Creatine Phosphotransferase Research Articles

Determination of total creatine kinase activity in blood serum using an amperometric biosensor based on glucose oxidase and hexokinase

Creatine kinase (CK: adenosine-5-triphosphate–creatine phosphotransferase) is an important enzyme of muscle cells; the presence of a large amount of the enzyme in blood serum is a biomarker of muscular injuries, such as acute myocardial infarction. This work describes a bi-enzyme (glucose oxidase and hexokinase based) biosensor for rapid and convenient determination of CK activity by measuring the rate of ATP production by this enzyme. Simultaneously the biosensor determines glucose concentration in the sample. Platinum disk electrodes were used as amperometric transducers. Glucose oxidase and hexokinase were co-immobilized via cross-linking with BSA by glutaraldehyde and served as a biorecognition element of the biosensor. The biosensor work at different concentrations of CK substrates (ADP and creatine phosphate) was investigated; optimal concentration of ADP was 1mM, and creatine phosphate – 10mM. The reproducibility of the biosensor responses to glucose, ATP and CK during a day was tested (relative standard deviation of 15 responses to glucose was 2%, to ATP – 6%, to CK – 7–18% depending on concentration of the CK). Total time of CK analysis was 10min. The measurements of creatine kinase in blood serum samples were carried out (at 20-fold sample dilution). Twentyfold dilution of serum samples was chosen as optimal for CK determination. The biosensor could distinguish healthy and ill people and evaluate the level of CK increase. Thus, the biosensor can be used as a test-system for CK analysis in blood serum or serve as a component of multibiosensors for determination of important blood substances. Determination of activity of other kinases by the developed biosensor is also possible for research purposes.

Quantitative determination of creatine kinase release from herring ( Clupeaharengus) spermatozoa induced by tributyltin

Creatine kinase (CK, ATP creatine phosphotransferase, EC 2.7.3.2) is an enzyme participating in ATP regeneration, which is the primary source of energy in living organisms. We demonstrated that CK from herring spermatozoa has high activity (∼452 μmol/min/g of fresh semen) and has a different electrophoretic mobility from isoenzymes present in skeletal muscle. In our study, we investigated toxic effect of tributyltin (TBT) on herring spermatozoa using a specific sperm viability kit to observe live and dead sperm cells with a confocal microscope. Treatment of herring spermatozoa with TBT caused a time-dependent decrease of viability: 35% nonviable cells with 5 μM TBT and more than 90% nonviable cells with 10 μM TBT after 6 h exposure. We also monitored CK release from damaged spermatozoa into surrounding medium containing different concentrations of TBT. The higher concentration of TBT was used the more CK release from spermatozoa was observed. We suggest that CK could be a good biomarker of sperm cell membranes degradation in the case when lactate dehydrogenase release from permeabilized cells is not possible for rapid determination of the effect of TBT.

Macro CK type 1 as a major component of serum creatine kinase activity in pregnant sheep

Electrophoretic separation of creatine kinase (ATP: creatine phosphotransferase, E.C. 2.7.3.2) isoenzymes in agarose gel was made in the serum of 93 sheep of three Slovenian domestic breeds (Solcavska, Pramenka and Bovska breeds). 44 sheep were pregnant between 112 and 135 days at the time of the blood collection. The median value of serum total CK activity for all animals investigated was 82 U/l. After direct immunoinhibition of CK with anti-M-CK monoclonal antibodies the total CK activity remained the same (88 U/l, P = 0.354). There were significant differences among breeds in CK activity for the Solcavska (101 U/l), Bovska (89 U/l) and Pramenka breeds (73 U/l), respectively (Kruskal–Wallis one way analysis of variance, P < 0.01), and between pregnant (105 U/l) and non-pregnant animals (76 U/l), irrespective of the breed (Mann–Whitney rank sum test, P < 0.05). According to electrophoresis, all non-pregnant sheep had exclusively free CK-BB serum bands activity. In all pregnant sheep coupled dimeric BB variant appeared as macro-CK type 1 in the range between 80% and 100% of total CK activity. The present study confirms the existence of an elimination mechanism for CK from the plasma abundance free CK-BB enzyme.

