Fluxes through cytosolic and mitochondrial creatine kinase, measured by P-31 NMR

The kinetic properties of the cytoplasmic and the mitochondrial iso-enzymes of creatine kinase from striated muscle were studied in vitro and in vivo. The creatine kinase (CK) iso-enzyme family has a multi-faceted role in cellular energy metabolism and is characterized by a complex pattern of tissue-specific expression and subcellular distribution. In mammalian tissues, there is always co-expression of at least two different CK isoforms. As a result, previous studies into the role of CK in energy metabolism have not been able to directly differentiate between the individual CK species. Here, we describe experiments which were directed at achieving this goal. First, we studied the kinetic properties of the muscle-specific cytoplasmic and mitochondrial CK isoforms in purified form under in vitro conditions, using a combination of P-31 NMR and spectrophotometry. Secondly, P-31 NMR measurements of the flux through the CK reaction were carried out on intact skeletal and heart muscle from wild-type mice and from transgenic mice, homozygous for a complete deficiency of the muscle-type cytoplasmic CK isoform. Skeletal muscle and heart were compared because they differ strongly in the relative abundance of the CK isoforms. The present data indicate that the kinetic properties of cytoplasmic and mitochondrial CK are substantially different, both in vitro and in vivo. This finding particularly has implications for the interpretation of in vivo studies with P-31 NMR. (Mol Cell Biochem 174: 33–42, 1997)     

The creatine kinase system in smooth muscle

Despite the energetic flux being much lower in smooth muscle compared to striated muscles (such as the heart and skeletal muscle) creatine kinase (CK) has been found present and active in all smooth muscles studied to date. A complete CK circuit has been identified, with CK found in the mitochondria, contractile elements, membrane pumps and the cytoplasm. CK isoenzymes are coupled to many cellular energetic processes and appears to be involved in energy production and consumption by acting as an energy transducer. The CK system responds to pathological insults and development (e.g. hypertrophy and gestation respectively) by changes in sub-cellular distribution localization, isoenzymes, and specific activity. The conclusion from these observations is that creatine kinase is intimately involved in the energetic system of smooth muscle.Abbreviations CK creatine kinase - Mi-CK mitochondrial creatine kinase - Cr creatine - PCr phosphocreatiner - NMR nuclear magnetic resonance - SHR spontaneously hypertensive rat - -GPA -guanidinopropionic acid     

Compartmentation of ATP synthesis and utilization in smooth muscle: roles of aerobic glycolysis and creatine kinase

The phosphocreatine content of smooth muscle is of similar magnitude to ATP. Thus the function of the creatine kinase system in this tissue cannot simply be regarded as an energy buffer. Thus an understanding of its role in smooth muscle behavior can point to CK function in other systems. From our perspective CK function in smooth muscle is one example of a more general phenomenon, that of the co-localization of ATP synthesis and utilization. In an interesting and analogous fashion distinct glycolytic cascades are also localized in regions of the cell with specialized energy requirements. Similar to CK, glycolytic enzymes are known to be localized on thin filaments, sarcoplasmic reticulum and plasma membrane. In this chapter we will describe the relations between glycolysis and smooth muscle function and compare and contrast to that of the CK system. Our goal is to more fully understand the significance of the compartmentation of distinct pathways for ATP synthesis with specific functions in smooth muscle. This organization of metabolism and function seen most clearly in smooth muscle is likely representative of many other cell types.     

Functional properties and [Ca(2+)](i) metabolism of creatine kinase--KO mice myocardium

One major function of the creatine kinase system is to maintain energy demand of myofibrillar contraction processes. Loss of the CK-system led to adaptations in skeletal muscle. To analyze the impact on myocardial function contractile parameters and intracellular calcium metabolism of transgenic mice lacking mitochondrial CK (ScCKmit(-/-)) alone or both mitochondrial and cytoplasmic ScCK (CK(-/-)) were investigated compared to wild type at various workload conditions using isolated intact muscle fibers. Force development at baseline conditions, force-frequency relationship (60-600/min), and rapid frequency switch (60-600/min) were unaltered in myocardium of transgenic mice compared to wild type. Intracellular calcium metabolism revealed unchanged amplitude of the intracellular calcium transients (ICT), refilling of the sarcoplasmic reticulum (calcium reuptake, post-rest behavior) in the ScCKmit(-/-) and CK(-/-) mice. The results demonstrate the effectiveness of myocardial energy-recruiting compensatory mechanisms at baseline as well as under stress conditions in CK depleted myocardium of transgenic mice.     

