Purification and some properties of two creatine kinase isoforms from herring (Clupea harengus) spermatozoa

Creatine kinase (CK, EC 2.7.3.2) isoforms play important role in energy homeostasis and together with easily diffusible compounds like creatine and phosphocreatine maintain a cellular energy buffer and intracellular energy transport system. The CK activity in spermatozoa is the highest from all studied tissues in herring. It was detected that the two CK isoforms, CK1 and CK2, are characteristic only for spermatozoa and are not expressed in other herring tissues. Isolation and purification procedures allowed obtaining purified enzymes with specific activity of the 345 micromol/min/mg for CK1 and 511 micromol/min/mg for CK2. Native Mr's of the CK1 and CK2 determined by gel permeation chromatography were about 330,000 and 90,000, respectively. These results indicate that CK1 form has octameric structure and CK2 is a dimer mostly characteristic for cytosolic CK enzymes. In immunoblotting studies with antisera against CK2, the response was observed for CK2 and there was no response for CK1 and two other isoforms from herring skeletal muscle. These findings make the herring isoforms an interesting model for studies on the fish CK biochemical properties.     

Characterization of isoforms of human mitochondrial creatine kinase by isoelectric focusing

High enzyme activity of mitochondrial creatine kinase (creatine-N-phosphotransferase, mCK, EC 2.7.3.2) was detected in serum from a patient with advanced carcinoma of the rectum and its isoforms were characterized by means of isoelectric focusing (IEF). Three forms of mCK, membrane-bound (pI 6.9–7.0), octameric (pI 7.0–7.9) and dimeric (pI 6.7, 6.8, 6.9 and 7.0), were detected in the fresh serum. These three forms of mCK were converted to five dimeric isoforms, and these were characterized as one reduced form (pI 7.0) and four oxidized (pI 6.6, 6.7, 6.8 and 6.9) forms upon treatment with urea, hydrogen peroxide or 2-mercaptoethanol (2-ME). The C-terminal of the mCKs was concluded to be a lysine residue because the mCKs treated with carboxypeptidase B migrated to positions closer to the anode than did those not treated with carboxypeptidase B. Therefore, four bands were concluded to represent one reduced-delysined isoform (pI 6.4) and three oxidized-delysined isoforms (pI 6.1, 6.2 and 6.3). The broad octameric mCK band disappeared and a narrow band focused at pI 6.8–6.9 appeared upon probable delysination of the mCKs. Thus, the number of lysine residues at the C-terminal of the octamer was concluded to be variable due to variable catalysis by carboxypeptidase N in the plasma. mCKs seemed to be inactivated during conversion from a membrane-bound form to dimeric oxidized-delysined forms via the octameric, dimeric reduced and oxidized forms.     

Modulation of creatine kinase activity by ruthenium complexes

Creatine kinase is a crucial enzyme for brain, heart and skeletal muscle energy homeostasis, and a decrease of its activity has been associated with cell death. Many biological properties have been attributed to ruthenium complexes. In this context, this work was performed in order to evaluate creatine kinase activity from rat brain, heart and skeletal muscle (quadriceps) after administration of ruthenium complexes, trans-[RuCl(2)(nic)(4)] (nic=3-pyridinecarboxylic acid) 180.7 micromol/kg (complex I), trans-[RuCl(2)(i-nic)(4)] (i-nic=4-pyridinecarboxylic acid) 13.6 micromol/kg (complex II), trans-[RuCl(2)(dinic)(4)] (dinic=3,5-pyridinedicarboxylic acid) 180.7 micromol/kg (complex III) and trans-[RuCl(2)(i-dinic)(4)] (i-dinic=3,4-pyridinedicarboxylic acid) 180.7 micromol/kg (complex IV). Our results showed that complex I caused inhibition of creatine kinase activity in hippocampus, striatum, cerebral cortex, heart and skeletal muscle. Besides, complex II did not affect the enzyme activity. complexes III and IV increased creatine kinase activity in hippocampus, striatum, cerebral cortex and heart, but not in skeletal muscle. Besides, none of the complexes in vitro altered creatine kinase activity, suggesting that enzymatic activity is indirectly affected by complexes I, III and IV. It is believed that diminution of creatine kinase in brain of rats caused by complex I may be related to results from other study reporting memory impairment caused by the same complex. Further research is necessary in order to elucidate the effects of ruthenium complexes in other important metabolic enzymes.     