Conformational Changes at the Active Site of Creatine Kinase During Denaturation of Urea

It has been previously reported that during the denaturation of creatine kinase (ATP: creatine phosphotransferase EC 2.7.3.2) by guanidine hydrochloride, urea or SDS, inactivation occurs before noticeable conformational change of the enzyme molecule as a whole can be detected, and the inactivation rate constants of the enzyme in the above denaturant solutions are several orders of magnitude faster than those of conformational

Innervation is required to stabilize and amplify creatine kinase activity in regenerated extensor digitorum longus muscles of rats

Neural control of creatine kinase (CK, adenosine 5′-triphosphate creatine phosphotransferase: EC 2.7.3.2) was investigated by measuring enzymatic activity and isoenzymatic representation of CK in extensor digitorum longus (EDL) muscles of adult rats during and after regeneration. Experimental models were ischemized EDLs, reversibly or permanently denervated. Results showed that, during regeneration, CK in muscle fibers was likewise modified in both reversibly and permanently denervated EDLs. After regeneration a clear dichotomy was observed between the regenerated EDLs which were innervated and recovering CK activity, and those in which innervation was prevented and were rapidly losing activity. Further to investigate the neural influence on CK turnover, merely denervated agematched EDLs were analysed and found to lose CK activity rapidly. The major conclusions are that during regeneration muscle CK is autonomously expressed, but following regeneration neural influence becomes an absolute requirement for stabilization and amplification of CK.

31P-saturation-transfer nuclear-magnetic-resonance measurements of phosphocreatine turnover in guinea-pig brain slices.

The technique of 31P saturation-transfer n.m.r. was used to determine the forward and the reverse rate constants of creatine phosphotransferase in superfused guinea-pig cerebral tissues in vitro. The calculated forward rate constant of 0.22 +/- 0.03s-1 compared well with a previously reported value for rat brain in vivo [Shoubridge, Briggs & Radda (1982) FEBS Lett. 140, 288-292]. The reverse rate constant was found to be 0.55 +/- 0.10s-1. 3. By using concentrations of ATP and phosphocreatine estimated previously for this superfused preparation [Cox, Morris, Feeney & Bachelard (1983) Biochem. J. 212, 365-370], forward and reverse flux rates were calculated to be 0.68 and 0.72 mumol X s-1 X g-1 respectively. The concordance of forward and reverse fluxes contrasts with the situation observed in vitro in other tissues, and suggests that the creatine phosphotransferase reaction is at equilibrium under the conditions used here. 4. Lowering the concentration of glucose in the superfusing medium from 10mM to 0.5mM had no significant effect on phosphocreatine concentration or on the forward (ATP-generating) flux through creatine phosphotransferase. The results indicate that a normal phosphocreatine content in the presence of lowered glucose availability is reflected by an unchanged turnover rate.

Creatine kinase isoenzymes in serum from cord blood and the blood of healthy full-term infants during the first three postnatal days.

Isoenzymes of creatine kinase (ATP:creatine phosphotransferase; EC 2.7.3.2; CK) were measured by electrophoresis in serum from cord blood and skin-puncture blood taken from 45 healthy full-term infants during the first three postnatal days. Mean total CK activities (in U/L at 30 degrees C) were 185 in cord samples, 536 in samples taken between 5--8 h postnatally, 494 between 24--33 h, and 288 in the 72-100 h samples. Values for all three isoenzymes increased to a peak over this period, with the highest values generally being found in the samples taken 5--33 h after birth; the subsequent decline was most rapid for CK-BB. Serum CK isoenzymes in cord samples and those taken at 72--100 h in the 11 babies delivered by cesarian section did not differ significantly from those of babies delivered vaginally. However the postnatal increases in total CK, CK-MM, and CK-MB (but not in CK-BB) were significantly greater in those patients born by vaginal delivery. The reasons for the increases in CK isoenzymes after birth are not clear, but our results and reported studies on the ontogeny of CK suggest that CK-MB cannot be regarded as a "cardiac-specific" isoenzyme in the neonatal period.

