Point amino acid substitutions in the Ca2+-binding centers of recoverin. I. Mechanism of successive filling of Ca2+-binding centers

The molecule of photoreceptor Ca(2+)-binding protein recoverin contains four potential Ca(2+)-binding sites of the EF-hand type, but only two of them (the second and the third) can actually bind calcium ions. We studied the interaction of Ca2+ with recoverin and its mutant forms containing point amino acid substitutions at the working Ca(2+)-binding sites by measuring the intrinsic protein fluorescence and found that the substitution of Gln for Glu residues chelating Ca2+ in one (the second or the third) or simultaneously in both (the second and the third) Ca(2+)-binding sites changes the affinity of the protein to Ca2+ ions in different ways. The Gln for Glu121 substitution in the third site and the simultaneous Gln substitutions in the second (for Glu85) and in the third (for Glu121) sites result in the complete loss of the capability of recoverin for a strong binding of Ca(2+)-ions. On the other hand, the Gln for Glu85 substitution only in the second site moderately affects its affinity to the cation. Hence, we assumed that recoverin successively binds Ca(2+)-ions: the second site is filled with the cation only after the third site has been filled. The binding constants for the third and the second Ca(2+)-binding sites of recoverin determined by spectrofluorimetric titration are 3.7 x 10(6) and 3.1 x 10(5) M-1, respectively.     

Conjugates of 2',3'-didehydro-3'-deoxythymidine with thymogen. Synthesis and anti-HIV activity

An effective synthesis of thymogen was developed. Conjugates of 2',3'-didehydro-3'-deoxythymidine (nucleoside d4T) with thymogen were prepared in which the nucleoside hydroxyl group was linked to the thymogen carboxyl group of either tryprophan or glutamic acid residues. It was shown that the anti-HIV activity of the d4T-thymogene conjugate with the tryptophan linkage was comparable to that of d4T, whereas its cytotoxicity was nil. The d4T-tryptophan conjugate also displayed high anti-HIV activity.     

Comparative study of the reactability of phosphoric acid residues in tRNA(Ser) and tRNA(Leu) from Thermus thermophilus

The reactivity of phosphates in the Thermus thermophilus tRNA(Ser) (GCU) and tRNA(Leu) (CAG) was studied using the ethylnitrosourea modification. It was shown that phosphates of nucleotides 58-60 (T loop), 20-22 (D loop), and 48 (at the junction of the variable and T stems) were poorly modified in both tRNAs. The most pronounced differences in the reactivity were observed for phosphates at the junctions of the variable stem with T-stem (47q, 49) and anticodon stem (45). This indicates differences in orientations of the long variable arm relative to the backbone in the tRNAs studied.     

Characteristics of some stages of energy metabolism and antioxidant system in bone marrow myeloid cells and leukocytes from piglets

The age changes of enzymes of activity catalyzing several links of energy metabolism (hexokinase, phosphofructokinase, pyruvate kinase, lactate dehydrogenase, glucose-6-phosphate dehydrogenase, NADP-isocitrate dehydrogenase, cytochrome c-oxidase) and antioxidant system (superoxide dismutase and glutathione reductase) in bone marrow myeloid cells and blood leukocytes of pig in the 10-day period after birth were investigated. The bone marrow cells and leukocytes of the new born piglets were characterized by low intensity of oxidative steps of energy metabolism as well as by low activity of antioxidant enzymes. In the period of neonatal adaptation reorganization of energy metabolism, particularly, intensification of oxidative processes in the investigated cells occurred. It included the pentose phosphate way and cytochrome c-oxidase activation. During the neonatal period of development the functional activity of antioxidant enzymes in the investigated cells of piglets increased.     

Immune sensor for determining myoglobin level

For the first time, the photoluminescence of porous silicon was used to create an immune sensor capable of detection of low concentrations of myoglobin important for the diagnostics of heart failure diseases which in both model solutions and human serum, being. It is shown that the sensitivity of the sensor was 10 ng/ml, time of 1 measurement--15-30 min. Duration of the analysis can be even shorter if measurements are performed in the kinetic regime. The comparison of the characteristics of the immune sensor and standard method ELISA is also given in the paper.