ISOLATION AND PURIFICATION OF PROTEIN THAT IS IMMUNOLOGICALLY SIMILAR TO HUMAN LACTOFERRIN

The study aimed at isolating a substance that is immunologically similar to human lactoferrin, hereinafter — microbial-derived lactoferrin (MdLF) isolated from K.pneumoniae liquid culture. Protein extraction started with isolation of ballast proteins in 2M ammonium sulphate (p.a.). Ion chromatography with cation-exchange agents was the basic method used for isolating MLF. Proteins from the isolated protein fractions were identified with AGID method. The isolated MLF is glucoprotein with the molecular mass of about 84,000, the agarose diffusion coefficient of 3.1 × 10–7 cm2 sec–1 and the relative electrophoretic mobility of 0.42. MLF is characterized by low hydrophobicity and elutes from phenylSepharose with 0.4 ammonium sulphate. Its isoelectric point equals to 9.2.

ASSESSMENT OF GENETIC STRUCTURE VARIABILITY OF RAINBOW TROUT Oncorhynchus mykiss (WALBAUM, 1792) OF UKRAINIAN LOCAL STOCKS USING POLYMORPHIC BLOOD PLASMA PROTEINS

Rainbow trout is a valuable species of aquaculture, which is characterized by a high level of variability. Aim. The goal was to study the peculiarities of genetic variability of rainbow trout based on polymorphism of transferrin (TF), posttransferrin (PTF), esterase (EST) (EC 3.1.1.1.) and albumin (ALB) loci. Methods. Electrophoretic separation of plasma proteins of rainbow trout from three local stocks (Chernivtsi, Kharkiv and Transcarpathian) was carried out in the native polyacrylamide gel. Results. Peculiarities of the distribution and relative electrophoretic mobility of allelic variants of the studied loci of rainbow trout of Ukrainian local stocks were established.The results demonstrated the effectiveness of the analysis using methods of biochemical genetics to establish the characteristics of the genetic structure, the level of heterozygosity of local stocks and to carry out their differentiation. Conclusion. The use of selected markers would allow monitoring the dynamics of changes in the state of local stocks under the established conditions of cultivation in the future. Key words: rainbow trout, polymorphic blood plasma proteins, transferrin, posttransferrin, esterase, albumin.

Phosphorylation and Dephosphorylation of Tau Protein by the Catalytic Subunit of PKA, as Probed by Electrophoretic Mobility Retard

Background: Tau is a microtubule associated protein that regulates the stability of microtubules and the microtubule-dependent axonal transport. Its hyperphosphorylated form is one of the hallmarks of Alzheimer’s disease and other tauopathies and the major component of the paired helical filaments that form the abnormal proteinaceous tangles found in these neurodegenerative diseases. It is generally accepted that the phosphorylation extent of tau is the result of an equilibrium in the activity of protein kinases and phosphatases. Disruption of the balance between both types of enzyme activities has been assumed to be at the origin of tau hyperphosphorylation and the subsequent toxicity and progress of the disease. Objective: We explore the possibility that, beside the phosphatase action on phosphorylated tau, the catalytic subunit of PKA catalyzes both tau phosphorylation and also tau dephosphorylation, depending on the ATP/ADP ratio. Methods: We use the shift in the relative electrophoretic mobility suffered by different phosphorylated forms of tau, as a sensor of the catalytic action of the enzyme. Results: The results are in agreement with the long-known thermodynamic reversibility of the phosphorylation reaction (ATP + Protein = ADP+Phospho-Protein) catalyzed by PKA and many other protein kinases. Conclusion: The results contribute to put the compartmentalized energy state of the neuron and the mitochondrial-functions disruption upstream of tau-related pathologies.

Vitamin C protects against and reverses specific hypochlorous acid- and chloramine-dependent modifications of low-density lipoprotein

Activated phagocytes produce the highly reactive oxidant hypochlorous acid (HOCl) via the myeloperoxidase-catalysed reaction of hydrogen peroxide with chloride ions. HOCl reacts readily with a number of susceptible targets on apolipoprotein B-100 of low-density lipoprotein (LDL), resulting in uncontrolled uptake of HOCl-modified LDL by macrophages. We have investigated the efects of vitamin C (ascorbate), an effective water-soluble antioxidant, on the HOCl- and chloramine-dependent modification of LDL. Co-incubation of vitamin C (25-200 μM) with LDL resulted in concentration-dependent protection against HOCl (25-200 μM)-mediated oxidation of tryptophan and lysine residues, formation of chloramines and increases in the relative electrophoretic mobility of LDL. Vitamin C also partially protected against oxidation of cysteine residues by HOCl, and fully protected against oxidation of these residues by the low-molecular-mass chloramines, Nα-acetyl-lysine chloramine and taurine chloramine, and to a lesser extent monochloramine (each at 25-200 μM). Further, we found that HOCl (25-200 μM)-dependent formation of chloramines on apolipoprotein B-100 was fully reversed by 200 μM vitamin C; however, the loss of lysine residues and increase in relative electrophoretic mobility of LDL were only partially reversed, and the loss of tryptophan and cysteine residues was not reversed. Time-course experiments showed that the reversal by vitamin C of HOCl-dependent modifications became less efficient as the LDL was incubated for up to 4 h at 37 °C. These data show that vitamin C not only protects against, but also reverses, specific HOCl- and chloramine-dependent modifications of LDL. As HOCl-mediated LDL modifications have been strongly implicated in the pathogenesis of atherosclerosis, our data indicate that vitamin C could contribute to the anti-atherogenic defence against HOCl.