Electrophoresis of reacting systems involving sulfhydryl oxidation of proteins

Numerical solution of the relevant continuity equations has been used to examine the possible effects of intramolecular sulfhydryl oxidation on the electrophoresis of proteins. Simulations of moving boundary electrophoresis, based on variants of a previous model [J. R. Cann, N. H. Fink, and D. J. Winzor (1983) Arch. Biochem. Biophys.221, 57–63], show that the Schlieren patterns for the ascending and descending limbs are likely to exhibit pronounced nonenantiography. Whereas the pattern for one limb may comprise essentially a single peak, that for the conjugate side can exhibit bimodality, the nature of which is time dependent. Bimodality of the Schlieren pattern can develop in either the ascending or descending limb of the electrophoresis cell, depending basically upon the number of sulfhydryl groups available for oxidation, and on the relative magnitudes of the rate constants describing the oxidation and the isomerization of the oxidized protein species. Whether the faster-moving or slower-moving peak grows with time is shown to depend upon the magnitude of the electrophoretic mobility of the resultant isomer in relation to that of the oxidized protein species. Schlieren patterns for fish muscle creatine kinase and rabbit muscle aldolase are then used to support the relevance of these predictions to moving boundary electrophoresis of proteins undergoing intramolecular sulfhydryl oxidation. Finally, numerical simulation of the zonal electrophoretic behavior of such systems serves to illustrate that bimodal patterns may also obtain, thereby giving a false impression of inherent protein heterogeneity. Emphasis is therefore placed on the importance of maintaining an adequate concentration of reducing agent throughout the medium in which the protein migrates, a potential problem in polyacrylamide gel electrophoresis at neutral pH.     

Anomalous electrophoretic behavior of creatine kinase: A thiol-dependent isomeric system with migration subject to kinetic control

Moving boundary electrophoresis of creatine kinase in 0.1 I diethylbarbiturate buffer, pH 8.9, has yielded anomalous migration behavior that indicates intereonversion between two coexisting states of the enzyme at a rate comparable with the rate at which the two enzymic forms tend to separate by differential migration. Whereas a single, symmetrical boundary is observed in the ascending limb, distinct bimodality of the descending pattern is evident in electrophoresis of enzyme isolated from skeletal muscle of either rabbit or fish (Mugil cephalus): pronounced changes in the nature of this bimodality with time are observed in the case of fish muscle creatine kinase. The abnormal migration behavior is eliminated by inclusion of dithiothreitol in the electrophoresis medium or by covalent modification of the enzyme with 5,5′-dithiobis(2-nitrobenzoic acid). Velocity and equilibrium sedimentation studies have been used to identify the macromolecular event as an isomerization, and studies of sulfhydryl content to implicate either reversible sulfhydryl oxidation or thiol-disulfide interchange in the isomerization mechanism.     

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 isoenzymes in Torpedo californica: absence of the major brain isoenzyme from nicotinic acetylcholine receptor membranes

Creatine kinase, actin, and nu 1 are three proteins of Mr 43 000 associated with membranes from electric organ highly enriched in nicotinic acetylcholine receptor. High levels of creatine kinase are required to maintain adequate ATP levels, while actin may play a role in maintaining the synaptic cytoskeleton. Previous investigations have prompted the conclusion that postsynaptic specializations at the receptor-enriched membrane domains in electroplax contain the brain form of creatine kinase rather than the form of creatine kinase predominantly found in muscle. We have examined this conclusion by purifying Torpedo brain creatine kinase to virtual homogeneity in order to examine its immunochemical, molecular, and electrophoretic properties. On the basis of immunological cross-reactivity and isozyme analysis, the receptor-associated creatine kinase is identified to be of the muscle type. When the molecular characteristics of Torpedo brain and muscle creatine kinase are compared, the brain enzyme is positioned at a more basic pH during chromatofocusing and on two-dimensional gel electrophoresis (pI = 7.5-7.9). Furthermore, electrophoretic mobilities of the brain and muscle forms of creatine kinase differ in sodium dodecyl sulfate electrophoresis: the brain isozyme of creatine kinase has lower apparent molecular weight (Mr 41 000) when compared with the muscle enzyme (Mr 43 000). On the basis of the results of our current investigations, the hypothesis that the brain isozyme of creatine kinase is a component of the postsynaptic specializations of the Torpedo californica electroplax must be abandoned. Recent sequence data have established close homology between Torpedo and mammalian muscle creatine kinases. On the basis of electrophoretic criteria, our results indicate that a lower degree of homology exists between the brain isozymes.     

