[Effect of x-irradiation on the structural and functional characteristics of rat erythrocytes and activity of individual enzymes of glycolysis]. / Doslidzhennia vplyvu renthenivs'koho vyprominiuvannia na strukturno-funktsional'ni kharakterystyky erytrotsytiv shchuriv i aktivnist' okremnykh fermentiv hlikolizu.

The activity of glycolytic enzymes (lactate dehydrogenase, pyruvate kinase, hexokinase), content of metabolites (lactate, pyruvate, ATP, 2,3-DPG) and haemolytic stability of rat erythrocytes at the action of chronic X-irradiation at a daily dose 0.258 mC/kg, 2.58 mC/kg, 5.16 mC/kg during 90, 60 and 30 days, accordingly, have been investigated. It was shown, that the glycolytic enzymes activity of female rats in different seasons may vary in wide limits. A general tendency to the decrease of lactate dehydrogenase activity is revealed, but the beginning effects under the irradiation at a daily dose of 2.58 mC/kg and 5.16 mC/kg may be differently directed. The analogous tendencies are found in the change of hexokinase activity. The changes in pyruvate kinase activity correlated with erythrocytes haemolytic stability. The data obtained prove that changes in the direction of glycolytic enzymes activity under the irradiation depend on the initial level, which is caused by seasonal peculiarities of physiological state.

[The individual glycolysis enzymes in the enterocytes of the rat small intestine studied during exposure to ionizing radiation]. / Issledovanie otdel'nykh fermentov glikoliza énterotsitov tonkogo kishechnika krys pri vozdeistvii ioniziruiushchei radiatsii.

Activity and isoenzyme composition of certain enzymes of glycolysis of rat small intestine enterocytes was studied in the course of exposure to X radiation. Hexokinase activity was shown to increase significantly throughout the entire period of observation. Activity of pyruvate kinase and lactate dehydrogenase was inhibited at early (days 1-3) and increased at later (days 5-10) times of observation.

Transferrin binding of bone marrow cells and metabolic activity of erythrocytes after 5 Gy irradiation.

The effect of total body irradiation (5 Gy) on functional mouse erythroid lineage has been studied. The transferrin binding capacity by bone marrow cells and the activity of glycolytic regulatory enzymes and intracellular levels of 2,3 bisphosphoglycerate in peripheral blood erythrocytes have been determined. Results obtained along one year post-irradiation period suggest a complete recovery in the erythroid cell lineage with respect to the biological endpoints investigated.

In vitro photosensitization of tumour cell enzymes by photofrin II administered in vivo.

The ability of injected Photofrin II, a preparation enriched in hydrophobic dihaematoporphyrin ethers and esters, to photosensitize selected mitochondrial and cytosolic enzymes during illumination in vitro was examined. Preparations of R3230AC mammary tumours, obtained at designated times after a single dose of Photofrin II, displayed a time-dependent photosensitivity. Maximum inhibition of mitochondrial enzymes occurred at 24 hours post-treatment, whereas no inhibition of the cytosolic enzyme, pyruvate kinase, was observed over the 168 hour time course. At the selected 24 hour time point, mitochondrial enzyme photosensitisation was found to be drug dose (5.25 mg kg-1 Photofrin II) and light dose dependent, the rank order of inhibition being cytochrome c oxidase greater than F0F1 ATPase greater than succinate dehydrogenase greater than NADH dehydrogenase. We conclude that porphyrin species contained in Photofrin II accumulate in mitochondria of tumour cells in vivo and produce maximum photosensitisation at 24-72 hours after administration to tumour-bearing animals. The time course observed here with Photofrin II is similar to that seen previously with the more heterogenous haematoporphyrin derivative preparation in this in vivo-in vitro model.

The apparent target size of rat brain benzodiazepine receptor, acetylcholinesterase, and pyruvate kinase is highly influenced by experimental conditions.