Characterization of two tropomyosin isoforms from the fast skeletal muscle of bluefin tuna Thunnus thynnusorientalis

Fast skeletal muscle tropomyosin (TM) of tunas is composed of nearly equimolar amount of two isoforms designated α-TM and β-TM expediently based on their migration behavior in SDS-PAGE, whereas corresponding TMs from the other fish species are homogenous (α-type). The presence of β-TM is thus specific to tunas so far. The amino acid sequence of β-TM from bluefin tuna Thunnus thynnus orientalis, which has not been revealed to date unlike α-TM, was successfully obtained in this study by cDNA cloning. The coding region of β-TM cDNA comprised of an open reading frame of 855 bp encoding 284 amino acid residues, like most of the TMs. Unexpectedly, the sequence of β-TM showed high similarity to those of other vertebrate α-type TMs including tuna α-TM. Phylogenetic analysis also showed that β-TM has the closest relationship with α-TM of tuna. This fact was quite unlike the relation of mammalian α- and β-TMs. Based on the distribution of amino acid substitutions, it was suggested that tuna TM isoforms are the products of different genes. By thermodynamic analysis of native and reconstituted TMs, it was demonstrated that β-TM is less thermostable than α-TM. Proteolytic digestion also supported the lower stability of the former.     

Creatine supplementation in health and disease. Effects of chronic creatine ingestion in vivo: Down-regulation of the expression of creatine transporter isoforms in skeletal muscle

Interest in creatine (Cr) as a nutritional supplement and ergogenic aid for athletes has surged over recent years. After cellular uptake, Cr is phosphorylated to phosphocreatine (PCr) by the creatine kinase (CK) reaction using ATP. At subcellular sites with high energy requirements, e.g. at the myofibrillar apparatus during muscle contraction, CK catalyzes the transphosphorylation of PCr to ADP to regenerate ATP, thus preventing a depletion of ATP levels. PCr is thus available as an immediate energy source, serving not only as an energy buffer but also as an energy transport vehicle. Ingestion of creatine increases intramuscular Cr, as well as PCr concentrations, and leads to exercise enhancement, especially in sprint performance. Additional benefits of Cr supplementation have also been noticed for high-intensity long-endurance tasks, e.g. shortening of recovery periods after physical exercise.The present article summarizes recent findings on the influence of Cr supplementation on energy metabolism, and introduces the Cr transporter protein (CreaT), responsible for uptake of Cr into cells, as one of the key-players for the multi-faceted regulation of cellular Cr homeostasis. Furthermore, it is suggested that patients with disturbances in Cr metabolism or with different neuro-muscular diseases may benefit from Cr supplementation as an adjuvant therapy to relieve or delay the onset of symptoms. Although it is still unclear how Cr biosynthesis and transport are regulated in health and disease, so far there are no reports of harmful side effects of Cr loading in humans. However, in this study, we report that chronic Cr supplementation in rats down-regulates in vivo the expression of the CreaT. In addition, we describe the presence of CreaT isoforms most likely generated by alternative splicing.     

Plasma accumulation of hypoxanthine, uric acid and creatine kinase following exhausting runs of differing durations in man

During exhausting exercise adenylate kinase in the muscle cells is activated and a degradation of adenosine 5'-diphosphate occurs. Consequently, degradation products of adenosine 5'-monophosphate including hypoxanthine and uric acid, accumulate in plasma. The aim of this study was to compare the concentration changes of hypoxanthine and uric acid in plasma following running of varying duration and intensity. In addition, plasma creatine kinase activity was measured to assess the possible relationship between metabolic stress and protein release. Four groups of competitive male runners ran 100 m (n = 7), 800 m (n = 11), 5000 m (n = 7) and 42,000 m (n = 7), respectively, at an exhausting pace. Subsequent to the 100 m event (mean running time 11 s) plasma concentrations of hypoxanthine and uric acid increased by 364% and 36% respectively (P less than 0.05), indicating a very high rate of adenine nucleotide degradation during the event. Following the 800-m event (mean running time 125 s), hypoxanthine and uric acid concentrations had increased by 1598% and 66%, respectively (P less than 0.05). Both the events of longer duration, 5000 m and 42,000 m, also caused a significant increase in plasma concentration of hypoxanthine (742% and 237% respectively, P less than 0.05) and plasma uric acid (54% and 34% respectively, P less than 0.05). Plasma activities of creatine kinase were significantly increased at 24 h only following the 5000 m and 42,000 m events (64% and 1186% respectively, P less than 0.05). Changes in plasma creatine kinase activity showed no correlation with changes in plasma concentration of either hypoxanthine or uric acid for the 5000 m and 42,000 m events (r = 0.00-0.45, P greater than 0.05).     

