Heart Creatine Kinase Research Articles

Cardiac expression and location of hexokinase changes in a mouse model of pure creatine deficiency.

Creatine kinase (CK) is considered the main phosphotransfer system in the heart, important for overcoming diffusion restrictions and regulating mitochondrial respiration. It is substrate limited in creatine-deficient mice lacking l-arginine:glycine amidinotransferase (AGAT) or guanidinoacetate N-methyltranferase (GAMT). Our aim was to determine the expression, activity, and mitochondrial coupling of hexokinase (HK) and adenylate kinase (AK), as these represent alternative energy transfer systems. In permeabilized cardiomyocytes, we assessed how much endogenous ADP generated by HK, AK, or CK stimulated mitochondrial respiration and how much was channeled to mitochondria. In whole heart homogenates, and cytosolic and mitochondrial fractions, we measured the activities of AK, CK, and HK. Lastly, we assessed the expression of the major HK, AK, and CK isoforms. Overall, respiration stimulated by HK, AK, and CK was ∼25, 90, and 80%, respectively, of the maximal respiration rate, and ∼20, 0, and 25%, respectively, was channeled to the mitochondria. The activity, distribution, and expression of HK, AK, and CK did not change in GAMT knockout (KO) mice. In AGAT KO mice, we found no changes in AK, but we found a higher HK activity in the mitochondrial fraction, greater expression of HK I, but a lower stimulation of respiration by HK. Our findings suggest that mouse hearts depend less on phosphotransfer systems to facilitate ADP flux across the mitochondrial membrane. In AGAT KO mice, which are a model of pure creatine deficiency, the changes in HK may reflect changes in metabolism as well as influence mitochondrial regulation and reactive oxygen species production.NEW & NOTEWORTHY In creatine-deficient AGAT-/- and GAMT-/- mice, the myocardial creatine kinase system is substrate limited. It is unknown whether subcellular localization and mitochondrial ADP channeling by hexokinase and adenylate kinase may compensate as alternative phosphotransfer systems. Our results show no changes in adenylate kinase, which is the main alternative to creatine kinase in heart. However, we found increased expression and activity of hexokinase I in AGAT-/- cardiomyocytes. This could affect mitochondrial regulation and reactive oxygen species production.

Effect of Nardostachys jatamansi DC. on Apoptosis, Inflammation and Oxidative Stress Induced by Doxorubicin in Wistar Rats.

The study aimed to investigate the protective action of jatamansi (Nardostachys jatamansi DC.) against doxorubicin cardiotoxicity. Methanolic extract of jatamansi (MEJ) was prepared and standardized using HPTLC fingerprinting, GC-MS chemoprofiling, total phenolic content, and antioxidant activity in vitro. Further in vivo activity was evaluated using rodent model. Animals were divided into five groups (n = 6) namely control (CNT) (Normal saline), toxicant (TOX, without any treatment), MEJ at low dose (JAT1), MEJ at high dose (JAT2), and standard desferrioxamine (STD). All groups except control received doxorubicin 2.5 mg per Kg intra-peritoneally for 3 weeks in twice a week regimen. After 3 weeks, the blood samples and cardiac tissues were collected from all groups for biochemical and histopathological evaluation. Treatment with MEJ at both dose levels exhibited significant reduction (p < 0.001 vs. toxicant) of serum CK-MB (heart creatine kinase), LDH (Lactate dehydrogenase) & HMG-CoA (3-hydroxy-3-methylglutaryl-coenzyme A) levels, and tissue MDA (melondialdehyde) level; insignificant difference was observed (p > 0.05) in TNF-alpha (tumour necrosis factor), IL-6 (interleukine-6) levels and caspase activity as compared to TOX. Histopathological evaluation of cardiac tissues of different treatment groups further reinforced the findings of biochemical estimation. This study concludes that jatamansi can protect cardiac tissues from oxidative stress-induced cell injury and lipid peroxidation as well as against inflammatory and apoptotic effects on cardiac tissues.

Interleukin-12p35 Knock Out Aggravates Doxorubicin-Induced Cardiac Injury and Dysfunction by Aggravating the Inflammatory Response, Oxidative Stress, Apoptosis and Autophagy in Mice.

