ATP-creatine Phosphotransferase Research Articles

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.

The pattern of ATP-creatine phosphotransferase activity in growing long bones of new-born mice.

The localization of creatine kinase (ATP-creatine phosphotransferase) in growing long bones of new-born mice was studied histochemically. Its activity was found to vary within and between different areas of the cartilagenous epiphyses of proximal tibia. Chondrocytes in the cartilaginous epiphysis showed a high cytoplamatic activity. Active enzyme was also apparent within the growth plate of the diaphysis (with the exception of degenerated cells), in the metaphyseal part of the growth plate within the matrix and in neighbouring areas of the metaphysis, and in areas containing lamella bone. Periosteum and perichondrium also showed signs of enzyme activity. The significance of these findings is discussed in relation to the growth 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.

Sleep disturbance and serum CPK activity in acute psychosis.

SINCE the nature and etiology of acute psychosis are still obscure, the establishment of any abnormal biological parameters for acutely psychotic patients, irrespective of whether such parameters relate to cause or effect of the psychosis, would be of considerable interest. Recently, severe sleep disturbances affecting both rapid-eye-movement (REM) sleep and nonrapid-eye-movement (NREM) sleep were reported in patients having psychotic depressions and acute schizophrenic episodes. 1 Secondly, increased activity of creatine-phosphokinase (CPK) (ATP-creatine phosphotransferase) in serum was found in patients having recent onsets of psychosis. 2-4 Both types of abnormalities seem to be present mainly at the onset of the acute psychotic episode and generally last from several days to two weeks, with occasional exceptions. It is of interest that both types of biological abnormalities are present in acutely psychotic patients of a variety of diagnostic types, including patients with manic-depressive psychosis, psychotic depressions, and

Site actif des ATP: Guanidine phosphotransférases: II. Mise en évidence de résidus histidine essentiels au moyen du pyrocarbonate d'éthyle

The active site of ATP: guanidine phosphotransferases. II. Evidence for a critical histidine residue through use of a specific reagent, diethylpyrocarbonate 1. 1. ATP-creatine phosphotransferase (EC 2.7.3.2) and ATP: l-arginine phosphotransferase (EC 2.7.3.3) are inhibited at pH 6.1 by diethylpyrocarbonate, their reactive sulfhydryl groups being reversibly masked or not. The extent of inactivation is first-order with respect to time and inhibitor concentration. The ability of the substrates, used separately or mixed, to protect arginine kinase against inhibition, suggests the involvement of the modified group in the enzymic reaction. Nevertheless, in the case of creatine kinase no protection has been observed. 2. 2. The carbethoxylated enzymes develop a difference ultraviolet spectrum with a maximum peak at 240 mμ, a specific feature of N-carbethoxyimidazole formation. From the kinetic data of spectrophotometric titrations and loss of activity, it is concluded that one histidine residue and two histidine residues are modified per mole of arginine kinase and creatine kinase respectively, causing total inactivation. 3. 3. Advantages of diethylpyrocarbonate as a protein-histidyl reagent are demonstrated by the specific titration of the histidine content in both denaturated enzymes. Furthermore it is shown that the number and reactivity of -SH groups are not modified in the carbethoxylated inhibited phosphokinases.

Some changes in the reactivity of enzymes resulting from their chemical attachment to water-insoluble derivatives of cellulose

1. Purified ficin was chemically attached to CM-cellulose, and partially purified ATP-creatine phosphotransferase was chemically attached to both CM-cellulose and p-aminobenzylcellulose. 2. The apparent K(m) with respect to ATP and Mg(2+) of ATP-creatine phosphotransferase was observed to increase about tenfold on attachment of the enzyme to CM-cellulose, and to increase by only 23% on its attachment to p-aminobenzylcellulose. 3. The reactivity of both ficin and ATP-creatine phosphotransferase with 5,5'-dithiobis-(2-nitrobenzoic acid) was observed to decrease on chemical attachment of these enzymes to water-insoluble derivatives of cellulose. With derivatives prepared from CM-cellulose, the extent of the reaction with 5,5'-dithiobis-(2-nitrobenzoic acid) was dependent on ionic strength, but with similar derivatives prepared from p-aminobenzylcellulose the extent of this reaction was independent of ionic strength. 4. The effect of diffusion and electrostatic interaction of charged enzyme substrates and charged enzyme supports on the apparent K(m) of a water-insoluble derivative of an enzyme is discussed. An equation is derived that satisfactorily describes the observed effects of these factors on the apparent K(m).

Spectrophotometric Determination of Tryptophan and Tyrosine in Atp-Creatine Phosphotransferase

Abstract A recent paper by Edelhoch1 has presented a spectrophotometric method for the rapid determination of the tryptophan and tyrosine content of unhydrolyzed proteins.

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 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.

KINETIC STUDIES OF THE REVERSE REACTION CATALYSED BY ADENOSINE TRIPHOSPHATE-CREATINE PHOSPHOTRANSFERASE. THE INHIBITION BY MAGNESIUM IONS AND ADENOSINE DIPHOSPHATE.

1. Kinetic investigations of the reaction catalysed by ATP-creatine phosphotransferase have been carried out. 2. No firm conclusions could be reached about the reaction of Mg(2+) at the nucleotide-binding site of the enzyme. The value of the kinetic constant for this reaction depends on the value used for the apparent stability constant of the metal ion-nucleotide complex and, to a smaller extent, on the method of plotting the results. 3. At higher concentrations Mg(2+) is a non-competitive inhibitor of the enzyme with respect to both MgADP(-) and phosphocreatine. 4. ADP(3-) is a competitive inhibitor of the enzyme with respect to MgADP(-) and a non-competitive inhibitor with respect to phosphocreatine. 5. The concentration of phosphocreatine has little, if any, effect on the kinetic constants for the nucleotide reactants.

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.

The effect of gamma radiation on the amino acid content and the enzymatic activity of ATP-creatine phosphotransferase

Whereas in the intact protein in solution histidine is most radiosensitive, in the corresponding amino acid hydrolyzate in solution, methionine is most labile. Phenylalanine and arginine are also faster destroyed if the protein is hydrolyzed before radiation exposure. Since rank in terms of radiation lability varies with individual proteins, stereochemical orientation (amino acid sequence, etc.) must be suspected as a determinant. Reconstitution of some amino acids by interconversion during radiation exposure enhances “apparent” stability of glutamic and aspartic acids, for instance, and their G values with reference to the protein will thus have a “destructive” and a “constructive” component. Sulfhydryl (SH) groups disappear at a faster rate than other amino acid side chains, and decline in enzymic activity is even more rapid than destruction of SH groups. No decision, however, can be reached as to whether destruction of SH side chains or disruption of protein configuration causes the loss of enzyme activity in the early stages of γ-irradiation.