[Effect of low-intensity 900 MHz frequency electromagnetic radiation on rat liver and blood serum enzyme activities].

The comparative analysis of the rat liver and blood serum creatine kinase, alanine aminotransferase, aspartate aminotransferase, alkaline phosphatase and purine nucleoside phosphorylase post-radiation activity levels after a total two-hour long single and fractional exposure of the animals to low-intensity 900 MHz frequency electromagnetic field showed that the most sensitive enzymes to the both schedules of radiation are the liver creatine kinase, as well as the blood serum creatine kinase and alkaline phosphatase. According to the comparative analysis of the dynamics of changes in the activity level of the liver and blood serum creatine kinase, alanine aminotransferase, aspartate aminotransferase and purine nucleoside phosphorylase, both single and fractional radiation schedules do not affect the permeability of a hepatocyte cell membrane, but rather cause changes in their energetic metabolism. The correlation analysis of the post-radiation activity level changes of the investigated enzymes did not reveal a clear relationship between them. The dynamics of post-radiation changes in the activity of investigated enzyme levels following a single and short-term fractional schedules of radiation did not differ essentially.

[Influence of ionizing radiation on enzymatic activity and state of nucleus-nucleolar apparatus in rat hepatocytes].

The effects of a single exposure of rats to the whole-body roentgen irradiation at the doses of 3.5 Gy and 4.5 Gy on the activity of creatine kinase, purine nucleoside phosphorylase, alanine aminotransferase, aspartate aminotransferase, as well as on the state of the nuclear-nucleolar apparatus in rat hepatocytes on the 6th and 13th days after radiation exposure have been studied. Irradiation at the above doses induced changes in the levels of enzymatic activity of different values and different directions within the same time periods, as well as oscillating changes in this type of enzymatic activity over time. This demonstrates various radiosensitivity and adaptation abilities of these enzymatic activities. The changes in the enzymatic activity significantly correspond to the changes in the morphometric indices of nuclear-nucleolar apparatus of hepatocytes, as well as the distribution of hepatocytes within the ploidy classes: in particular, stabilization of the enzymatic activity on the 13th day after irradiation correlates with the increased transcriptional activity, which is detectable through the increased number of nucleoli per nucleus and the expanded space of a hepatocyte nucleus. The compensation mechanisms are likely to be targeted at the changes in the functional activity of surviving hepatocytes, rather than at the replacement of the damaged cells by the new ones.

[Some inhibitors of purine nucleoside phosphorylase].

Purine nucleoside phosphorylase (PNP) catalyzes reversible phosphorolysis of purine deoxy- and ribonucleosides with formation (d)Rib-1-P and corresponding bases. PNP plays a leading role in the cell metabolism of nucleosides and nucleotides, as well as in maintaining the immune status of an organism. The major aim of the majority of studies on the PNP is the detection of highly effective inhibitors of this enzyme, derivatives ofpurine nucleosides used in medicine as immunosuppressors, which are essential for creating selective T-cell immunodeficiency in a human body for organ and tissue transplantation. The present work is devoted to the study of the effects of some synthetic derivatives of purine nucleosides on activity of highly purified PNP from rabbit spleen and also from human healthy and tumor tissues of lung and kidneys. Purine nucleoside analogues modified at various positions of both the heterocyclic base and carbohydrate residues have been investigated. Several compounds, including 8-mercapto-acyclovir, 8-bromo-9-(3,4-hydroxy-butyl)guanine, which demonstrated potent PNP inhibition, could be offered for subsequent study as immunosuppressors during organ and tissue transplantation.

[Purine nucleoside phosphorylase inhibitors and their clinical significance].

PNP catalyzes a reversible phosphorolysis of purine deoxy- and ribonucleosides with formation of (d)Rib-1-P and appropriate bases. PNP plays a leading role in the cell metabolism of nucleosides and nucleotides, as well as in maintaining the immune status of an organism. The major purpose of the majority of studies on the PNP is the detection of high-performance enzyme inhibitors, derivatives of the purine nucleosides, which are used in medicine as immunosuppressors. It is well known that the latter are necessary for creating a selective T-cell immunodeficiency in a human body under organs and tissue transplantation. The review discusses the issues related to deliberate synthesis of effective, metabolically inert, and low-toxic PNP inhibitors. It also analyzes the available studies on substrate and inhibitory properties of the analogues of purine nucleosides, as well as research on the structural factors which reinforce the inhibitor activity of those analogues. The inhibitors which are either used in medical practice or are currently at a stage of preclinical testing are described. The inhibitors which are more efficient in their influence on the PNF from tumorous tissues are of special interest. Using PNP inhibitors in case of a number of pathologies denotes the importance and promise of research on both the enzyme and the compounds affecting its activity.

[Role of individual phospholipids in the functional activity of human myocardial creatine kinase]. / Rol' individual'nykh fosfolipidov v funktsional'noi aktivnosti kreatinkinazy serdechnoi myshtsy cheloveka.

Regulating effect of individual phospholipids on the activity of creatine kinase MM from human myocardium was studied. Cardiolipin, phosphatidic acid, phosphatidyl serine and phosphatidyl choline (dipalmitoyl) stimulated the enzymatic activity, while phosphatidyl inositol and lysophosphatidyl choline inhibited the creatine kinase MM activity by 80-100%. When mechanisms of the phospholipids inhibitory effects were studied, mixed type of inhibition was detected in the presence of phosphatidyl inositol and non-competitive type--in presence of lysophosphatidyl choline if guanidine was used as a substrate. Phosphatidyl inositol and lysophosphatidyl choline inhibited creatine kinase MM by the uncompetitive type if ADP was used as a substrate.