BackgroundRecent evidence has demonstrated that interleukin 12p35 knockout (IL-12p35 KO) is involved in cardiac diseases by regulating the inflammatory response. The involvement of inflammatory cells has also been observed in doxorubicin (DOX)-induced cardiac injury. This study aimed to investigate whether IL-12p35 KO affects DOX-induced cardiac injury and the underlying mechanisms.MethodsFirst, the effect of DOX treatment on cardiac IL-12p35 expression was assessed. In addition, to investigate the effect of IL-12p35 KO on DOX-induced cardiac injury, IL-12p35 KO mice were treated with DOX. Because IL-12p35 is the mutual subunit of IL-12 and IL-35, to determine the cytokine that mediates the effect of IL-12p35 KO on DOX-induced cardiac injury, mice were given phosphate-buffered saline (PBS), mouse recombinant IL-12 (rIL-12) or rIL-35 before treatment with DOX.ResultsDOX treatment significantly increased the level of cardiac IL-12p35 expression. In addition, IL-12p35 KO mice exhibited higher serum and heart lactate dehydrogenase levels, higher serum and heart creatine kinase myocardial bound levels, and greater cardiac dysfunction than DOX-treated mice. Furthermore, IL-12p35 KO further increased M1 macrophage and decreased M2 macrophage differentiation, aggravated the imbalance of oxidants and antioxidants, and further activated the mitochondrial apoptotic pathway and endoplasmic reticulum stress autophagy pathway. Both rIL-12 and rIL-35 protected against DOX-induced cardiac injury by alleviating the inflammatory response, oxidative stress, apoptosis and autophagy.ConclusionsIL-12p35 KO aggravated DOX-induced cardiac injury by amplifying the levels of inflammation, oxidative stress, apoptosis and autophagy. (234 words).

α-Bisabolol abrogates isoproterenol-induced myocardial infarction by inhibiting mitochondrial dysfunction and intrinsic pathway of apoptosis in rats.

Mitochondrial dysfunction plays crucial role in the pathologenesis of myocardial infarction (MI). The present study evaluated the protective effect of α-bisabolol against isoproterenol (ISO)-induced mitochondrial dysfunction and apoptosis in rats. Male albino Wistar rats were pre- and co-treated with intraperitoneal injection of α-bisabolol (25mg/kg body weight) daily for 10days. To induce experimental MI, ISO (85mg/kg body weight) was injected subcutaneously to the rats at an interval of 24h for 2days (9th and 10thday). ISO-induced MI was indicated by the decreased activities of heart creatine kinase and lactate dehydrogenase in rats. ISO administration also enhanced the concentrations of heart mitochondrial lipid peroxidation products and decreased the activities/concentrations of mitochondrial antioxidants, Kreb's cycle dehydrogenases and mitochondrial electron transport chain complexes I, II + III and IV in rats. Furthermore, ISO triggers calcium overload and ATP depletion in the rat's heart mitochondria followed by the mitochondrial cytochrome-C release and the activation of intrinsic pathway of apoptosis by upregulating the myocardial pro-apoptotic Bax, P53, APAF-1, active caspase-3, active caspase-9 and down regulating the expressions of anti-apoptotic Bcl-2. α-Bisabolol pre and co-treatment showed considerable protective effects on all the biochemical and molecular parameters studied. Transmission electron microscopic study and mitochondrial swelling assay confirmed our biochemical and molecular findings. The in vitro study on hydroxyl radical also revealed the potent free radical scavenging activity of α-bisabolol. Thus, α-bisabolol attenuates mitochondrial dysfunction and intrinsic pathway of apoptosis in ISO-induced myocardial infarcted rats.

Marcadores psicológico, fisiológico e bioquímico para determinação dos efeitos da carga de treino e do overtraining

Athletic training is a process aimed at breaking the internal balance of the human organism and thus to improve the athlete’s performance. However, training stress may result in negative responses such as overtraining syndrome. This maladaptation can be avoided by monitoring the effects of training using psychological, physiological and biochemical variables. No consensus exists regarding the efficiency of some markers, but studies are unanimous that s single marker is unable to monitor and prevent this syndrome. Further studies are necessary to clarify doubts and to broaden the knowledge about this topic, which is of marked interest to researchers involved in high performance sports. Thus, the purpose of this review of the speciali-zed literature was to discuss how psychological, physiological and biochemical markers, such as mood state, overtraining score, resting heart rate variability and creatine kinase, are associated with training load and overtraining.