Radiation inactivation is a method to determine the apparent target size of molecules. In this report we examined whether radiation inactivation of various enzymes and brain receptors is influenced by the preparation of samples preceding irradiation. The apparent target sizes of endogenous acetylcholinesterase and pyruvate kinase from rat brain and from rabbit muscle and benzodiazepine receptor from rat brain were investigated in some detail. In addition the target sizes of alcohol dehydrogenase (from yeast and horse liver), beta-galactosidase (from Escherichia coli), lactate dehydrogenase (endogenous from rat brain), and 5-HT2 receptors, acetylcholine muscarine receptors, and [35S] butyl bicyclophosphorothionate tertiary binding sites from rat brain were determined. The results show that apparent target sizes are highly influenced by the procedure applied for sample preparation before irradiation. The data indicate that irradiation of frozen whole tissue as opposed to lyophilized tissue or frozen tissue homogenates will estimate the smallest and most relevant functional target size of a receptor or an enzyme.

Glucose-6-phosphate dehydrogenase from Leuconostoc mesenteroides is a reliable internal standard for radiation-inactivation studies of membranes in the frozen state.

The target size of four soluble enzymes (beta-galactosidase, pyruvate kinase, alcohol dehydrogenase, and glucose-6-phosphate dehydrogenase) in the presence or absence of subcellular membrane fractions has been determined by the radiation-inactivation method using samples in the frozen state. For each of the four enzymes, full activity was recovered after freezing and thawing in the absence of radiation. We found minimal (less than 20%) binding of the enzymes to either submitochondrial vesicles or sarcoplasmic reticulum vesicles. Under the conditions tested, beta-galactosidase, pyruvate kinase, and alcohol dehydrogenase exhibited target sizes which varied according to the experimental conditions, i.e., the buffer selected and also the presence or absence of membrane preparations. For these tetrameric enzymes, the target sizes were generally comparable to either a monomer or a dimer. By contrast, the target size of glucose-6-phosphate dehydrogenase from Leuconostoc mesenteroides was found to be essentially invariant when frozen in a variety of buffers and in the presence or absence of either cryoprotectant (sucrose or glycerol) or different membrane preparations. The target size from 19 separate determinations gave an average value of 104 +/- 16 kDa, which is comparable to the molecular weight of the enzyme (104 kDa). We conclude that glucose-6-phosphate dehydrogenase from L. mesenteroides is a reliable internal standard for radiation-inactivation studies of membrane preparations in the frozen state.

Photo-oxidation of histidine residues in rat M1- and L-type pyruvate kinases.

Rat M1- and L-type pyruvate kinases were inactivated by photo-oxidation mediated by methylene blue at pH 8.0 and O degrees C according to first-order kinetics. The pH profiles of the inactivation rates of these isozymes showed that amino acid residues having a pK value of 7.0-7.5 were involved in the inactivation. Three histidine residues per subunit of M1- or L-type enzyme were destroyed by the photo-oxidation with complete loss of the enzyme activities. However, two of the three photo-oxidized histidine residues in the L-type enzyme were more important in the inactivation reaction. The kinetics of the partially inactivated L-type enzyme suggests that complete inactivation is achieved via an intermediate form having low affinity for phosphoenolpyruvate (PEP). These observations revealed the involvement of essential histidine residues of two different kinds in the catalytic mechanism of the L-type enzyme. In the photo-oxidation of M1-type enzyme, no intermediate form was observed. Addition of PEP or pyruvate to the reaction mixture markedly prevented the photo-oxidative inactivation of only the M1-type enzyme in the presence of K+ and Mg2+; the addition of ADP or ATP was ineffective even in the presence of both metal ions. This protective effect of PEP was counteracted by further addition of ATP but not by ADP. However, photo-oxidative inactivation of the L-type enzyme was not prevented even by the addition of PEP in the presence of both metal ions, owing to the low affinity for PEP at 0 degrees C, in spite of the presence of fructose 1,6-bisphosphate (Fru-1,6-P2).(ABSTRACT TRUNCATED AT 250 WORDS)