Triphosphate-creatine Phosphotransferase Research Articles

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.

Purification and properties of turtle heart creatine kinase: role for the enzyme in glycolytic control

1. 1. Adenosine triphosphate-creatine phosphotransferase (EC 2.7.3.2) from turtle ( Pseudemys scriptd) heart occurs in two major electrophoretic forms: one cytoplasmic ( pI = 6.67) and one mitochondrial (pI = 7.37). 2. 2i. In the forward direction (phosphocreatine + ADP → ATP + creatine), NADH actevates the purified cytosol enzyme by lowering the K m for phosphocreatine almost thr e-fold. 3. 3. From these studies it is concluded that in vivo the rising levels of NADH known to occur in the hypoxic heart of the diving turtle serve as the signal for rapid depletion of phosphocreatine and the subsequent autocatalytic activation of glycolysis. 4. 4. The role if creatine kinase in the overall anaerobic metabolism of turtle heart is discussed.

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.

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.

The significance of anions in the protection by substrates of adenosine triphosphate-creatine phosphotransferase against inhibition by iodoacetamide.

The significance of anions in the protection by substrates of adenosine triphosphate-creatine phosphotransferase against inhibition by iodoacetamide.

Further evidence for the role of the essential thiol groups in adenosine triphosphate-creatine phosphotransferase from a comparison of the human and rabbit enzymes.

Further evidence for the role of the essential thiol groups in adenosine triphosphate-creatine phosphotransferase from a comparison of the human and rabbit enzymes.

Relaxation spectra of adenosine triphosphate-creatine phosphotransferase.

ADVERTISEMENT RETURN TO ISSUEPREVArticleNEXTRelaxation spectra of adenosine triphosphate-creatine phosphotransferaseGordon G. Hammes and James K. HurstCite this: Biochemistry 1969, 8, 3, 1083–1094Publication Date (Print):March 1, 1969Publication History Published online1 May 2002Published inissue 1 March 1969https://doi.org/10.1021/bi00831a040RIGHTS & PERMISSIONSArticle Views51Altmetric-Citations53LEARN ABOUT THESE METRICSArticle Views are the COUNTER-compliant sum of full text article downloads since November 2008 (both PDF and HTML) across all institutions and individuals. These metrics are regularly updated to reflect usage leading up to the last few days.Citations are the number of other articles citing this article, calculated by Crossref and updated daily. Find more information about Crossref citation counts.The Altmetric Attention Score is a quantitative measure of the attention that a research article has received online. Clicking on the donut icon will load a page at altmetric.com with additional details about the score and the social media presence for the given article. Find more information on the Altmetric Attention Score and how the score is calculated. Share Add toView InAdd Full Text with ReferenceAdd Description ExportRISCitationCitation and abstractCitation and referencesMore Options Share onFacebookTwitterWechatLinked InReddit PDF (1 MB) Get e-Alerts Get e-Alerts

Formation of an unusual hybrid in the development of human adenosine 5'-triphosphate-creatine phosphotransferase.

1. Starch-gel analysis of extracts from adult human muscle, heart and brain reveals a hybrid creatine kinase with an abnormally high electrophoretic mobility. 2. Hybridization in vitro confirms that the postulated hybrid is formed from a combination of brain- and muscle-type enzyme sub-units. 3. The relative electrophoretic mobility of the hybrid is not affected by changing the starch concentration in the gel or by the buffer system used, or by electrophoresis in thin layers of Sephadex. 4. It is concluded that hybrid formation results in a net increase on the dimeric enzyme of from 4 to 6 negative charges. 5. During development a sharp increase in the rate of production of muscle-type enzyme sub-units occurs in heart at 10-13 weeks' gestation and in muscle at 18-20 weeks' gestation. The latter change is accompanied by a relative decrease in the concentration of brain-type sub-units.

Ion Binding by Adenosine Triphosphate-Creatine Phosphotransferase

Abstract The binding of K+, Na+, Cl-, Ca++, and Mg++ by deionized creatine kinase (ATP-creatine phosphotransferase, EC 2.7.3.2) was investigated as a function of pH at 25° with the use of permselective membrane electrodes. Whereas significant binding of K+ to creatine kinase occurs, no binding of Na+ can be detected. Cl- is also bound. Graphs of v/c with respect to v give a linear relationship for K+ and Cl-; association constants and number of maximal binding sites are deduced from such plots for these ions. The number of binding sites on the protein for Mg++ is essentially the same as that for Ca++, but the affinity of the protein for Mg++ is only about 0.6 of that for Ca++. The combining sites for each of these 2 divalent ions cannot be considered as belonging to a single class with identical association constants.