Kinetics of the creatine kinase reaction in neonatal rabbit heart: an empirical analysis of the rate equation

Here we define the kinetics of the creatine kinase (CK) reaction in an intact mammalian heart containing the full range of CK isoenzymes. Previously derived kinetic constants [Schimerlik, M. I., & Cleland, W. W. (1973) J. Biol. Chem. 248, 8418-8423] were refit for the reaction occurring at 37 degrees C. Steady-state metabolite concentrations from 31P NMR and standard biochemical techniques were determined. 31P magnetization transfer data were obtained to determine unidirectional creatine kinase fluxes in hearts with differing total creatine contents and differing mitochondrial CK activities during KCl arrest and isovolumic work for both the forward reaction (MgATP synthesis) and reverse reaction (phosphocreatine synthesis). The NMR kinetic data and substrate concentration data were used in conjunction with a kinetic model based on MM-CK in solution to determine the applicability of the solution-based kinetic models to the CK kinetics of the intact heart. Our results indicated that no single set of rate equation constants could describe both the KCl-arrested and working hearts. We used our experimental data to constrain the solution-derived kinetic model and derived a second set of rate equation constants, which describe the isovolumic work state. Analysis of our results indicates that the CK reaction is rate limited in the direction of ATP synthesis, the size of the guanidino substrate pool drives the measured CK flux in the intact heart, and during isovolumic work the CK reaction operates under saturating conditions; that is, the substrate concentrations are at least 2-fold greater than the Km or Kim for each substrate.(ABSTRACT TRUNCATED AT 250 WORDS)     

Tetracycline-Inducible System for Regulation of Skeletal Muscle-Specific Gene Expression in Transgenic Mice

Tightly regulated control of over-expression is often necessary to study one aspect or time point of gene function and, in transgenesis, may help to avoid lethal effects and complications caused by ubiquitous over-expression. We have utilized the benefits of an optimized tet-on system and a modified muscle creatine kinase (MCK) promoter to generate a skeletal muscle-specific, doxycycline (Dox) controlled over-expression system in transgenic mice. A DNA construct was generated in which the codon optimized reverse tetracycline transactivator (rtTA) was placed under control of a skeletal muscle-specific version of the mouse MCK promoter. Transgenic mice containing this construct expressed rtTA almost exclusively in skeletal muscles. These mice were crossed to a second transgenic line containing a bi-directional promoter centered on a tet responder element driving both a luciferase reporter gene and a tagged gene of interest; in this case the calpain inhibitor calpastatin. Compound hemizygous mice showed high level, Dox dependent muscle-specific luciferase activity often exceeding 10,000-fold over non-muscle tissues of the same mouse. Western and immunocytochemical analysis demonstrated similar Dox dependent muscle-specific induction of the tagged calpastatin protein. These findings demonstrate the effectiveness and flexibility of the tet-on system to provide a tightly regulated over-expression system in adult skeletal muscle. The MCKrtTA transgenic lines can be combined with other transgenic responder lines for skeletal muscle-specific over-expression of any target gene of interest.     