Pretreatment with a combination of quercetin and α-tocopherol ameliorates adenosine triphosphatases and lysosomal enzymes in myocardial infarcted rats

Aims Membrane bound adenosine triphosphatases (ATPases) and lysosomal enzymes play an important role in the pathology of myocardial infarction. This study was aimed to evaluate the combined preventive effects of quercetin and α-tocopherol on membrane bound ATPases and lysosomal enzymes in isoproterenol induced myocardial infarcted rats. Main methods Male Wistar rats were pretreated with a combination of quercetin (10 mg/kg) and α-tocopherol (10 mg/kg) daily for 14 days. After the pretreatment period, isoproterenol (100 mg/kg) was injected to rats at an interval of 24 h for two days to induce myocardial infarction. The activities of ATPases and lysosomal enzymes were assayed. Key findings Isoproterenol treated rats showed decreased levels of heart creatine kinase and lactate dehydrogenase. The activity of sodium potassium adenosine triphosphatase was decreased and the activities of magnesium adenosine triphosphatase and calcium adenosine triphosphatase were increased in isoproterenol treated rats. Also, the activities of β-glucuronidase, β-N-acetylglucosaminidase, β-galactosidase, cathepsin-B and D were increased (serum and heart), but the activities of β-glucuronidase and cathepsin-D were decreased in lysosomal fraction and increased in cytosolic fraction of the heart in isoproterenol treated rats. Furthermore, the heart lipid peroxidation products were increased in isoproterenol treated rats. Combined pretreatment with quercetin and α-tocopherol to isoproterenol treated rats normalized all the biochemical parameters studied. The observed effects are due to their membrane stabilizing property and this property might be due to decreased lipid peroxidation. Significance Our study demonstrated that combined pretreatment was better than single pretreatment. This study may have significant impact on myocardial infarcted patients.

Protective Effect of Piper longum Fruits against Experimental Myocardial Oxidative Stress Induced Injury in Rats

The present study investigated the protective effect of methanolic extract of fruits of Piper longum L. (PLM) on isoproterenol (ISO) induced myocardial infarction in rats. PLM was administered to Wistar albino rats in two different doses, by gastric gavage for 28 days. Myocardial infarction was induced by subcutaneous administration of ISO for 2 days at the end of the dosing period. ISO administration resulted in significant decrease in the activities of marker enzymes aspartate transaminase (AST), alanine transaminase (ALT), lactate dehydrogenase (LDH) and creatine kinase (CK) in heart with a concomitant increase in their activities in serum. A significant increase in lipid peroxide levels (TBARS) in hearts of ISO-induced rats was also observed. Activities of myocardial antioxidant enzymes like catalase (CAT), superoxide dismutase (SOD), glutathione peroxidase (GPOX) and glutathione reductase (GR) along with reduced glutathione (GSH) were significantly lowered owing to myocardial infarction in ISO treated rats. PLM pretreatment was found to ameliorate the effect of ISO on lipid peroxide formation and retained activities of marker enzymes. It also prevented ISO-induced decrease in antioxidant enzymes in the heart. The histopathological studies of heart revealed a protective role PLM in ISO treated rats. The results exhibited that the pretreatment with methanolic extract of Piper longum fruits may be useful in preventing the damage induced by ISO in rat heart.

Preventive effect of S-allylcysteine on lipid peroxides and antioxidants in normal and isoproterenol-induced cardiotoxicity in rats: A histopathological study

The consumption of diets rich in plant foods are associated with a reduced risk of cardiovascular diseases. This study was aimed to evaluate the role of S-allylcysteine (SAC) in isoproterenol (ISO)-induced myocardial infarction (MI) in rats. Subcutaneous injection of ISO (150 mg/kg) to Wistar rats showed a significant decrease in the activities of marker enzymes such as creatine kinase, lactate dehydrogenase, aspartate and alanine transaminases in heart and a significant increase in the levels of thiobarbituric acid reactive substances and lipid hydroperoxides in plasma and heart. ISO-induced rats also showed a significant decrease in the activities of superoxide dismutase, catalase, glutathione peroxidase, glutathione reductase and glutathione S-transferase in heart and the levels of glutathione and ascorbic acid in plasma and heart. Oral administration of SAC (100 and 150 mg/kg) to ISO-treated rats daily for a period of 45 days caused a significant increase in the activities of marker enzymes and improved the antioxidant status by decreasing lipid peroxidative products and increasing the activities of antioxidant enzymes and the levels of nonenzyomic antioxidants. Administration of SAC to normal rats did not show any significant effect. Histopathological findings of the myocardial tissue showed a protective role of SAC in ISO-treated rats. The effect at a dose of 150 mg/kg of SAC was more pronounced than that of the dose 100 mg/kg and brought back all the parameters to near normal. The effect exerted by 100 mg/kg of SAC was similar to that of α-tocopherol (60 mg/kg). The results of our study show that SAC possesses antioxidant activity in ISO-induced experimental MI.