The Reaction of Nucleotide Substrate Analogues with Adenosine Triphosphate-Creatine Phosphotransferase

Abstract The reverse reaction catalyzed by creatine kinase has been studied at pH 8.0 to determine the effects of modifying separately the base, sugar, and phosphate moieties of the magnesium-adenosine diphosphate complex. On the basis that the reaction mechanism is rapid equilibrium, random, irrespective of the nucleotide substrate, dissociation constants have been determined for the reaction of nucleotide analogues with both the free enzyme and the enzyme-phosphocreatine complex. The following magnesium-nucleotide complexes were found to function as substrates with maximum velocities in the order MgADP- g MgdADP- ≃ MgIDP- g MgCDP- ≃ MgUDP- g MgGDP-. The affinity of free enzyme for these complexes was shown to decrease in the order MgADP- ≃ MgdADP- g MgIDP- ≃ MgGDP- ≃ MgCDP- ≃ MgUDP-. The substrates studied can be grouped into three classes according to whether interaction with the free enzyme is greater, equal to, or less than the interaction with the enzyme-phosphocreatine complex. MgCDP-, MgUDP-, and MgGDP- were also studied as inhibitors of the reaction. The dissociation constants determined by this approach were essentially similar to those evaluated from their reactions as substrates. The role of the phosphate chain in the binding of MgADP- to the enzyme has been studied by investigating adenosine, AMP, and magnesium-inorganic pyrophosphate as inhibitors of the reaction. The results are in accord with the hypothesis that a conformational change occurs at the active site of the enzyme as a result of the combination of substrate.

Adenosine 5 -triphosphate--creatine phosphotransferase from dystrophic mouse skeletal muscle. A genetic lesion associated with the catalytic-site thiol group.

1. A column procedure for the purification of creatine kinase from normal and dystrophic mouse muscle is described. 2. The native enzymes are indistinguishable by various physical criteria and have mol.wt. about 80000. 3. The purified enzyme from dystrophic muscle is only half as active as the normal, contains only one thiol group readily alkylated by iodoacetamide instead of two and has one less free thiol group/mol. 4. Michaelis constants for MgATP and creatine are the same for both preparations. 5. The inhibitor constant for ADP at pH9.0 is different in the two enzymes and this may account for the different degrees of inhibition observed in vitro with the drug Laevadosin. 6. The enzyme from dystrophic muscle is protected by an equilibrium mixture of substrates against inhibition by iodoacetamide to a greater extent than the normal enzyme. 7. ;Fingerprinting' suggests one peptide difference between creatine kinases from normal and dystrophic muscle. 8. The possibility that this finding represents the primary lesion in dystrophy is discussed.

Changes in Phosphate Compounds during the Development and Maintenance of Rigor Mortis

Abstract Rigor mortis was induced in frog sartorii from female Rana pipiens by incubating the isolated muscles in 0.38 mm 2,4-dinitrofluorobenzene Ringer's (frog) solution for 90 min at 0°. This compound completely inhibits adenosine triphosphatecreatine phosphotransferase in vivo and eventually induces rigor mortis as characterized by shortening of the muscle to 50% of its rest length and the development of stiffness. As the muscle began to shorten the rate of 45Ca efflux increased and the ATP level fell from 3.80 to 0.0 µmoles per g. There was a lack of correspondence between ATP breakdown and appearance of inorganic phosphate, which can be explained by leakage of inorganic phosphate from the muscle and by an increase in the concentration of glucose 6-phosphate, fructose 6-phosphate, and glucose 1-phosphate. The frog sartorius muscle breaks down ATP during a working contraction and in the shortening phase of rigor mortis. There is no ATP requirement for tension maintenance in rigor mortis after the muscle has finished shortening.

The mechanism of the reaction catalysed by adenosine triphosphate-creatine phosphotransferase

1. The forward and reverse reactions catalysed by ATP-creatine phosphotransferase have been studied kinetically at pH8.0 in the presence and absence of products, under conditions in which the free Mg(2+) concentration was maintained constant at 1mm. Thus at fixed pH the reaction may be considered as being bireactant and expressed as:MgATP(2-)+creatine(0)right harpoon over left harpoonMgADP(-)+phosphocreatine(2-)2. The initial-velocity pattern in the absence of products and the product-inhibition pattern have been determined. These are consistent with a random mechanism in which all steps are in rapid equilibrium except that concerned with the interconversion of the central ternary complexes, and in which two dead-end complexes (enzyme-MgADP-creatine and enzyme-MgATP-phosphocreatine) are formed. The results are in accord with previous suggestions that the enzyme possesses distinct sites for the combination of the nucleotide and guanidino substrates. 3. Values have been determined for the Michaelis and dissociation constants involved in the combination of each substrate with various enzyme forms. Although these values cannot be regarded as absolute, they appear to indicate that the presence of one substrate on the enzyme enhances the combination of the second substrate. In addition, it would seem that in the formation of the enzyme-MgADP-creatine complex the concentration of one reactant does not affect the combination of the other. This contrasts with the formation of the enzyme-MgATP-phosphocreatine complex, where each reactant hinders the combination of the other.

