MEMBRANE-ASSOCIATED THIAMINE KINASE FROM PIG'S BRAIN: DISTRIBUTION IN SUBCELLULAR FRACTIONS AND BRAINS, AMINO ACID COMPOSITION

Background. Thiamine kinase is a medical enzyme, genetically determined "breakdowns" during the synthesis of which lead to a number of neurodegenerative diseases. Knowledge of the distribution of the enzyme in the compartments of the brain, the regulatory capabilities of the globule in the formation of the coenzyme form of vitamin B1 – thiamine diphosphate, will enable to perform targeted correction of pathological conditions. Purpose of the study. To determine the localization of the enzyme in the pig's brain, the role of hydrophobic and hydrophilic interactions in association on membranes, the nature of amino acid residues that determine the structure of the protein. Material and methods. To obtain membranes, fresh brain, cleaned from membranes and vessels, filled with 4 volumes of chilled Tris-HCl buffer (50 mM, pH 7.4) with 0.2 mM EDTA and homogenized (2000 rpm–1) for 5 cycles. The homogenate was centrifuged for (60 min at 30,000 g), the supernatant was discarded into the membranes remaining in the pellet and subjected to solubilization with the initial buffer containing 0.05-1 % of detergents. The enzyme activity was assessed by the rate of thiamine diphosphate formation. Protein hydrolysis was carried out in evacuated ampoules with 6 M HCl at 110 °C for 18, 22, 48, and 72 h. The isoelectric point (pI) was calculated from the amino acid composition and using the isoelectric focusing method. The isoionic point, was determined by dialysis of thiamine kinase, followed by measuring the pH in the dialysate. Results. It was found that hydrophobic detergents have a more pronounced solubilizing effect compared to hydrophilic ones. At high values of the critical concentration of micelle formation (1 %), both of them change the conformational state of the macromolecule, affecting its affinity for substrates and effectors. Thiamine kinase is fairly evenly dispersed in all parts of the brain. However, the subcellular localization is different. Low enzymatic activity is observed in the mitochondrial fractions. The enzyme is characterized by an increased concentration of amino acids, contributing to the α-helicalization of the protein globule, while at the same time a low content of residues that bind polypeptide chains and high - carry out its sharp rotation by 130 °, incompatible with the course of the α-helix. Conclusions. Brain thiamine kinase is a membrane-associated protein. Hydrophobic forces are mainly involved in the interaction with the lipid bilayer of membranes. Depending on the concentration of the detergent, the solubilization process is accompanied by a change in the conformation of the globule. The main amount of thiamine kinase is concentrated in mitochondrial membranes.