Kinetic analysis of multi enzyme systems: A case study of the closed system of creatine kinase, hexokinase and glucose 6-phosphate dehydrogenase

This paper describes a general methodology to handle closed multi enzyme systems using mixture of symbolic (which depends on the Grobner Basis technique) and numerical computation methods. The applicability of the proposed method has been examined for the closed three-enzyme system of rabbit heart creatine kinase (EC 2.7.3.2), yeast hexokinase (EC 2.7.1.1) and human erythrocyte glucose 6-phosphate dehydrogenase (EC 1.1.1.49) using experimental data.

Developmental changes in regulation of mitochondrial respiration by ADP and creatine in rat heart in vivo.

In saponin-skinned muscle fibers from adult rat heart and m. soleus the apparent affinity of the mitochondrial oxidative phosphorylation system for ADP (Km = 200-400 microM) is much lower than in isolated mitochondria (Km = 10-20 microM). This suggests a limited permeability of the outer mitochondrial membrane (OMM) to adenine nucleotides in slow-twitch muscle cells. We have studied the postnatal changes in the affinity of mitochondrial respiration for ADP, in relation to morphological alterations and expression of mitochondrial creatine kinase (mi-CK) in rat heart in vivo. Analysis of respiration of skinned fibers revealed a gradual decrease in the apparent affinity of mitochondria to ADP throughout 6 weeks post partum that indicates the development of mechanism which increasingly limits the access of ADP to mitochondria. The expression of mi-CK started between the 1st and 2nd weeks and reached the adult levels after 6 weeks. This process was associated with increases in creatine-activated respiration and affinity of oxidative phosphorylation to ADP thus reflecting the progressive coupling of mi-CK to adenine nucleotide translocase. Laser confocal microscopy revealed significant changes in rearrangement of mitochondria in cardiac cells: while the mitochondria of variable shape and size appeared to be random-clustered in the cardiomyocytes of 1 day old rat, they formed a fine network between the myofibrils by the age of 3 weeks. These results allow to conclude that in early period of development, i.e. within 2-3 weeks, the diffusion of ADP to mitochondria becomes progressively restricted, that appears to be related to significant structural rearrangements such as formation of the mitochondrial network. Later (after 3 weeks) the control shifts to mi-CK, which by coupling to adenine nucleotide translocase, allows to maximally activate the processes of oxidative phosphorylation despite limited access of ADP through the OMM.

Different changes in the cytosole creatine kinase isoenzymes from heart of adult and old rats during stress.

We have shown that immobilization stress causes changes in the spectrum of the heart's cytosole creatine kinase isoenzymes. In myocardium adult immobilized rats, MM isoenzyme is decreased and MB and BB isoenzymes are increased compared to intact animals. The pattern of isoenzyme response in old and adult rats was different. Immobilization of old rat caused an increase of MM and a decrease of MB isoenzyme in the heart's creatine kinase isoenzyme spectrum. We suggest that this change in the creatine kinase isoenzyme spectrum in the myocardium of adult rats plays an adaptive role, increasing the effectiveness of its performance during stress. In old rats, apparent changes in the creatine kinase isoenzyme spectrum promote a disturbance of myocardial energy metabolism.

PH and temperature effects on kinetics of creatine kinase in aqueous solution and in isovolumic perfused heart. A 31P nuclear magnetization transfer study

Phosphorylated metabolites concentrations and creatine kinase kinetics are measured by 31P NMR in solution and in isovolumic perfused rat hearts submitted to hypo- and hyperthermia as well as to acidosis (37 degrees C). In the organ, temperature variation from 40 to 25 degrees C induces an increase of phosphocreatine (PCr) stores, a decrease of Pi and ADP concentrations, but does not affect the ATP pool. Creatine kinase forward flux (Vfor) is gradually reduced when the temperature is lowered both in vitro and in perfused heart. In normothermic and hypothermic conditions, a relationship is found between cardiac performance (rate pressure product, RPP), Vfor and ATP synthesis estimated through the myocardial oxygen consumption rate (MVO2). At 40 degrees C however, the RPP is reduced although both Vfor and MVO2 increase. In vitro experiments show an optimum pH of 7.7 for the forward creatine kinase reaction. In perfused heart submitted to acidosis, a decrease of PCr concentration is observed, whereas ATP and ADP contents remain unchanged. Heart creatine kinase flux increased as in hyperthermia. These high fluxes are attributed to the coupling of the creatine kinase reaction with energy consuming or producing reactions: the increase of energy demand related to non-contractile processes could explain the high MVO2 and Vfor observed in those conditions.