The sarcoplasmic proteins of white muscle of an antarctic hemoglobin-free fish, Champsocephalus gunnari.

1. The protein composition of the sarcoplasm of Champsocephalus gunnari white muscle has been examined by ultracentrifugation and starch-gel electrophoresis. 2. The extracts have been fractionated by several methods in order to compare them more closely to similar extracts of other fish species and to isolate creatine kinase and the parvalbumins IV and V. 3. The creatine kinase does not appear to differ from other fish creatine kinases. Both parvalbumins are also very similar to other parvalbumins except that they are more easily oxidized than all the parvalbumins described so far.     

Identification of a 43-kDa polypeptide associated with acetylcholine receptor-enriched membranes as MM creatine kinase

Creatine kinase isoenzymes from Torpedo californica electric organ, skeletal muscle, and brain were purified and characterized. Torpedo electric organ and skeletal muscle creatine kinase have identical apparent Mr, electrophoretic mobility, and cyanogen bromide fragments. The electrophoretic mobility of the Torpedo creatine kinase was anodal as compared to mammalian MM creatine kinase. No creatine kinase isoenzyme with an electrophoretic mobility similar to mammalian BB creatine kinase was seen in any of the Torpedo tissues examined. Hybridization studies demonstrate the Torpedo electric organ creatine kinase to be composed of identical subunits and capable of producing an enzymatically active heterodimer when combined with canine BB creatine kinase. Creatine kinase from sucrose gradient-purified Torpedo electric organ acetylcholine receptor-rich membranes has an electrophoretic mobility identical with the cytoplasmic isoenzyme and an apparent Mr identical with mammalian MM creatine kinase. Western blot analysis showed Torpedo electric organ skeletal muscle creatine kinase and acetylcholine receptor-enriched membrane creatine kinase reacted with antiserum specific for canine MM creatine kinase. NH2-terminal amino acid sequence determinations show considerable sequence homology between human MM, Torpedo electric organ, chicken MM, and porcine MM creatine kinase. The acetylcholine receptor-associated creatine kinase is, therefore, identical with the cytoplasmic form from the electric organ and is composed of M-subunits.     

Human ''creatine kinase conversion factor'' identified as a carboxypeptidase.

The effect of partially purified 'creatine kinase conversion factor' on rabbit muscle creatine kinase is shown to be that of a carboxypeptidase, removing the C-terminal lysine residue from both subunits. These changes fully explain the three-banded electrophoretic patterns of the partially and the fully modified rabbit and human enzymes. The factor also produces a similar electrophoretic pattern with haemoglobin A; comparison with the effects of carboxypeptidases A and B permits the inference that the C-terminal residues of both alpha- and beta-subunits are removed. Small synthetic peptides are poor or non-substrates. A low activity with hippuryl-L-lysine may be due to contamination of the preparation with carboxypeptidase N. The possibility has been excluded that the action of conversion factor on creatine kinase involves modification of the protein thiol groups. Mr, substrate-specificity, pH-activity profile and the effects of metal ions distinguish creatine kinase conversion factor from carboxypeptidases A, B and N. On the basis of this evidence it is proposed to give the conversion factor the provisional name of carboxypeptidase K.     