Influence of mitochondrial creatine kinase on the mitochondrial/extramitochondrial distribution of high energy phosphates in muscle tissue: evidence for a leak in the creatine shuttle

The influence of mitochondrial creatine kinase on subcellular high energy systems has been investigated using isolated rat heart mitochondria, mitoplasts and intact heart and skeletal muscle tissue.In isolated mitochondria, the creatine kinase is functionally coupled to oxidative phosphorylation at active respiratory chain, so that it catalyses the formation of creatine phosphate against its thermodynamic equilibrium. Therefore the mass action ratio is shifted from the equilibrium ratio to lower values. At inhibited respiration, it is close to the equilibrium value, irrespective of the mechanism of the inhibition. The same results were obtained for mitoplasts under conditions where the mitochondrial creatine kinase is still associated with the inner membrane.In intact tissue increasing amounts of creatine phosphate are found in the mitochondrial compartment when respiration and/or muscle work are increased. It is suggested that at high rates of oxidative phosphorylation creatine phosphate is accumulated in the intermembrane space due to the high activity of mitochondrial creatine kinase and the restricted permeability of reactants into the extramitochondrial space. A certain amount of this creatine phosphate leaks into the mitochondrial matrix.This leak is confirmed in isolated rat heart mitochondria where creatine phosphate is taken up when it is generated by the mitochondrial creatine kinase reaction. At inhibited creatine kinase, external creatine phosphate is not taken up. Likewise, mitoplasts only take up creatine phosphate when creatine kinase is still associated with the inner membrane. Both findings indicate that uptake is dependent on the functional active creatine kinase coupled to oxidative phosphorylation.Creatine phosphate uptake into mitochondria is inhibited with carboxyatractyloside. This suggests a possible role of the mitochondrial adenine nucleotide translocase in creatine phosphate uptake.Taken together, our findings are in agreement with the proposal that creatine kinase operates in the intermembrane space as a functional unit with the adenine nucleotide translocase in the inner membrane for optimal transfer of energy from the electron transport chain to extramitochondrial ATP-consuming reactions.     

The activity of creatine kinase in frog skeletal muscle studied by saturation-transfer nuclear magnetic resonance.

1. The activity of creatine kinase in intact anaerobic frog muscle at 4 degrees C at rest and during contraction was investigated by using saturation-transfer 31P n.m.r. 2. At rest, the measured forward (phosphocreatine to ATP) reaction flux was 1.7 X 10(-3) M . s-1 and the backward flux was 1.2 X 10(-3) M . s-1. The large magnitude of both fluxes shows that creatine kinase is active in resting muscle, so the observed constancy of [phosphocreatine] demonstrates that the enzyme and its substrates are at equilibrium. 3. The apparent discrepancy between the fluxes must arise largely from an underestimation of the backward flux resulting from interaction of ATP with other systems, e.g. via adenylate kinase. For purposes of further calculation we have therefore adopted 1.6 X 10(-3) M . s-1 as an estimate of both fluxes. 4. During contraction, when the creatine kinase reaction is no longer at equilibrium, the net rate of phosphocreatine breakdown, estimated directly from the change in area of the inorganic phosphate peak, was 0.75 X 10(-3) M . s-1. Saturation transfer indicates that the forward reaction flux remains at approx. 1.6 X 10(-3) M . s-1 and the backward flux decreases to about 0.85 X 10(-3) M . s-1. 5. The activity of creatine kinase during contraction is large enough to account for the well-established observation that, during contraction, the concentration of ATP falls by less than 2-3%. The reaction catalysed by creatine kinase is driven forward during contraction by the large relative increase in the concentration of free ADP, which is more than doubled. 6. The observation that the forward flux does not increase during contraction and that the backward flux decreases can most simply be explained on the basis of competition of reactants for a limited amount of enzyme.     

Kinetic characterization of human heart and skeletal muscle CK isoenzymes

The purpose of this study was to investigate the kinetic properties of human creatine kinase (CK) isoenzymes partially purified from heart and skeletal muscle. Utilizing the backward CK-catalyzed reaction of creatine phosphate + ADP in equilibrium creatine + ATP, Km values for heart and skeletal muscle CK MM (3.7 mmol/l) were significantly (p less than 0.05) greater than CK MB (2.1 mmol/l) which were significantly (p less than 0.05) greater than mitochondrial CK (1.8 mmol/l) at variable creatine phosphate and fixed ADP concentrations. However, Km values for similar isoenzymes from the two different tissues, i.e., CK MB from heart vs. skeletal muscle, were not different. These results show that kinetic analysis of CK isoenzymes cannot differentiate the tissue source of elevated blood CK isoenzymes after the acute stress of long distance running or after acute myocardial infarction.     