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.

Improved quantification with validation of multiple mRNA species by polymerase chain reaction: application to human myocardial creatine kinase M and B

Quantitation by polymerase chain reaction (PCR) has not been validated by an independent assay and is not easily applicable to multiple samples. The aim of this study was therefore to attempt to quantify human heart creatine kinase M (CKM) and B (CKB) mRNA simultaneously and to validate the method with a quantitative S1 nuclease protection assay. Conditions were optimised to achieve high efficiency of reverse transcription and PCR amplification of a short target sequence using total human heart RNA and an in vitro transcribed RNA standard as a substrate. A nested radiolabelled primer was used for specific detection and quantification of the amplified DNA sequence. The amplification efficiency for CKM and CKB mRNA were 0.98 (SD 0.07) and 1.11 (0.04) respectively. CKM and CKB mRNA levels were determined in 42 samples from 24 human hearts and found to be 156.7(36.2) and 19.9(5.2) amol.microgram-1 RNA, respectively. Parallel quantitative S1 nuclease protection assay yielded results of 131.2(64.2) and 9.4(5.2) amol.microgram-1 RNA. The cardiac CKB, but not CKM mRNA level, was twofold higher using the quantitative PCR method. However this discrepancy was abolished when compared to a higher stringency S1 nuclease protection assay. The cardiac CKB mRNA was 12.7% of the CKM level. This proportion remained the same from hearts with end stage cardiomyopathies of various aetiologies. This validated quantitative PCR method offers advantages over the S1 nuclease protection assay in that less RNA is required, the procedure is less dependent on RNA integrity and secondary structure, and multiple RNA species can be quantified simultaneously. The results also suggest that the abundance of the CKM and CKB mRNA level are coordinately regulated in the human heart.

Experience with phase extension and ab initio phase determination in macromolecular crystallography using maximum-entropy methods.

Three procedures, or 'tools', have been developed and tested for applying maximum-entropy methods to phase extension and to ab initio phase determination. The phase expander tool has been used in connection with the solution of two previously unknown macromolecular structures. An efficient algorithm for the determination of an electron-density distribution that is everywhere positive and that agrees with observed structure amplitudes (tools II and III) has been used to determine the phases of X-ray diffraction data from recombinant bovine chymosin, a protein with 323 amino-acid residues in the molecular chain, the structure of which was recently determined using replacement methods. By use of the same maximum-entropy methods, the structure amplitudes from the unknown structure of bovine heart creatine kinase, a protein with 381 amino-acid residues, have been phased ab initio to 2.7 A resolution. The phases of the centric reflections have also been confirmed by a satisfactory solution of the Patterson map of a mercury derivative. The current status of the structure interpretation is presented. This technique has also been applied to a test case where 48 centric reflections from bovine prothrombin fragment 1 data were phased ab initio and subsequently used in the determination of Patterson solutions for a heavy-atom derivative data set.

Maturational changes in respiratory control through creatine kinase in heart in vivo.

The role of creatine kinase in regulation of myocardial respiration was studied in vivo as a function of maturation. Unidirectional creatine kinase flux (JCK), phosphocreatine to gamma-ATP, was measured in newborn lambs (age 3-9 days, n = 8) and mature sheep (age 30-60 days, n = 6) using 31P saturation transfer techniques, and total creatine kinase activity was measured using standard methods. Myocardial oxygen consumption (MVO2) was measured simultaneously via an extracorporeal shunt from the coronary sinus as cardiac work was increased via epinephrine (1-3 micrograms.kg-1.min-1). Findings were as follows: 1) baseline newborn JCK was markedly lower than in mature sheep despite higher levels of MVO2, and this could be related to a decrease in total creatine kinase activity; 2) JCK was substantially higher than the rate of ATP synthesis in both groups at baseline rates of oxygen consumption; and 3) JCK decreased significantly in newborns during increases in MVO2, whereas there was no change in flux rate in the mature sheep during even larger relative changes in work and oxygen consumption. These data imply that creatine kinase does not limit oxidative phosphorylation. However, this enzyme system probably maintains at least an indirect role in respiratory control that is a function of the myocardial developmental state.