AMINO-ACID SEQUENCE AROUND THE REACTIVE THIOL GROUPS OF ADENOSINE TRIPHOSPHATE--CREATINE PHOSPHOTRANSFERASE.

Amino-Acid Sequence Around the Reactive Thiol Groups of Adenosine Triphosphate–creatine Phosphotransferase

Adenosine triphosphatase activity of adenosine triphosphate-creatine phosphotransferase

Abstract Creatine kinase (ATP-creatine phosphotransferase, EC 2.7.3.2)1 has been found to have a low but constant amount of ATPase activity2. The ratio of ATPase activity to creatine kinase activity remains the same during repeated crystallization and during repeated precipitations. Loss of kinase activity by a variety of treatmentsusing physical and chemical agents has been found to result in a proportionate loss of ATPase activity. Twice-crystallized preparations show no evidence of protein or activity heterogeneity on DEAE-cellulose column chromatography. Kinetics studies like those carried out for the kinase activity3 lead to the identical conclusion that MgATP2− is also the true substrate for ATPase activity. It may be postulated that the trace ATPase activity is due to a water molecule acting as a nucleophilic agent instead of the creatine molecule.

Adenosine triphosphate-creatine phosphotransferase from ox brain: purification and isolation.

Adenosine triphosphate-creatine phosphotransferase from ox brain: purification and isolation.

The conversion of adenosine 5'-phosphate into adenosine triphosphate as catalysed by adenosine triphosphate--creatine phosphotransferase and adenosine triphosphate--adenosine monophosphate phosphotransferase in the presence of phosphocreatine.

Research Article| February 01 1963 The conversion of adenosine 5′-phosphate into adenosine triphosphate as catalysed by adenosine triphosphate–creatine phosphotransferase and adenosine triphosphate–adenosine monophosphate phosphotransferase in the presence of phosphocreatine MD DOHERTY; MD DOHERTY Search for other works by this author on: This Site PubMed Google Scholar JF MORRISON JF MORRISON Search for other works by this author on: This Site PubMed Google Scholar Biochem J (1963) 86 (2): 344–350. https://doi.org/10.1042/bj0860344 Views Icon Views Article contents Figures & tables Video Audio Supplementary Data Peer Review Share Icon Share MailTo Twitter LinkedIn Cite Icon Cite Get Permissions Citation MD DOHERTY, JF MORRISON; The conversion of adenosine 5′-phosphate into adenosine triphosphate as catalysed by adenosine triphosphate–creatine phosphotransferase and adenosine triphosphate–adenosine monophosphate phosphotransferase in the presence of phosphocreatine. Biochem J 1 February 1963; 86 (2): 344–350. doi: https://doi.org/10.1042/bj0860344 Download citation file: Ris (Zotero) Reference Manager EasyBib Bookends Mendeley Papers EndNote RefWorks BibTex toolbar search Search Dropdown Menu toolbar search search input Search input auto suggest filter your search All ContentAll JournalsBiochemical Journal Search Advanced Search This content is only available as a PDF. © 1963 The Biochemical Society1963 Article PDF first page preview Close Modal You do not currently have access to this content.

A study of the 'reactive' sulphydryl groups of adenosine 5'-triphosphate-creatine phosphotransferase.

Research Article| December 01 1962 A study of the ‘reactive’ sulphydryl groups of adenosine 5′-triphosphate-creatine phosphotransferase DC WATTS; DC WATTS Search for other works by this author on: This Site PubMed Google Scholar BR RABIN BR RABIN Search for other works by this author on: This Site PubMed Google Scholar Biochem J (1962) 85 (3): 507–516. https://doi.org/10.1042/bj0850507 Views Icon Views Article contents Figures & tables Video Audio Supplementary Data Peer Review Share Icon Share Facebook Twitter LinkedIn MailTo Cite Icon Cite Get Permissions Citation DC WATTS, BR RABIN; A study of the ‘reactive’ sulphydryl groups of adenosine 5′-triphosphate-creatine phosphotransferase. Biochem J 1 December 1962; 85 (3): 507–516. doi: https://doi.org/10.1042/bj0850507 Download citation file: Ris (Zotero) Reference Manager EasyBib Bookends Mendeley Papers EndNote RefWorks BibTex toolbar search Search Dropdown Menu toolbar search search input Search input auto suggest filter your search All ContentAll JournalsBiochemical Journal Search Advanced Search This content is only available as a PDF. © 1962 The Biochemical Society1962 Article PDF first page preview Close Modal You do not currently have access to this content.