Experimental Clarification of PRPS-1 Structural Essentials.

Phosphoribosyl pyrophosphate synthetase-1 (PRPS-1; EC 2.7.6.1.) catalyzes the binding of phosphate-group to ribose 5-phosphate, forming the 5-phosphoribosyl-1-pyrophosphate, which is necessary for the salvage pathways of purine and pyrimidine, pyridine nucleotide cofactors - NAD and NADP, the amino acids histidine and tryptophan biosynthesis. We aimed to investigate the impact of the different effectors on the activity of PRPS-1, to check the activity of the enzyme in vitro in a wide range of pHs and investigate some structural essentials of the enzyme, isolated from brain and liver. Molecular docking analyses were used to delineate the essentials of PRPS-1 structure, to find out the existence of enzyme effectors. Previously created by us kit was used for determination of the activity of PRPS-1 based on the formation of the inorganic phosphates (λ = 700 nm, Cary 60, Agilent, USA). Effectors impact on the activity of PRPS-1 was evaluated. In silico results of the effectors were later proven by in vitro experiments. For the first time biochemical essentials, including the existence of the multiple pockets, involvement of the amino acids into the processes of interactions with the effectors, evolutional of the sequence conservation, tissue depended Vmax differences were identified.

Dependence of cell survival on correlative activities of xanthine oxidase and dihydopyrimidine dehydrogenase in human brain-derived cell culture.

Xanthine Oxidase (XO; EC. 1.1.3.22) and Dihydropyrimidine Dehydrogenase (DPD; EC. 1.3.1.2) are two enzymes responsible for the last steps of purine and pyrimidine catabolism, and hydroxylation of a wide variety of pyrimidine, pterin, and aldehyde substrates. Elion showed that purine isomers can be converted to various nucleotides, which influence pyrimidine metabolism (Elion, 1978). The current study is devoted to delineating the correlation between survival of human brain derived cells in culture and the activities of XO and DPD. Cultivation of (E90) brain cells was performed by the modified method of Mattson (1990). XO activity was measured by the formation of uric acid in the tissue. DPD activity was evaluated by the reduction of NADPH and the associated absorbance decrease at 320 nm. Cell death was detected by Trypan Blue dye leakage. During our investigation, we noticed a reversed correlation between the activities of XO and DPD over 12 days under normal conditions as well as in the presence of the XO and DPD inhibitors, allopurinol and dipyridamole. During the treatment period of 12 days, as well as from days 7-12 with the inhibitors, we observed cell protection, whereas treatment from days 1-7 elevated the percentage of dead cells in culture. A low dosage of allopurinol over 12 days also stimulated cell growth and increased their number in culture. We concluded that timely inhibition of XO as well as DPD activities might initiate cell growth and prevent their death. However, the main influence as the final enzyme of purine metabolism in the processes of cell proliferation belongs to XO in contrast to DPD.

The influence of hypothalamic cytokine PRP on protein synthesis in brain subcellular compartments in crush syndrome.

Crush-syndrom (CS) was characterized by Bywaters E.G.L. in 1941 after London blitz. The soft tissues is followed by acute hemodynamic shock, myoglobinuria, acute renal insufficiency, and lethal endotoxicity. Data of CS pathogenesis study has shown that the largest changes in Crush occur during decompression and are accompanied by acute alteration of brain protein synthesis and strong morphological changes of brain structures. The period of decompression might be characterized by the proteolytic breakdown of the myoglobine and formation of toxic peptides. In our current work we have identified four newly formed peptides in the brain of the animals subjected to the experimental muscle tissue injury. Our investigations related with the CS experimental model have demonstrated that during the 2-hours compression protein synthesis was decreased in cytosol (32,7%) and mitochondria (49%), after 5-h compression there were registered non-significant changes in the level of protein synthesis. Intraperitoneal administration of Proline-rich peptide, ((PRP), 1 mcg/100g weight of rats), originating from proteolysis of C-terminal glycoprotein a neurophysin II along with vasopressin and oxytocin and transferring from the hypothalamus to the neurohypophysis by axonal transport, initiates activation of the protein synthesis in all studied cellular subcomponents of brain cells. The positive effect of the peptide is conditioned, most probably, by activation of the immune system and adaptation mechanisms, including mobilization of endogen-protective mechanisms of the organism.