Comparative enzymology of AMP deaminase,adenylate kinase,and creatine kinase in vertebrate heart and skeletal muscle: the characteristic AMP deaminase levels of skeletal versus cardiac muscle are reversed in the North American toad

The specific activity of three characteristic enzymes, adenylate deaminase, adenylate kinase, and creatine kinase, in the skeletal muscles and heart of a variety of vertebrate land animals, including the human, are surveyed. Data from this study and available studies in the literature suggest that adenosine monophosphate deaminase in land vertebrates is quite high in white skeletal muscle, usually somewhat lower in red muscle, and 15-to 500-fold lower in cardiac muscle. Adenosine monophosphate deaminase is active primarily under ischemic or hypoxic conditions which occur frequently in white muscle, only occasionally in red muscle, and ought never occur in heart muscle, and this may therefore account for observed enzyme levels. The common North American toad, Bufo americanus, provides a striking exception to the rule with cardiac adenosine monophosphate deaminase as high as in mammalian skeletal muscle, whereas its skeletal muscle level of adenosine monophosphate deaminase is several times lower. The exceptional levels in the toad are not due to a change in substrate binding and are not accompanied by comparable change in the level of adenylate or creatine kinase. Nor do they signal any major change in isozyme composition, since a human muscle adenosine monophosphate deaminase-specific antiserum reacts with toad muscle adenosine monophosphate deaminase, but not with toad heart adenosine monophosphate deaminase. They do not represent any general anuran evolutionary strategy, since the bullfrog (Rana catesbeiana) and the giant tropic toad (Bufo marinus) have the usual vertebrate pattern of adenosine monophosphate deaminase distribution. Lower skeletal muscle activities in anurans may simply represent the contribution of tonic muscle fiber bundles containing low levels of adenosine monophosphate deaminase, but the explanation for the extremely high adenosine monophosphate deaminase levels in heart ventricular muscle is not apparent.Abbreviations AK adenylate kinase - AMP adenosine monophosphate - AMPD, AMP deaminase - CPK creatine (phospho)kinase - EHNA erythro-9-(2-hydroxy-3-nonyl)-adenine-HCl     

Transgenic and tissue culture analyses of the muscle creatine kinase enhancer Trex control element in skeletal and cardiac muscle indicate differences in gene expression between muscle types

The muscle creatine kinase (MCK) gene is expressed at high levels only in differentiated skeletal and cardiac muscle. The activity of the cloned enhancer–promoter has previously been shown to be dependent on the Trex element which is specifically bound by a yet unidentified nuclear factor, TrexBF. We have further characterized the function of the Trex site by comparing wild-type and Trex-mutated MCK transgenes in five mouse skeletal muscles: quadriceps, extensor digitorum longus (EDL), soleus, diaphragm, and distal tongue, as well as in heart ventricular muscle. Several types of statistical analysis including analysis of variance (ANOVA) and rank sum tests were used to compare expression between muscle types and between constructs. Upon mutation of the Trex site, median transgene expression levels decreased 3- to 120-fold in the muscles examined, with statistically significant differences in all muscles except the EDL. Expression in the largely slow soleus muscle was more affected than in the EDL, and expression in the distal tongue and diaphragm muscles was affected more than in soleus. Median expression of the transgene in ventricle decreased about 18-fold upon Trex mutation. Transfections into neonatal rat myocardiocytes confirmed the importance of the Trex site for MCK enhancer activity in heart muscle, but the effect is larger in transgenic mice than in cultured cells.     

Phosphoenolpyruvate-dependent protein kinase activity in rat skeletal muscle

Phosphoenolpyruvate-dependent protein kinase activity has been demonstrated in the soluble fraction of rat skeletal muscle. The reaction was not due to the formation of ATP in the incubation mixture. Cyclic AMP, calcium, ATP and a number of phosphate acceptor proteins did not stimulate the reaction. One 32P-labelled protein (Mr 25000) was observed on SDS gels. The phosphorylated protein contained acid stable phosphoserine as a major phosphorylated amino acid. The phosphorylation reaction in crude extracts was not directly proportional to the amount of protein, but typical of a two-component system; i.e., kinase and substrate. The chromatography of soluble proteins on Ultrogel AcA44 separated the phosphate acceptor protein(s) from the phosphoenolpyruvate-dependent protein kinase activity.     