最大等速离心运动诱发肌肉损伤程度与肌酸激酶水平的关系

为探讨人体进行最大等速离心运动(ECC)诱发血液肌酸激酶(CK)水平变化、血清肌酸激酶水平与肌肉损伤(EIMD)的关系,本研究筛选出150名"缺乏运动"的健康大学生为受试者,进行血样采集,进行前测包括血清肌酸激酶(CK)、最大等长肌力(MVC)、肘关节活动角度(ROM)、上臂围(CIR)、肌肉感受(VAS)。受试者进行5组×12次最大等速离心运动,运动后恢复期,将全部受试者血清肌酸激酶值进行排序:血清肌酸激酶值最高和最低20%样本,高肌酸激酶水平组(HCK组)和低肌酸激酶水平组(low LCK组),利用SPSS18.0统计学软件,以方差分析和多元回归分析进行统计分析。本研究发现全部受试者、高肌酸激酶水平组、低肌酸激酶水平组在最大等速离心运动后各评估指标均显著高于比前测结果,p<0.05。全部受试者、高肌酸激酶水平组受试者在最大等速离心运动后各指标变化皆明显大于低肌酸激酶水平组受试者,p<0.05。受试者血清肌酸激酶峰值与最大等长肌力、肘关节活动角度、上臂围、肌肉感受最大变化值有相关,p<0.05。本研究认为肌肉损伤程度与肌酸激酶水平具有显著相关,尤其高血清肌酸激酶水平者肌酸激酶水平较大程度反映肌肉损伤程度趋势。本研究表明,肘关节活动角度、上臂围具有预测肌酸激酶峰值的效果。     

Adenylate kinase: Kinetic behavior in intact cells indicates it is integral to multiple cellular processes

Monitoring the kinetic behavior of adenylate kinase (AK) and creatine kinase (CK) in intact cells by ¹⁸O-phosphoryl oxygen exchange analysis has provided new perspectives from which to more fully define the involvement of these phosphotransferases in cellular bioenergetics. A primary function attributable to both AK and CK is their apparent capability to couple ATP utilization with its generation by glycolytic and/or oxidative processes depending on cell metabolic status. This is evidenced by the observation that the sum of the net AK- plus CK-catalyzed phosphoryl transfer is equivalent to about 95% of the total ATP metabolic flux in non-contracting rat diaphragm; under basal conditions almost every newly generated ATP molecule appears to be processed by one or the other of these phosphotransferases prior to its utilization. Although CK accounts for the transfer of a majority of the ATP molecules generated/consumed in the basal state there is a progressive, apparently compensatory, shift in phosphotransfer catalysis from the CK to the AK system with increasing muscle contraction or graded chemical inhibition of CK activity. AK and CK appear therefore to provide similar and interrelated functions. Evidence that high energy phosphoryl transfer in some cell types or metabolic states can also be provided by specific nucleoside mono- and diphosphate kinases and by the phosphotransfer capability inherent to the glycolytic system has been obtained. Measurements by ¹⁸O-exchange analyses of net AK- and CK-catalyzed phosphoryl transfer in conjunction with 31P NMR analyses of total unidirectional phosphoryl flux show that each new energy-bearing molecule CK or AK generates subsequently undergoes about 50 or more unidirectional CK-or AK-catalyzed phosphotransfers en route to an ATP consumption site in intact muscle. This evidence of multiple enzyme catalyzed exchanges coincides with the mechanism of vectorial ligand conduction suggested for accomplishing intracellular high energy phosphoryl transfer by the AK and CK systems. AK-catalyzed phosphotransfer also appears to be integral to the transduction of metabolic signals influencing the operation of ion channels regulated by adenine nucleotides such as ATP-inhibitable K⁺ channels in insulin secreting cells; transition from the ATP to ADP liganded states closely coincides with the rate AK-catalyzes phosphotransfer transforming ATP (+AMP) to (2) ADP.     