Effect of curare on the development of chicken embryo skeletal muscle in ovo

Injection of curare in ovo between days 7 and 14 of embryonic development delayed the differentiation of myoneural junctions, and greatly reduced the mass of pectoralis and leg muscles. Although the administration of curare was discontinued at 14 days of development, muscle paralysis persisted and prevented normal hatching at 21 days. The protein composition of muscles from curare-treated embryos was characterized by a decrease in myosin, Ca 2+ transport ATPase, and ATP: creatine phosphotransferase. No significant differences were found in the total Ca 2+ content of muscle, in the Ca 2+ uptake measured in whole muscle homogenates, and in muscle ultrastructure as evident from electron microscopy and histochemical analysis.

Synthesis of the calcium transport ATPase of sarcoplasmic reticulum and other muscle proteins during development of muscle cells in vivo and in vitro

The effect of medium Ca 2+ concentration upon the concentration and the rate of synthesis of muscle proteins was investigated in chicken pectoralis muscle cultures. There is an easily identifiable class of muscle protein which includes the Ca 2+-ATPase of sarcoplasmic reticulum, myosin, troponin C, ATP : creatine phosphotransferase, muscle specific actin, tropomyosin 1 and 2, and muscle hemagglutinin, which show a large increase in concentration during normal development. The increased synthesis of these proteins was inhibited, without inhibition of cell proliferation, in culture media of relatively low Ca 2+ concentration, 0.05–0.3 mM, where fusion was prevented. Similar medium Ca 2+ concentration was required for the expression of all these proteins, suggesting their coordinate regulation. The proteins are denoted as ‘calcium-modulated proteins’. The increased Ca 2+ transport activity of sarcoplasmic reticulum in cultured chicken pectoralis muscle cells during development at 1.8 mM medium calcium concentration represents de novo synthesis of the Ca 2+ transport ATPase, as shown by immunoprecipitation, active site labeling and direct identification of the Ca 2+ transport ATPase on two-dimensional gel electropherograms of whole muscle homogenates. The concentration and the turnover rate of the majority of the muscle proteins is not affected significantly by medium Ca 2+ concentration between 0.06 and 1.8 mM. It is proposed that increase in cytoplasmic free Ca 2+ concentration during fusion plays a central role in the regulation of the synthesis of calcium-modulated proteins.

ATP synthesis from ADP by the filtrate of washed rat platelet suspension through cellulose ester membrane: liberation of platelet creatine kinase.

Abstract The filtrate of washed rat platelet suspension through cellulose ester membrane was found to contain a soluble protein which possessed a potent activity to synthesize [ 14 C]ATP from added [ 14 C]ADP. This ATP synthetic activity was detectable even at low temperature, 2°C, stimulated by Mg 2+ or Ca 2+ , and was maximal at pH 8.6. Simultaneous production of AMP was not observed. Inhibitors and substrates of glycolysis had no effect, while creatine phosphate caused increase of the ATP synthesis. These results suggest the presence in platelets of potent creatine kinase (ATP: creatine phosphotransferase, EC 2.7.3.2) and the prompt leakage of this protein from the cells under the specified condition.

Cellulose acetate electrophoresis of creatine phosphokinase isoenzymes in the diagnosis of myocardial infarction.

A method for rapid separation and visualization of creatine phosphokinase (EC 2.7.3.2 ATP: creatine phosphotransferase) isoenzymes on cellulose acetate has been developed. The technic uses the Beckman microzone electrophoresis system, and is applicable to other cellulose acetate systems. Incubation is with a commercially available substrate solution, and the isoenzyme bands are visualized by fluorescence using a long-wavelength ultraviolet light. An intermediate migrating isoenzyme band (CPK-MB) was detected in sera of 67 of 68 patients with myocardial infarction, and was not present in patients with other disorders that might clinically be confused with myocardial infarction.