Geographic distribution of xanthine oxidase, free radical scavengers, creatine kinase, and lactate dehydrogenase enzyme systems in rat heart and skeletal muscle

The geographic distribution of the following enzyme systems is described in the rat heart (left and right ventricles) and in different skeletal muscles (soleus, plantaris, and red and white gastrocnemius): xanthine oxidase and dehydrogenase, creatine kinase isoenzymes, lactate dehydrogenase isoenzymes, and the free radical scavenger enzymes superoxide dismutase, glutathione reductase, and glutathione peroxidase. No substantial difference in enzyme activities was observed between the left and right ventricles. Skeletal muscles showed a clear distinction between enzyme activities depending on their composition of oxidative fibers and glycolytic fibers.

Perinatal development of coupled mitochondrial creatine kinase in rabbit heart

Perinatal development of coupled mitochondrial creatine kinase in rabbit heart

The heart creatine kinase system in cardiomyopathy

The heart creatine kinase system in cardiomyopathy

Investigation of the Stability of Bovine Heart Creatine Kinase

In order to stabilise an enzyme against irreversible thermoinactivation, it is important to know what mechanisms cause destabilization of the enzyme and what structural features actually contribute to the inherent stability of the enzyme. Features responsible for stabilising dimeric creatine kinase from bovine heart (EC 2.7.3.2.) were examined by initially modifying the enzyme with crosslinking and other specific group modifying reagents. Crosslinking the enzyme with dimethyl suberirnidate (2rng DMS/mg enzyme in 0.1M phosphate buffer, pH 8.0 for 3hr) resulted in a vastly increased thermostable enzyme at 55°C [l]. In this reaction, the positive charge of the original amino groups of creatine kinase are retained. However, crosslinking with N-hydroxysuccinimide esters [2] at a concentration of Img/mg of enzyme in 0.1M phosphate buffer, pH 10 (a process whereby the positive charge of the original amino groups is lost), resulted in a very dramatic loss of activity upon heating at 55°C. Similarly, when reductive alkylation of the enzyme was performed [3] using a p r o t e i n c o n c e n t r a t i o n of 1.6mg/ml, t h e resu l t ing carboxyrnethylated amino groups also gave rise to a destabilised enzyme at 5 5 ~ From these results it was obvious that the type of reagent used to modify creatine kinase was extremely important and could give valuable clues as to the identity of groups needed to maintain enzyme structure and to stabilise the protein. The role of lysine residues was therefore thought to play an important part in stabilising creatine kinase through electrostatic interactions. To determine if salt bridges were responsible for maintaining some degree of enzyme structure in creatine kinase, the effect of a concentrated inorganic salt, which is known to weaken electrostastic forces [4] on the thermal inactivation of the enzyme was examined. The rates of inactivation of the enzyme in the resence and absence of 1M KCL were 1.82min-' and 0.6minrespectively, at 55OC. This 3-fold difference in the rates implies that salt bridges are involved as stabilising forces in native creatine kinase and may explain why modification leading to the removal of the positive charge on the amino groups resulted in destabilisation of the enzyme. The second part of this work was concerned with determining whether irreversible inactivation of the enzyme was caused by conformational or covalent processes. The following approach was adopted [5]. Concentrated solutions of strong denaturants disrupt non-covalent interactions in proteins [6]. These agents should maintain the enzyme molecules in a highly unfolded form and thereby prevent formation of incorrectly folded structures. Hence, heating enzymes in the presence of denaturants should stabilise them against irreversible thermoinactivation due to conformational processes. This procedure was performed on creatine kinase (0.2mg/ml) at pH 4.0, 6.7 (Fig.1.) and 8.0. In all cases, the presence of 6M guanidine-HCI significantly reduced the rate of irreversible thermoinactivation at 55OC, indicating that conformational processes were involved and that incorrect structure formation was responsible for the inactivation of the enzyme. Further investigation revealed that the presence of copper ions ( 1 0 m M ) a t p H 6.7 acce lera ted t h e thermoinactivation of the enzyme. It is known that copper ions catalyse 0 2 oxidation of thiols [7] allowing disulphide bond formation to occur. It seems therefore, that this process may be P 4 0 2 4 6 8 10