Purification of mitochondrial creatine kinase: Two interconvertible forms of the active enzyme

The purification of creatine kinase from beef heart mitochondria is described. The purified enzyme appears as a single band after electrophoresis on SDS gels. Electrophoresis on cellulose acetate followed by staining for creatine kinase activity shows two forms of the enzyme. The slower migrating (m-1) form upon concentration is converted to the more rapidly migrating form (m-2). The reverse conversion occurs if the m-2 is incubated with β-mercaptoethanol. These results are consistent with a reversible oxidation of protein sulfhydryl group (s).     

Modification of contractile proteins by oxygen free radicals in rat heart

This study was undertaken to investigate the effects of oxygen free radicals on myofibrillar creatine kinase activity. Isolated rat heart myofibrils were incubated with xanthine+xanthine oxidase (a superoxide anion radical-generating system) or hydrogen peroxide and assayed for creatine kinase activity. To clarify the involvement of changes in sulfhydryl groups in causing alterations in myofibrillar creatine kinase activity, 1) effects of N-ethylmaleimide (sulfhydryl groups reagent) on myofibrillar creatine kinase activity, 2) effect of oxygen free radicals on myofibrillar sulfhydryl groups content, and 3) protective effects of dithiothreitol (sulfhydryl groups-reducing agent) on the changes in myofibrillar creatine kinase activity due to oxygen free radicals were also studied. Xanthine+xanthine oxidase inhibited creatine kinase activity both in a time-and a concentration-dependent manner. Superoxide dismutase (SOD) showed a protective effect on the depression in creatine kinase activity caused by xanthine+xanthine oxidase. Hydrogen peroxide inhibited creatine kinase activity in a concentration-dependent manner; this inhibition was prevented by the addition of catalase. N-ethylmaleimide reduced creatine kinase activity in a dose-dependent manner. The content of myofibrillar sulfhydryl groups was decreased by xanthine+xanthine oxidase; this reduction was protected by SOD. Furthermore, the depression in myofibrillar creatine kinase activity by xanthine+xanthine oxidase was protected by the addition of dithiothreitol. Oxygen free radicals may inhibit myofibrillar creatine kinase activity by modifying sulfhydryl groups in the enzyme protein. The reduction of myofibrillar creatine kinase activity may lead to a disturbance of energy utilization in the heart and may contribute to cardiac dysfunction due to oxygen free radicals.     

Proteomic analysis of slow- and fast-twitch skeletal muscles

Skeletal muscles are composed of slow- and fast-twitch muscle fibers, which have high potential in aerobic and anaerobic ATP production, respectively. To investigate the molecular basis of the difference in their functions, we examined protein profiles of skeletal muscles using sodium dodecyl sulfate-polyacrylamide gel electrophoresis and two-dimensional gel electrophoresis with pH 4-7 and 6-11 isoelectric focusing gels. A comparison between rat soleus and extensol digitorum longus (EDL) muscles that are predominantly slow- and fast-twitch fibers, respectively, showed that the EDL muscle had higher levels of glycogen phosphorylase, most glycolytic enzymes, glycerol 3-phosphate dehydrogenase, and creatine kinase; while the soleus muscle had higher levels of myoglobin, TCA cycle enzymes, electron transfer flavoprotein, and carbonic anhydrase III. The two muscles also expressed different isoforms of contractile proteins including myosin heavy and light chains. These protein patterns were further compared with those of red and white gastrochnemius as well as red and white quadriceps muscles. It was found that metabolic enzymes showed a concerted regulation dependent on muscle fiber types. On the other hand, expression of contractile proteins seemed to be independent of the metabolic characteristics of muscle fibers. These results suggest that metabolic enzymes and contractile proteins show different expression patterns in skeletal muscles.     