Serum enzyme and tissue changes in shaven rabbits exposed to cold

Shaven rabbits were exposed to −5 °C from 6 to 16 hr. With maximum exposure 50% became severely hypothermic, and the rest either became moderately hypothermic or remained normothermic. In severely hypothermic rabbits, marked elevations in serum concentrations of glutamic oxalacetic transaminase, glutamic pyruvic transaminase, aldolase, creatine phosphotransferase, and lactic dehydrogenase developed within 10–16 hr. Serum enzyme concentrations reached peak levels within 1 day after removal from cold and returned to normal within 6 days. Serum levels of isoenzyme lactic dehydrogenase 5 (SLDH 5) greatly increased and SLDH 1 decreased in severely hypothermic rabbits. This indicated that striated muscle tissue contributed to serum concentration of lactic dehydrogenase. About one-half of the rabbits showed foci of cardiac necrosis multiple pulmonary hemorrhages, and fatty changes in the liver, kidney, and striated and heart muscles. It is apparent that serum enzymes reached peak concentrations within the first day after cold exposure at a time when fatty changes and necrosis were found in the heart and other organs. The tissue changes were most frequent and severe in the hypothermic rabbits with the highest serum enzyme concentrations. Within 3–6 days fatty changes had largely disappeared, but the myocardial lesions persisted in the form of granulation and scar tissue.

Diagnostic efficacy of myocardial creatine phosphokinase using polyacrylamide disk gel electrophoresis.

A method for separating and quantitating the isoenzymes of creatine phosphokinase (EC2.7.3.2ATP: creatine phosphotransferase) using polyacrylamide disk gel electrophoresis is described. Electrophoresis of either tissue homogenates or serum yields clear, well-defined bands representing enzymic activity. Damage to either myocardium or skeletal muscle results in serum isoenzyme patterns similar to those obtained on homogenates of the tissues themselves. An intermediate “cardiac” band was found in specimens from 40 patients with myocardial infarcts documented by clinical, electrocardiographic and other laboratory evidence. In two of these patients the cardiac bands were transient and disappeared within 48 hours after the infarction occurred. The cardiac band was present also in specimens from patients wiuh carbon monoxide poisoning and those with malignant hyperthermia, probably as a result of myocardial damage in these conditions.

Characterization of the aspartate carbamoyltransferase subunit obtained from a multienzyme aggregate in the pyrimidine pathway of yeast. Activity and physical properties

The carbamoylphosphate synthase (ATP:carbamate phosphotransferase, dephosphorylating, EC 2.7.2.5)-aspartate carbamoyltransferase (ATP:creatine phosphotransferase, EC 2.1.3.2) aggregate of Saccharomyces cerevisiae was dissociated by heat and the resulting aspartate carbamoyltransferase subunit purified to homogeneity as determined by polyacrylamide gel electrophoresis and ultracentrifugation. The molecular weight of the subunit was estimated to be 138 000. Treatment with sodium dodecyl sulphate gave a single band on gel electrophoresis corresponding to a molecular weight of 21 000. The pH dependence and Michaelis constants for both substrates were determined and found to be identical with those parameters previously determined for the enzyme aggregate. Antibody to the enzyme aggregate caused the precipitation of aspartate carbamoyltransferase activity when it was reacted with either the aggregate or the subunit. The purified subunit, probably a hexamer, retains many of the properties associated with the intact multienzyme aggregate.