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.     

Mitochondrial creatine kinase functional development in post-natal rat skeletal muscle. A combined polarographic/31P NMR study

Mitochondrial creatine kinase (Mi-CK) function in viable mitochondria from developing rat skeletal muscle was assessed both by polarographic measurements of creatine-induced respiration and 31P NMR spectroscopy measurements of phosphocreatine (PCr) synthesis. Creatine-induced respiration was observed in very young rats and increased by 50% to 35 days of age. PCr synthesis was present in 7 day old animals and increased by 300% reaching levels measured in 35 day and adult muscle. Unlike reports showing Mi-CK enzymatic activities but no mitochondrial function in several situations, a concomitant progression of enzymatic activity and mitochondrial function was evidenced during the developmental stages of skeletal muscle Mi-CK in altricious animals. These results correlated with the progressive pattern of muscle differentiation during development of motricity in such animals. The observation that Mi-CK is functional in skeletal muscle mitochondria very early after birth, strongly favors the notion that adaptations in skeletal muscle of Mi-CK knock-out mice occur early.     

Oxidation and structural perturbation of redox-sensitive enzymes in injured skeletal muscle

Molecular events that control skeletal muscle injury and regeneration are poorly understood. However, inflammation associated with oxidative stress is considered a key player in modulating this process. To understand the consequences of oxidative stress associated with muscle injury, inflammation, and regeneration, hind-limb muscles of C57Bl/6J mice were studied after injection of cardiotoxin (CT). Within 1 day post-CT injection, polymorphonuclear neutrophilic leukocyte accumulation was extensive. Compared to baseline, tissue myeloperoxidase (MPO) activity was elevated eight- and fivefold at 1 and 7 days post-CT, respectively. Ubiquitinylated protein was elevated 1 day postinjury and returned to baseline by 21 days. Cysteine residues of creatine kinase (CK) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were irreversibly oxidized within 1 day post-CT injection and were associated with protein conformational changes that fully recovered after 21 days. Importantly, protein structural alterations occurred in conjunction with significant decreases in CK activity at 1, 3, and 7 days post-CT injury. Interestingly, elevations in tissue MPO activity paralleled the time course of conformational changes in CK and GAPDH. In combination, these results demonstrate that muscle proteins in vivo are structurally and functionally altered via the generation of reactive oxygen species produced during inflammatory events after muscle injury and preceding muscle regeneration.     

蛋白激酶C对胰岛素抵抗骨骼肌细胞的影响

目的探讨蛋白激酶C(Protein Kinase C,PKC)在棕榈酸(Palmitic Acid,PA)诱导的骨骼肌细胞胰岛素抵抗(Isulin Resistance,IR)中的作用。方法免疫荧光鉴定原代大鼠骨骼肌细胞,氧化酶-过氧化物酶偶联法(GOD-POD法)检测培养液中葡萄糖浓度。设立对照组、棕榈酸组(PA组)、罗格列酮组(Rosiglitazone,Ros组),每组一分为二,分别加PKC抑制剂白屈莱红碱(Chelerythrine Chloride,CC)与正常培养液作用1h,Western Blot检测PKB及P-Ser473 PKB表达水平。结果 90%以上的细胞-αsarcometric actin免疫荧光染色呈阳性反应,表明培养的细胞为骨骼肌细胞;0.6mmol/L的PA作用24h可诱导骨骼肌细胞产生胰岛素抵抗;PA组与对照组相比P-Ser473 PKB水平显著降低,与本组未加CC相比显著升高。同时,罗格列酮组及本组加CC中P-Ser473PKB水平均高于PA组。结论在PA诱导的骨骼肌细胞IR方面PKC起重要作用,罗格列酮与PKC抑制剂CC均能改善PA引起的IR。     

Cholecystokinin (CCK) in Atlantic herring (Clupea harengus L.) - ontogeny and effects of feeding and diurnal rhythms