[Creatine kinase from rabbit skeletal muscles: formation of O-acyltyrosine as a result of the activation of the carboxylic group of the enzyme active site by affinity reagents, nucleotide imidazolides]. / Kreatinkinaza iz skeletnykh myshts krolika: obrazovanie O-atsiltirozina v rezul'tate aktivatsii karboksil'noi gruppy aktivnogo tsentra fermenta pod deistviem affinnykh reagentov-- imidazolidov nukleotidov.

Creatine kinase from skeletal muscle (EC 2.7.3.2) was inactivated by means of imidazolides of AMP, ADP, ATP. Rates of the inactivation of the enzyme's M- and M'-subunits differ 50-100 fold and decrease in the presence of ADP and ATP. Differential spectrum of the native and modified enzymes corresponds to the spectrum of N,O-diacetyltyrosine. Kinetic curves of hydroxylamine-dependent destruction of N,O-diacetyltyrosine and of alteration of differential spectrum of the modified and native enzymes essentially coincide. The enzyme's inactivation appears to be caused mainly by the formation of a bond between nucleotide imidazolides activated carboxyl group of the active centre and OH-group of Tyr residue arranged in the close proximity. The stoichiometry of acyltyrosine formation is evaluated as 2.1 +/- 0.2 mole per mole of the functional dimer. Along with formation of ester bond between amino acid residues, a covalent attachment of 0.03-0.06 mole of [14C]nucleotides per mole of enzyme is observed. As the data of acid hydrolysis show, Im-ATP and Im-AMP block epsilon-amino group of Lys and guanidine group of Arg, respectively. Reasons of the multiple modification of creatine kinase by affinity reagents are discussed. The results obtained and literature data are summarised in the hypothetical scheme of disposition of various amino acid residues in the active centre of creatine kinase.

[Purification and various properties of creatine kinase MM isoenzyme from the human heart muscle]. / Ochistka i nekotorye svoistva izofermenta MM kreatinkinazy iz serdechnoi myshtsy cheloveka.

Homogeneous preparation of creatine kinase MM isoenzyme was isolated from human heart muscle using affinity chromatography on Sepharose containing immobilized Cibachron blue F3G-A. The enzyme was active at a wide range of pH 5.0-8.0 exhibiting maximal activity at pH 6.0-6.7. Dependence of the initial rate of creatine kinase reaction on the ADP concentration at pH 6.6 in presence or absence of Mg2+ did not follow the Michaelis-Menten kinetics, while hyperbolic dependence was found at pH 7.6 and pH 5.2. In presence of Mg2+ Km value for ADP at pH 7.6 and pH 5.2 was decreased 4-fold and 1.3-fold, respectively, whereas Vmax was increased 2-fold and 2.5-fold, respectively. Besides, Km value for Mg2+-ADP at pH 7.6 was 3-fold higher than at pH 5.2, while these Km values were similar for ADP. The data obtained suggest that in human heart functional dissimilarity of creatine kinase MM subunits appears to occur, which is of importance in regulation of transphosphorylation.

[Affinity modification of creatine kinase from rabbit skeletal muscle by 2',3'-dialdehyde derivatives of ADP and ATP]. / Afinnaia modifikatsiia kreatinkinazy iz skeletnykh myshts krolika s pomoshch'iu 2',3'-dial'degidnykh proizvodnykh ATP i ADP.

Periodate-oxidized ADP and ATP (oADP and oATP) are substrates and affinity reagents for creatine kinase from rabbit skeletal muscle. oADP and oATP modified a lysine epsilon-amino group in the nucleotide-binding site of the enzyme. Complete inactivation is observed upon binding 2 moles oADP per 1 mole of the enzyme dimer. Modification with oADP is described by a liner dependence of the log of enzyme activity on time, testifying to a pseudo-first-order of the reaction. The reaction rate constant (ki = 8.10(3) min-1) and dissociation constant for the reversible enzyme-oADP complex (Kd = 62 microM) were determined. ADP protected the enzyme from inactivation and covalent binding of the analog, whereas oADP covalently bound to the enzyme was phosphorylated by phosphocreatine. The data obtained allow to suggest that the epsilon-amino group of a lysine residue of the active site is located in close proximity to ribose of ATP and ADP forming a complex with the enzyme. This group seems essential for correct orientation of the nucleotide polyphosphate chain in the enzyme active center, but take no immediate part in the transphosphorylation process.

[Interaction of rabbit muscle creatine kinase with a reactive ATP derivative-ATP gamma-4(N-2-chloroethyl-N-methyl-amino)-benzylamidate]. / Vzaimodei kreatinkinazy iz myshts krolika s reaktsionnosposobnym proizvodnym ATP-4-(N-2-khlorztil-N-metilamino)-benzil-gamma-amidom ATP.

The interaction of AGP gamma-4(N-2-chloroethyl-N-methyl-amino)-benzylamidate with rabbit muscle creatine kinase was studied. ATP gamma-4-(N-2-hydroxyethyl-N-methyl-amino)-benzylamidate acts as competitive inhibitor of creatine kinase. The Km value for ATP and the Ki value for the gamma-analog have been determined. A complete inactivation is observed when 1 mole of the reagent binds per 1 mole of the enzyme. The modification of the second subunit of creatine kinase is achieved at higher concentrations of the reactive ATP analog. The reactive ATP derivative is shown to be an affinity reagent for this enzyme. The possibility of interaction between the subunits of creatine kinase is discussed.