Multiple effects of ions on ATP: l-arginine and ATP: Creatine phosphotransferases

1. 1. Cations inactivate Sipunculus arginine kinase (mol. wt 84 000) (ATP: l-arginine phosphotransferase, EC 2.7.3.3). The order of increasing effectiveness is K +, NH 4 +, Li +, guanidinium cation, tetravalent spermine action. The action of lithium acetate and guanidine · HCl is expressed by a two-step inhibition: an initial immediate inhibition which is of the non-competitive type with respect to l-arginine and Mg 2+- ATP, and a slower inactivation. While the first stage can be observed at 10°C, the rate of the secondary one increases with temperature. This inhibition is similar to that observed with anions. The modification of the protein absorption spectrum which characterizes the action of anions is displayed with guanidine · HCl and spermine · 4HCl but not with K +, NH 4 +, Li +. 2. 2. The effects of KCl on Sipunculus arginine kinase in H 2O and in 2H 2O are qualitatively similar but quantitatively different. In 2H 2O the decrease of V by KCl is greater than in H 2O. However, the modification of the protein absorption spectrum is less sharp in 2H 2O than in H 2O. 3. 3. Rabbit muscle creatine kinase (ATP:creatine phosphotransferase, EC 2.7.3.2) shows a susceptibility towards anions and cations comparable to that of Sipunculus arginine kinase. The order of effectiveness of anions and cations on both enzymes is the same. The kinetic behaviour of the action of Cl − is identical for both enzymes. Nevertheless no modification of the protein absorbance spectrum is observed by the interaction of Cl −.

Purification and properties of adenosine triphosphate–creatine phosphotransferase from muscle of the dogfish Scylliorhinus canicula

1. Creatine kinase occurs in high concentration in the soluble proteins of dogfish muscle. A fourfold purification gives essentially pure enzyme but with a low specific activity. This appears to be a property of the native enzyme and not a result of the isolation procedures used. 2. The amino acid composition is similar to that of other phosphagen kinases, but the enzyme differs from mammalian creatine kinases in having four thiol groups readily reactive towards 5,5'-dithiobis-(2-nitrobenzoic acid). Titration of two thiol groups is accompanied by almost complete loss of activity. The remaining two thiol groups react at different rates, suggesting that modifying the third thiol group affects the reactivity of the fourth thiol group. 3. The enzyme is markedly protected against inactivation by iodoacetamide by MgATP or MgADP. Addition of creatine to MgADP decreases protection, but the further addition of Cl(-) restores protection to the original value. The quaternary MgADP-creatine-enzyme-nitrate complex protects very strongly as is found for the rabbit enzyme. The involvement of the conformational state of the enzyme in such effects is discussed. 4. Creatine kinase from both dogfish and rabbit is equally sensitive to urea denaturation. Urea protects the dogfish enzyme by about 9% against inhibition by iodoacetamide. 5. The formation of a hybrid between the dogfish and rabbit enzymes in vitro has been demonstrated. 6. At high substrate concentrations the dogfish enzyme shows apparent ordered kinetics. The effect of temperature on V(max.) and the Michaelis constants for MgATP and creatine were determined. These and changes in the apparent activation energy suggest that limited adaptation has occurred commensurate with physiological need.

Sequence of dodecapeptide containing active lysine from chicken ATP: Creatine phosphotransferase

The particularly reactive two ε-NH 2 groups of lysine in chicken ATP: creatine phosphotransferase were labeled with 1-dimethylaminonaphthalene -5- sulfonylchloride and subsequently the protein was hydrolysed with trypsin. A single peptide composed of 12 residues was isolated and its sequence was established (Leu-Leu-Val-Pro-Asp-Ser-Lys-Leu-Phe-Ser-Val-Arg) by using Edman degradation and also hydrolysis with carboxypeptidases A and B before and after exposure to α chymotrypsin. α chymotrypsin catalyses rupture of the peptide bond between phenylalanine and serine.