Multiple disc gel bands of mitochondrial creatine and adenylate kinases

Abstract— The activities and electrophoretic patterns of creatine and adenylate kinases in the mitochondrial and high speed supernatant fractions of adult mouse brain were determined. Approximately 22 per cent of the activities of both kinases is firmly bound to the mitochondria. On acrylamide gel electrophoresis of creatine kinase, in addition to the major band previously described, there were several other bands found. Although present in both the mitochondrial and supernatant fractions these additional protein bands with creatine kinase activity were significantly more intense in the mitochondrial fraction. There was only onesecondary band of adenylate kinase activity in the mitochondrial fraction but additional bands were found in the soluble fraction.     

The amino acid sequence of the peptide containing the thiol group of creatine kinase from normal and dystrophic chicken breast muscle. Comparison of some of the immunological properties of the antibodies developed in rabbits against these enzymes

The major (14)C-labelled peptides from creatine kinase from normal and dystrophic chicken muscle obtained by carboxymethylating the reactive thiol groups with iodo[2-(14)C]acetic acid and digestion with trypsin were purified by ion-exchange chromatography on Dowex-50 (X2) and by paper electrophoresis. The chromatographic characteristics of the (14)C-labelled peptides, their electrophoretic mobilities at pH6.5, and their amino acid compositions were identical for the two enzymes. The sequence of amino acids around the essential thiol groups of creatine kinase from normal and dystrophic chicken muscle was shown to be Ile-Leu-Thr-CmCys-Pro-Ser-Asn-Leu-Gly-Thr-Gly-Leu-Arg (CmCys, carboxymethylcysteine). This sequence is almost identical with that for the creatine kinases in human and ox muscle and bovine brain and is very similar to that of arginine kinase from lobster muscle. Antibodies to the enzymes were raised in rabbits and their reaction with the creatine kinase from normal and dystrophic muscles in interfacial, immunodiffusion and immunoelectrophoretic experiments was studied. The cross-reaction between normal muscle creatine kinase and antisera against the dystrophic muscle enzyme (or vice versa) observed by immunodiffusion and by immunoelectrophoretic experiments further suggests that the enzymes from normal and dystrophic chicken muscle are similar in structure. The results of the present study, the identical amino acid sequence of the peptides containing the reactive thiol group from both the normal and dystrophic chicken muscle enzymes and the immunological similarities of the two enzymes are in accord with the similarity of the two enzymes observed by Roy et al. (1970).     

Purification and localization of brain-type creatine kinase in sodium chloride transporting epithelia of the spiny dogfish, Squalus acanthias.

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.     

Geographic variation of muscle adenylate kinase in the loach Misgurnus anguillicaudatus

The electrophoretic pattern of the loach muscle adenylate kinase was composed of one or two major bands. Each major band was preceded by two minor bands. Three codominant alleles were postulated to segregate in loach. Each allele coded for one major band with different mobility.
Adenylate kinase (AK. E.C. 2.7.4.3.) catalyses the reaction 2ADP ATP + AMP. and is known as a heat stable protein constituent of skeletal muscle. Electrophoretic variation of AK has been reported in the pika Ochotona r. rufescens (Vergnes et al. 1974). the teleostean fish Zoarces viviparus (Frydenberg & Si-monsen, 1973), the mussel Mvtilus edulis (Ahmad et al. 1977). and the tunicate Ciona intestinalis (Schmidtke & Engel. 1980). In this note, individual variation of AK in muscle extracts of the fresh-water fish Misgurnus anguillicaudatus is described.
Loach were collected in ponds or purchased from fish shops (Table 1 & Fig. 1). Three populations (OS. AS and KN) were purchased from fish shops in Osaka. Akashi and Kanazawa cities, respectively. Their exact sampling locations were not known. For reference, the locations of these cities are indicated by an open circle in Fig. i. Fish were collected and stored frozen at – 20 ^oC at the sampling time given in Table 1. Muscle extracts were prepared and examined in the period February-April 1981.The method for preparing muscle extract and-the starch gel electrophoretic procedures were the same as those reported previously (Kimura, 1976). The amine-citrate buffer system as described by Clayton & Tretiak (1972) was used. AK was stained by the method of Allendorf et al. (1977). After electrophoresis, an inhibition test was also performed by immersing the gel in 10^-3 M 5.5'-dithiobis-(2-nitrobenzoate) solution for 30 minutes at room temperature.
Under the electrophoretic condition used in the present study, all of the AK     

Decrease in heart mitochondrial creatine kinase activity due to oxygen free radicals.