Role of phosphocreatine in energy transport in skeletal muscle of bullfrog studied by 31P-NMR

To evaluate the energy-shuttle hypothesis of the phosphocreatine/creatine kinase system, diffusion rates for ATP, phosphocreatine and flux through the creatine kinase reaction were determined by 31P-NMR in resting bullfrog biceps muscle. The diffusion coefficient of phosphocreatine measured by 31P-pulsed gradient NMR was 1.4-times larger than ATP in the muscle, indicating the advantage of phosphocreatine molecules for the intracellular energy transport. The flux of the creatine kinase reaction measured by 31P-saturation transfer NMR was 3.6 mmol/kg wet wt. per s in the resting muscle. The flux is equal to the turnover rate of ATP, ADP, phosphocreatine and creatine molecules, therefore, the life-times of these substrates and the average distance traversed after the life-times by the diffusing molecules were calculated using the diffusion coefficients obtained by 31P-NMR. The mean square length of one-dimensional diffusion was 22 microns in ATP molecules and the minimum diffusion length was 1.8 microns in ADP molecules. The latter was calculated using free ADP concentration, 30 mumol/kg wet wt., obtained from the equilibrium constant of the creatine kinase reaction and the diffusion coefficient assumed to be the same of ATP in muscle. Similar diffusion lengths of ADP were calculated using the reported values for the flux of the creatine kinase reaction in heart and smooth-muscle. The diffusion lengths of all substrates involved in the creatine kinase reaction were larger than the radii of myofibrils. Therefore, in the muscles with an alternating arrangement of mitochondria and myofibrils, such as heart and certain skeletal muscles, ATP and ADP molecules can move freely between myofibrils and mitochondria without the aid of the creatine kinase reaction; thus, we conclude that the energy-shuttle hypothesis is not obligatory for energy transport between the mitochondria and the myofibrils.     

Evolution of isozyme loci and their differential tissue expression

Summary The phylogeny of the creatine kinase (CK, EC 2.7.3.2) isozyme loci and their differential tissue expressions were determined for representatives of 65 families of vertebrates, with emphasis on the fishes. The transition from the single creatine kinase locus, characteristic of certain echinoderms, to the two creatine kinase loci which are orthologous to those present in all vertebrates, occurred early in the chordate line. The majority of pre-teleostean fishes possesses only these two CK loci (A and C). These loci are relatively generalized in their tissue expressions which are variable among species of primitive fishes. The third and fourth creatine kinase loci (B and D) arose separately in the ancestors of the bony fishes and appear to be the result of regional genome duplications. Concomitant with the increase in the number of isozyme loci has been an increase in the specificity of their tissue expression. In the advanced teleost fishes the four CK loci are differentially expressed in a characteristic manner. The A₂ isozyme predominates in skeletal muscle, the B₂ isozyme in eye and brain, the C₂ isozyme in stomach muscle, and the D₂ isozyme is found exclusively in testis. We propose a phylogeny of the creatine kinase genes in the lower chordates based on the time of appearance of new CK loci, the sequence in which the loci achieve a tissue restricted expression, and the immunochemical relatedness of the orthologous and paralogous gene products.     

Muscle creatine kinase isoenzyme expression in adult human brain.

Previous studies have suggested that MM creatine kinase is a muscle-specific protein and is not present in adult brain tissue. We have isolated a protein from human brain with an apparent molecular weight of 43,000 as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis which is identical to the muscle M creatine kinase isoenzyme subunit at all 30 sequenced amino acid residues and possesses creatine kinase enzymatic activity following nondenaturing agarose-gel electrophoresis. Immunohistochemistry localizes M creatine kinase to discrete areas of adult human brain. Northern blot analysis of both total and poly(A)-selected RNA isolated from brain did not detect M creatine kinase mRNA. However, polymerase chain reaction amplification of cDNA synthesized from human placenta, heart, and brain mRNA detected M creatine kinase message in both heart and brain but not placenta which contains no detectable M creatine kinase protein. N1E115 and NS20Y, mouse neuroblastoma cell lines which have been used as models of neural cell differentiation, were found also to express MM creatine kinase. Moreover, a transiently transfected reporter gene with 4,800 base pairs of M creatine kinase upstream region fused to chloramphenicol acetyltransferase was expressed during differentiation of these neural cell lines. In summary, MM creatine kinase is present in human brain and we suggest the M creatine kinase upstream region is sufficient to modulate M creatine kinase expression in certain neuronal cells and may be regulated independently from other muscle genes.     