Inhibition of adenosine 5′-triphosphate–creatine phosphotransferase by substrate–anion complexes. Evidence for the transition-state organization of the catalytic site

1. The substrate combination creatine-MgADP does not significantly protect creatine kinase against inhibition by iodoacetamide in the absence of small anions. 2. Small anions can be divided into three groups according to the way in which they affect creatine kinase: I, acetate reversibly increases enzyme activity in the forward reaction but does not affect the rate of inhibition by iodoacetamide in the presence of creatine plus MgADP; II, planar anions and some halides (HCO(3) (-), HCO(2) (-), NO(3) (-), NO(2) (-), Cl(-), Br(-), F(-)) in the presence of creatine plus MgADP protect the enzyme from inhibition by iodoacetamide; III, tetrahedral anions (SO(4) (2-), HPO(4) (2-), ClO(4) (-), BF(4) (-)) and iodide do not affect the rate of inhibition by iodoacetamide in the presence of creatine plus MgADP but may decrease the protection by class II anions under these conditions. Anions of class II and class III also reversibly inhibit enzyme activity. 3. It is concluded that class II anions form a stable and inactive quaternary enzyme-creatine-MgADP-anion complex and this is responsible for the effect attributed by previous workers to the ternary complex lacking anion. Formation of this complex, particularly in the forward reaction, can lead to markedly non-linear enzyme progress curves. Some previous observations are re-appraised in the light of these findings. 4. From the behaviour of chloride and nitrate ions, and the marked lowering of the K(i) values for creatine and MgADP they produce, it is inferred that planar or monoatomic anions act in the quaternary complex by simulating the transferable phosphoryl group in the transition state (or another intermediate state) of the reaction. 5. It is suggested that, in the course of the reaction, the tetrahedral phosphate-binding site for the transferable phosphoryl group of the substrate (that also binds class II and class III anions) changes into a trigonal bipyramid site (also occupied by class II anions). This strains the phosphoryl group to adopt the transitional sp(3)d hybridized state and must contribute significantly to the low activation energy of the reaction. 6. Catalysis is deduced to proceed by an ;in line' transfer reaction and from the effects of class II anions it is possible to estimate the approximate dimensions of the anionic site in the transition-state complex. 7. The specific protecting effect of an equilibrium mixture of substrates against inhibition by iodoacetamide provides further evidence for the conformational change suggested above as a step in the catalytic process.

Brain adenosine 5'-triphosphate-creatine phosphotransferase.

1. The purification of creatine kinase (ATP-creatine phosphotransferase, EC 2.7.3.2) from ox brain by a method that is quicker, simpler and gives much higher yields than other published procedures is described. 2. Stoicheiometric inhibition studies with iodoacetate showed that the enzyme, like that from muscle, has two reactive thiol groups that are essential for enzyme activity. 3. The amino acid sequence around the essential thiol groups was determined and found to be virtually identical with that in creatine kinases from rabbit and ox muscle, and very similar to that found in arginine kinase; the evolutionary significance of this is discussed. 4. The identification of DNS-amino acids on thin layers of silica gel was found to have, in many cases, distinct advantages over that on polyamide layers.

Studies on the inhibition of ATP: Creatine phosphotransferase by NaCl

Kinetic experiments have been undertaken to determine the apparent inhibition constants for the reaction of Cl − with various forms of creatine kinase. The conditions wer similar to those previously used for initial-velocity and isotope-exchange studies of the reaction. It has been shown that NaCl is a non-competitive inhibitor with respect to both substrates of the forward and reverse reactions. With the lower concentrations of the inhibitor used in studying the forward reaction, both the slopes and vertical intercepts of double-reciprocal plots as appeared to be linear functions of the concentration of NaCl. On the other hand, with the higher concentrations of NaCl used to inhibit the reverse reaction, the non-competitive inhibition was of the sloep-parabolic, intercept-linear type. From the values obtained for the apparent inhibition constants, it appears that Cl − may well react at sites on the enzyme at which substrate normally combines, and that the presence of a substrate on the enzyme affects the combination of Cl −. Whereas the interaction of Cl − with the ternary enzyme complex is very weak, two Cl − can react with free enzyme. The inhibition of the reaciton by sodium acetate is considerably less than that by NaCl.