This study was undertaken to examine the effects of oxygen free radicals on mitochondrial creatine kinase activity in rat heart. Xanthine plus xanthine oxidase (superoxide anion radical generating system) reduced mitochondrial creatine kinase activity both in a dose- and a time-dependent manner. Superoxide dismutase showed a protective effect on depression in creatine kinase activity due to xanthine plus xanthine oxidase. Hydrogen peroxide inhibited creatine kinase activity in a dose-dependent manner, this inhibition was protected by the addition of catalase. In order to understand the detailed mechanisms by which oxygen free radicals inhibit mitochondrial creatine kinase activity, the effects of oxygen free radicals on mitochondrial sulfhydryl groups were examined. Mitochondrial sulfhydryl groups contents were decreased by xanthine plus xanthine oxidase or hydrogen peroxide; this depression in sulfhydryl groups contents was prevented by the addition of superoxide dismutase or catalase. N-Ethylmaleimide (sulfhydryl group reagent) expressed inhibitory effects on the creatine kinase activity both in a dose- and a time-dependent manner; dithiothreitol or cysteine (sulfhydryl group reductant) showed protective effects on the creatine kinase activity depression induced by N-ethylmaleimide. Dithiothreitol or cysteine also blocked the depression of mitochondrial creatine kinase activity caused by xanthine plus xanthine oxidase or hydrogen peroxide. These results lead us to conclude that oxygen free radicals may inhibit mitochondrial creatine kinase activity by modifying sulfhydryl groups in the enzyme protein.     

Purification and characterization of creatine kinase isozymes from the nurse shark Ginglymostoma cirratum

Creatine kinase from nurse shark brain and muscle has been purified to apparent homogeneity. In contrast to creatine kinases from most other vertebrate species, the muscle isozyme and the brain isozyme from nurse shark migrate closely in electrophoresis and, unusually, the muscle isozyme is anodal to the brain isozyme. The isoelectric points are 5.3 and 6.2 for the muscle and brain isozymes, respectively. The purified brain preparation also contains a second active protein with pI 6.0. The amino acid content of the muscle isozyme is compared with other isozymes of creatine kinase using the Metzger Difference Index as an estimation of compositional relatedness. All comparisons show a high degree of compositional similarity including arginine kinase from lobster muscle. The muscle isozyme is marginally more resistant to temperature inactivation than the brain isozyme; the muscle protein does not exhibit unusual stability towards high concentrations of urea. Kinetic analysis of the muscle isozyme reveals Michaelis constants of 1.6 mM MgATP, 12 mM creatine, 1.2 mM MgADP and 50 mM creatine phosphate. Dissociation constants for the same substrate from the binary and ternary enzyme-substrate complex do not differ significantly, indicating limited cooperatively in substrate binding. Enzyme activity is inhibited by small planar anions, most severely by nitrate. Shark muscle creatine kinase hybridizes in vitro with rabbit muscle or monkey brain creatine kinase; shark brain isozyme hybridizes with monkey brain or rabbit brain creatine kinase. Shark muscle and shark brain isozymes, under a wide range of conditions, failed to produce a detectable hybrid.     

Multiple forms of human phosphoglycerate kinase.

