimate of in vivo conditions when based only on measured concentrations of phosphocreatine, creatine, and total adenosine phosphates. Changes in phosphocreatine concentration cause rapid changes in the calculated concentrations of ADP and AMP that are consistent with postulated regulatory effects of these compounds on the Embden-Meyerhof pathway. The amount of ADP and AMP formed by a given discharge of high energy phosphate depends upon the H+ concentration, and physiological changes in pH are sufficient to cause significant changes in the rate of glycolysis in this way. The physiological significance of phosphocreatine as an effector is less clear.

A procedure was developed for the purification of phosphofructokinase from rabbit brain extracts. The enzyme was purified more than 100-fold with a yield of greater than 50 %. It was found to have the same distribution of multiple molecular forms as found in the total brain extract, including one molecular species that is different from the more extensively studied muscle (heart) and liver (erythrocyte) isozymes. The regulatory properties of brain phosphofructokinase were compared with those of muscle and liver phosphofructokinases at pH 7.1 and 7.4. All three enzymes were inhibited less at pH 7.4 than at 7.1. At pH 7.1 the sensitivity to ATP as an inhibitor decreased in the order liver g muscle g brain. At pH 7.4, the relative sensitivities of brain and muscle phosphofructokinases were reversed with the brain enzyme being inhibited at slightly lower concentrations of ATP. Other inhibitors acted synergistically with ATP. Sensitivity to inhibition by 3-phosphoglycerate and phosphoenolpyruvate decreased in the order muscle g brain g liver. With 2,3-diphosphoglycerate as an inhibitor, the sensitivity decreased in the order liver g muscle g brain. Muscle phosphofructokinase was the most sensitive to citrate inhibition. Creatine phosphate, a potent inhibitor of muscle phosphofructokinase, was completely ineffective as an inhibitor of liver and brain phosphofructokinase. The actions of activators were evaluated at pH 7.4 in the presence of inhibitory concentrations of ATP. The three enzymes were almost equally responsive to the deinhibiting action of AMP and cyclic adenosine 3':5'-monophosphate, although the enzyme from liver requires slightly higher concentrations. Muscle phosphofructokinase was the least sensitive to deinhibition by inorganic phosphate. The results are discussed with respect to the varying modes of carbohydrate metabolism in the different tissues taking into consideration the tissue concentrations of the effectors and the variations of concentration in different physiological states. The enzymes from brain and liver are apparently chiefly controlled by the relative amounts of the adenine nucleotides and inorganic phosphate. An exception may be the erythrocyte, which has the same isozyme that is present in liver. Here, 2,3-diphosphoglycerate may play an important role in regulation. Muscle phosphofructokinase also responds to varying levels of adenine nucleotides but in vivo it is most likely that the principal regulatory effectors are creatine phosphate, citrate, and possibly, 3-phosphoglycerate.

Electrical shock treatment produces a rapid loss of high energy phosphates in rat brain. The [ATP]/[ADP] ratio decreases to one-third of its control value within 10 s. The ammonia content increases 3-fold during the first minute after starting the stimulus. The total adenine nucleotide plus adenosine content of brain decreases an equivalent amount of hypoxanthine-containing compounds appears. Adenosine, inosine, and hypoxanthine accumulate, and there is a transitory accumulation of adenylosuccinate. The contents of ATP and creatine phosphate, and the [ATP]/[ADP] ratio, are rapidly restored to control values, but other metabolite contents are restored more slowly. The transient rise in adenylosuccinate and IMP provides evidence that the ammonia production is due in part, and possibly in whole, to the operation of the purine nucleotide cycle.

The steady state kinetics of creatine kinase have been studied at pH 7 by initial velocity, product inhibition, and dead end inhibition techniques using chromium ATP and chromium ADP. The mechanism appears to be rapid equilibrium random in the MgADP plus phosphocreatine direction and equilibrium ordered with MgATP adding before creatine in the other direction. This degenerate form of the rapid equilibrium random mechanism found in both directions at pH 8 may be caused by protonation of a group on the enzyme at pH 7 that will not permit binding of the positively charged portion of creatine until the MgATP is already bound. The chromium nucleotides were competitive inhibitors versus the magnesium nucleotides and noncompetitive versus the guanido substrates. Except for MgATP, chromium and magnesium nucleotides have about the same affinity for the enzyme at pH 7. The 10-fold greater affinity of CrATP over MgATP for the enzyme may reflect an equilibrium between doubly (β,γ) and triply (α,β,γ) coordinated MgATP in solution with the triply coordinated nucleotide being the actual substrate. Nitrate synergizes creatine inhibition of reaction in the MgADP plus phosphocreatine direction, but was ineffective in synergizing CrADP inhibition in the MgATP plus creatine direction. This may be because the planar PO3 group which nitrate mimics in the E-MgADP-creatine-NO3 complex is coordinated to the Mg2+, but while nitrate can displace the water which is replacing PO3 from Mg2+, it cannot displace the water molecule covalently coordinated to chromium in the corresponding E-CrADP-creatine complex.

Treatment of rat heart mitochondria with phosphate or mersalyl releases a number of proteins, including the mitochondrial creatine kinase (mt-CK). Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the released proteins showed that phosphate is more selective than mersalyl in releasing mt-CK. The rebinding of mt-CK to mitochondria was selectively inhibited by adriamycin, which complexes membrane-bound cardiolipin. mt-CK activity and binding experiments have shown that intact mitochondria are able to bind approximately twice the amount of mt-CK they originally contain. Liver mitochondria bound heart mitochondria mt-CK to the same extent as creatine kinase-depleted heart mitochondria. mt-CK was bound by liposomes but only if they contained cardiolipin. The binding of mt-CK to cardiolipin-containing liposomes was inhibited by adriamycin. Phosphatidylcholine liposomes reconstituted with the purified ADP/ATP translocator failed to bind mt-CK.