Creatine kinase equilibrium and lactate content compared with muscle pH in tissue samples obtained after isometric exercise

Muscle biopsies taken from the musculus quadriceps femoris of man were analysed for pH, ATP, ADP, AMP, creatine phosphate, creatine, lactate and pyruvate. Biopsies were taken at rest, after circulatory occlusion and after isometric contraction. Muscle pH decreased from 7.09 at rest to 6.56 after isometric exercise to fatigue. Decrease in muscle pH was linearly related to accumulation of lactate plus pyruvate. An increase of 22mumol of lactate plus pyruvate per g of muscle resulted in a fall of 0.5pH unit. The apparent equilibrium constant of the creatine kinase reaction (apparent K(CK)) increased after isometric contraction and a linear relationship between log(apparent K(CK)) and muscle pH was obtained. The low content of creatine phosphate in muscle after contraction as analysed from needle-biopsy samples is believed to be a consequence of an altered equilibrium state of the creatine kinase reaction. This in turn is attributed mainly to a change in intracellular pH.     

Smooth muscle and NMR review: An overview of smooth muscle metabolism

Nuclear magnetic resonance (NMR) is a non-invasive technique which allows us to examine the biochemical, physiological and metabolic events occurring inside living tissue; such as vascular and other smooth muscles.It has been found that the smooth muscle metabolism is compartmented such that mitochondrial function fuels contraction and that much glycolytic ATP production is used for membrane pumps. Using NMR we have been able to observe the ATP and phosphocreatine (PCr) concentrations and estimate the ADP concentration, as well as flux through the creatine kinase (CK) system. It has also been found that the smooth muscle metabolism is able to maintain ATP concentration in the absence of mitochondrial function (cyanide inhibition). Therefore, the vessels are able to adapt to metabolic demands as necessary.NMR is versatile in the information it can provide because it has also yielded important contributions with regard to the intracellular pH and ionic status. For example, the intracellular free Mg²⁺ ([Mg²⁺]_i) can be measured with NMR simultaneously with ATP concentrations and NMR has shown us that the [Mg²⁺]_i is highly protected in the muscle (within confined range), but also responds to the environment around it.In this review we conclude that NMR measurements of smooth muscle research is a useful technique for assessing chronic and acute changes that occur in the tissue and during diseases.     

The principal islet of the coho salmon (Oncorhyncus kisutch) contains the BB isoenzyme of creatine kinase

The importance of creatine kinase (E.C. 2.7.3.2) in endocrine tissues has been generally overlooked. Using a specific radiometric assay, we have demonstrated the existence of CK in the Brockmann body (principal islet) of the Coho salmon. We have purified this protein from insular tissue and concurrently purified CK from brain and muscle of the salmon. Purification characteristics, immunological cross-reactivity, and N-terminal sequence analysis have demonstrated that the predominant cytosolic CK from the Brockmann body is indistinguishable from the BB (brain) isoenzyme. Immunocytochemical studies indicated that the enzyme resides in the endocrine parenchyma. Phosphocreatine may serve as a reservoir of energy in the islet and augment its capacity to secrete hormones. The induction of CK-BB in the islet by other hormones could influence the secretion of insular hormones. Interorgan flux of the substrate creatine may be an undescribed mechanism of physiological regulation.     