Phosphoglycerate kinase was isolated by affinity chromatography from human skeletal muscle and erythrocytes. As in the tissue extracts, the purified enzyme showed in Cellogel electrophoresis one major and two minor bands with phosphoglycerate kinase activity. The multiple forms were separated by chromatography on CM-Sepharose. From the three separated forms, A, B, and C, the latter was not detectable in electrophoresis of tissue extracts or in the purified unresolved phosphoglycerate kinase. The faintest, most anodically migrating form observed in the tissue extracts could not be isolated in pure form by chromatography on CM-Sepharose. The electrophoretic mobility of the phosphoglycerate kinase forms depended strongly on the buffer systems used. The different forms had identical molecular weight, substrate affinity, and heat stability and were inhibited to the same extent by antibody. They could also not be separated by column affinity chromatography. Small differences were found in thiol group content and in the specific activity, the latter being a consequence of diminished free sulfhydryl residues. Exposure to either reductive or oxidative conditions changed the specific activity, but did not result in interconversion among the pure forms. The multiple forms probably arise as a result of epigenetic factors occurring after the primary polypeptide chain has been synthesized.     

Creatine kinase isoenzyme associated with synaptosomal membrane and synaptic vesicles

Brain creatine kinase is principally of soluble cytoplasmic origin (anodal electrophoretic mobility). However, synaptosomal membranes and synaptic vesicles are enriched in an isoenzyme electrophoretically similar to muscle type creatine kinase (cathodal electrophoretic mobility), but which can be distinguished from muscle type by other means.     

Purification and Characterization of Sperm Creatine Kinase and Guanylate Cyclase of the Sea Urchin Hemicentrotus pulcherrimus

Creatine kinase and guanylate cyclase were purified from Hemicentrotus pulcherrimus spermatozoa. The molecular weight of the purified sperm tail creatine kinase was estimated to be 137,000 by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Sperm tail guanylate cyclase was purified by chromatography on a WGA-Sepharose column connected to a Concanavalin A-Sepharose column, and a Superose 12 HR column. The molecular weight of the tail guanylate cyclase was estimated to be 128,000 by SDS-PAGE. The specific activity of the purified enzyme was 8.25 μmol of cGMP formed/min/mg protein. Sperm-activating peptide I (SAP-I) causes an electrophoretic mobility shift of H. pulcherrimus sperm guanylate cyclase from 131 kDa to 128 kDa. The 131 kDa form of guanylate cyclase was co-purified with a 76 kDa protein, whose molecular mass is similar to that of a SAP-I receptor. The purified 131 kDa form of guanylate cyclase had higher activity than the 128 kDa form. The 131 kDa and 128 kDa forms of guanylate cyclase contained 23.83 ± 0.65 and 4.16 ± 0.45 moles of phosphate per mol protein (mean ± S.D.; n = 3), respectively. The activities of guanylate cyclase and creatine kinase increased during the testis development. During spermatogenesis, sperm tail creatine kinase was detected immunohistochemically only in mature spermatozoa.     

Effects of free radicals on cytosolic creatine kinase activities and protection by antioxidant enzymes and sulfhydryl compounds

The main purpose of this study was to investigate the effect of free radicals and experimental diabetes on cytosolic creatine kinase activity in rat heart, muscle and brain. Hydrogen peroxide decreased creatine kinase activity in a dose dependent manner which was reversed by catalase. Xanthine/xanthine oxidase, which produces superoxide anion, lowered the creatine kinase activity in the same manner whose effect was protected by superoxide dismutase. N-acetylcysteine and dithiothreitol also significantly ameliorated the effect of Xanthine/xanthine oxidase and hydrogen peroxide. Experimental diabetes of twenty-one days (induced by alloxan), also caused a similar decrease in the activity of creatine kinase. This led us to the conclusion that the decrease in creatine kinase activity during diabetes could be due to the production of reactive oxygen species. The free radical effect could be on the sulfhydryl groups of the enzyme at the active sites, since addition of sulfhydryl groups like N-acetylcysteine and dithiothreitol showed a significant reversal effect.