Neural and alimentary cholecystokinin (CCK) levels in Atlantic herring, Clupea harengus, were analyzed from hatching to 40days after hatching (DAH). The head compartment representing the neural pool was quantitatively dominant (>80% of the total CCK content) while the digestive tract pool represented 6-10%. During ontogeny the CCK level in whole larvae increased almost 15-fold from 0 to 40 DAH, being particularly marked from 14 to 20 DAH. Larvae of 24 to 26 DAH were examined for potential occurrence of a circadian rhythm and to analyze the effects of feeding. Fed and fasted larvae were significantly different, where fed larvae showed higher CCK levels. There were large fluctuations in CCK levels analyzed at 3h intervals without an apparent diurnal pattern. Shorter sampling intervals of 1h in the morning when lights were switched gradually on and food was offered to the larvae demonstrated a marked drop in the relative gut CCK levels and a concurrent increase in the CCK carcass to gut ratio, 1h after introduction of food followed by a return to prefeeding levels after 2h. This response probably results from a release and re-synthesis of CCK in the gut after initiation of feeding. Taken together, these results support earlier reports that CCK participates in the regulation of digestive processes in herring larvae, but CCK does not seem to have a circadian rhythm independent of feeding.     

Spermatogenesis-related change in the synthesis of the creatine kinase B-type and M-type isoforms in human spermatozoa

We have demonstrated earlier that the per sperm creatine-N-phosphotransferase (CK) activity was increased in oligospermic vs. normospermic men. The increased sperm CK activity is related to higher concentrations of cellular CK, which may indicate a defect of cytoplasmic extrusion during spermatogenesis. In the present work, we examined whether in spermatozoa, similar to muscle, there is a change in the synthesis of B-CK and M-CK isoforms during cellular differentiation. In 109 normospermic and 50 oligospermic specimens (sperm concentrations 60.6 +/- 3.7 vs. 8.8 +/- 1.3 million sperm/ml; all values expressed as mean +/- SEM), the relative concentrations of the M-CK isoform (M-CK/M-CK + B-CK) were 27.2% +/- 2.1% vs. 6.7% +/- 0.9% (P less than 0.001). The per sperm CK activities showed comparable differences (0.21 +/- 0.02 vs. 0.89 +/- 0.1 CK IU/100 million sperm; P less than 0.001) in the two groups, and there was a close correlation between per sperm CK activities and M-CK concentrations (R = 0.69, P less than 0.001, N = 159). This indicates that the loss of cytoplasm and the commencement of M-CK isoform synthesis are related events during the last phase of spermatogenesis, also that the incidence of spermatozoa with incomplete cellular maturation is higher in oligospermic specimens. In characterizing the M-CK, we found that sperm (unlike muscle tissue) lack the MB hybrid of CK dimers. However, in the presence of muscle M-CK, the muscle-sperm MB-CK hybrid has formed. Thus in sperm and muscle the M-CK isoforms are structurally different, whereas the B-CKs are apparently homologous.(ABSTRACT TRUNCATED AT 250 WORDS)     

Isolation,characterization and nucleotide sequence of the muscle isoforms of creatine kinase from the Antarctic teleost Chaenocephalus aceratus

Creatine kinase (CK) was isolated from the white muscle of the Antarctic icefish Chaenocephalus aceratus, which is deficient in glycolytic capacity. C. aceratus white myotomal creatine kinase (MMCK) displayed an apparent K_m at 0.5 °C of 0.06 mM for ADP and 17 mM for Phosphocreatine. These K_m values are similar to those reported for other vertebrate MMCKs at their physiologically relevant body temperatures. C. aceratus MMCK exhibited optimal activity at pH of 7.6–7.7 at 0.5 °C, in contrast to rabbit MMCK which had optimum activity at pH 6.2 at 30 °C. The apparent V_max of C. aceratus MMCK at 0.5 °C is 94±4 S.D. (n=9) μmol ATP/min/mg (i.e. U/mg), which is comparable to rabbit MMCK assayed at 20 °C and 8-fold greater than rabbit MMCK measured at 0.5 °C. DEAE chromatography of C. aceratus white muscle CK resolved two distinct activity peaks. Cloning and sequencing of C. aceratus CK cDNAs confirmed that two muscle-specific isoforms of CK were expressed that were distinct from the mitochondrial and brain isoforms. Icefish MMCK was sensitive to transient temperature elevation, and the DEAE-fractionated forms were highly unstable. These results indicate that C. aceratus MMCK displays significant activity at physiological temperature and intracellular pH of icefish muscle that could contribute to sustaining energy charge during burst-swimming.     