Mathematical model of compartmentalized energy transfer: Its use for analysis and interpretation of 31P-NMR studies of isolated heart of creatine kinase deficient mice

A mathematical model of the compartmentalized energy transfer in cardiac cells is described and used for interpretation of novel experimental data obtained by using phosphorus NMR for determination of the energy fluxes in the isolated hearts of transgenic mice with knocked out creatine kinase isoenzymes. These experiments were designed to study the meaning and importance of compartmentation of creatine kinase isoenzymes in the cells in vivo. The model was constructed to describe quantitatively the processes of energy production, transfer, utilization, and feedback between these processes. It describes the production of ATP in mitochondrial matrix space by ATP synthase, use of this ATP for phosphocreatine production in the mitochondrial creatine kinase reaction coupled to the adenine nucleotide translocation, diffusional exchange of metabolites in the cytoplasmic space, and use of phosphocreatine for resynthesis of ATP in the myoplasmic creatine kinase reaction. It accounts also for the recently discovered phenomenon of restricted diffusion of adenine nucleotides through mitochondrial outer membrane porin pores (VDAC). Practically all parameters of the model were determined experimentally. The analysis of energy fluxes between different cellular compartments shows that in all cellular compartments of working heart cells the creatine kinase reaction is far from equilibrium in the systolic phase of the contraction cycle and approaches equilibrium only in cytoplasm and only in the end-diastolic phase of the contraction cycle.Experimental determination of the relationship between energy fluxes by a ³¹P-NMR saturation transfer method and workload in isolated and perfused heart of transgenic mice deficient in MM isoenzyme of the creatine kinase, MM -/- showed that in the hearts from wild mice, containing all creatine kinase isoenzymes, the energy fluxes determined increased 3-4 times with elevation of the workload. By contrast, in the hearts in which only the mitochondrial creatine kinase was active, the energy fluxes became practically independent of the workload in spite of the preservation of 26% of normal creatine kinase activity. These results cannot be explained on the basis of the conventional near-equilibrium theory of creatine kinase in the cells, which excludes any difference between creatine kinase isoenzymes. However, these apparently paradoxical experimental results are quantitatively described by a mathematical model of the compartmentalized energy transfer based on the steady state kinetics of coupled creatine kinase reactions, compartmentation of creatine kinase isoenzymes in the cells, and the kinetics of ATP production and utilization reactions. The use of this model shows that: (1) in the wild type heart cells a major part of energy is transported out of mitochondria via phosphocreatine, which is used for complete regeneration of ATP locally in the myofibrils - this is the quantitative estimate for PCr pathway; (2) however, in the absence of MM-creatine kinase in the myofibrils in transgenic mice the contraction results in a very rapid rise of ADP in cytoplasmic space, that reverses the mitochondrial creatine kinase reaction in the direction of ATP production. In this way, because of increasing concentrations of cytoplasmic ADP, mitochondrial creatine kinase is switched off functionally due to the absence of its counterpart in PCr pathway, MM-creatine kinase. This may explain why the creatine kinase flux becomes practically independent from the workload in the hearts of transgenic mouse without MM-CK. Thus, the analysis of the results of studies of hearts of creatine kinase-deficient transgenic mice, based on the use of a mathematical model of compartmentalized energy transfer, show that in the PCr pathway of intracellular energy transport two isoenzymes of creatine kinase always function in a coordinated manner out of equilibrium, in the steady state, and disturbances in functioning of one of them inevitably result in the disturbances of the other component of the PCr pathway. In the latter case, energy is transferred from mitochondria to myofibrils by alternative metabolic pathways, probably involving adenylate kinase or other systems.     

The principal islet of the coho salmon (Oncorhyncus kisutch) contains the BB isoenzyme of creatine kinase

The importance of creatine kinase (E.C. 2.7.3.2) in endocrine tissues has been generally overlooked. Using a specific radiometric assay, we have demonstrated the existence of CK in the Brockmann body (principal islet) of the Coho salmon. We have purified this protein from insular tissue and concurrently purified CK from brain and muscle of the salmon. Purification characteristics, immunological cross-reactivity, and N-terminal sequence analysis have demonstrated that the predominant cytosolic CK from the Brockmann body is indistinguishable from the BB (brain) isoenzyme. Immunocytochemical studies indicated that the enzyme resides in the endocrine parenchyma. Phosphocreatine may serve as a reservoir of energy in the islet and augment its capacity to secrete hormones. The induction of CK-BB in the islet by other hormones could influence the secretion of insular hormones. Interorgan flux of the substrate creatine may be an undescribed mechanism of physiological regulation.