Theoretical modelling of some spatial and temporal aspects of the mitochondrion/creatine kinase/ myofibril system in muscle

After discussing approaches to the modelling of mitochondrial regulation in muscle, we describe a model that takes account, in a simplified way, of some aspects of the metabolic and physical structure of the energy production/usage system. In this model, high-energy phosphates (ATP and phosphocreatine) and low energy metabolites (ADP and creatine) diffuse between the mitochondrion and the myofibrillar ATPase, and can be exchanged at any point by creatine kinase. Creatine kinase is not assumed to be at equilibrium, so explicit account can be taken of substantial changes in its activity of the sort that can now be achieved by transgenic technology in vivo. The ATPase rate is the input function. Oxidative ATP synthesis is controlled by juxtamitochondrial ADP concentration. To allow for possible functional coupling between the components of creatine kinase associated with the mitochondrial adenine nucleotide translocase and the myofibrillar ATPase, we define parameters and that set the fraction of the total flux carried by ATP rather than phosphocreatine out of the mitochondrial unit and into the ATPase unit, respectively. This simplification is justified by a detailed analysis of the interplay between the mitochondrial outer membrane porin proteins, mitochondrial creatine kinase and the adenine nucleotide translocase. As both processes of possible coupling are incorporated into the model as quantitative parameters, their effect on the energetics of the whole cell model can be explicitly assessed. The main findings are as follows: (1) At high creatine kinase activity, the hyperbolic relationship of oxidative ATP synthesis rate to spatially averaged ADP concentration at steady state implies also a near-linear relationship to creatine concentration, and a sigmoid relation to free energy of ATP hydrolysis. At high creatine kinase activity, the degree of functional coupling at either the mitochondrial or ATPase end has little effect on these relationships. However, lowering the creatine kinase activity raises the mean steady state ADP and creatine concentrations, and this is exaggerated when or is near unity (i.e. little coupling). (2) At high creatine kinase activity, the fraction of flow at steady state carried in the middle of the model by ATP is small, unaffected by the degree of functional coupling, but increases with ADP concentration and rate of ATP turnover. Lowering the creatine kinase activity raises this fraction, and this is exaggerated when or is near unity. (3) Both creatine and ADP concentrations show small gradients decreasing towards the mitochondrion (in the direction of their net flux), while ATP and phosphocreatine concentration show small gradients decreasing towards the myosin ATPase. Unless = 0 (i.e. complete coupling), there is a gradient of net creatine kinase flux that results from the need to transform some of the adenine nucleotide flux at the ends of the model into creatine flux in the middle; the overall net flux is small, but only zero if = . A reduction in cytosolic creatine kinase activity decreases ADP concentration at the mitochondrial end and increases it at the ATPase end. (4) During work-jump transitions, spatial average responses exhibit exponential kinetics similar to those of models of mitochondrial control that assume equilibrium conditions for creatine kinase. (5) In response to a step increase in ATPase activity, concentration changes start at the ATPase end and propagate towards the mitochondrion, damped in time and space. This simplified model embodies many important features of muscle in vivo, and accommodates a range of current theories as special cases. We end by discussing its relationship to other approaches to mitochondrial regulation in muscle, and some possible extensions of the model.     

Isoenzyme-specific interaction of muscle-type creatine kinase with the sarcomeric M-line is mediated by NH(2)-terminal lysine charge-clamps

Creatine kinase (CK) is located in an isoenzyme-specific manner at subcellular sites of energy production and consumption. In muscle cells, the muscle-type CK isoform (MM-CK) specifically interacts with the sarcomeric M-line, while the highly homologous brain-type CK isoform (BB-CK) does not share this property. Sequence comparison revealed two pairs of lysine residues that are highly conserved in M-CK but are not present in B-CK. The role of these lysines in mediating M-line interaction was tested with a set of M-CK and B-CK point mutants and chimeras. We found that all four lysine residues are involved in the isoenzyme-specific M-line interaction, acting pair-wise as strong (K104/K115) and weak interaction sites (K8/K24). An exchange of these lysines in MM-CK led to a loss of M-line binding, whereas the introduction of the very same lysines into BB-CK led to a gain of function by transforming BB-CK into a fully competent M-line-binding protein. The role of the four lysines in MM-CK is discussed within the context of the recently solved x-ray structures of MM-CK and BB-CK.