Effect of General Anesthesia Duration on Recovery of Secretion and Biochemical Properties of Tear Fluid in the Post-Anesthetic Period.

Changes in the biochemical composition of the tear film is a critical risk factor for the development of chronic perioperative dry eye syndrome, because increasing the duration of general anesthesia did not affect the dynamics of tear secretion recovery, but slowed down normalization of its structure and antioxidant activity in the post-anesthetic period.

[Visual transduction and calcium ions]. / Zritel'naia transduktsiia i iony kal'tsiia.

A general scheme of visual induction and its regulation in mammalian retinal rods and the main signal proteins involved in the functioning of the visual signaling apparatus were considered. The role of calcium ions and calcium-binding proteins in the switching off of the visual signal and the transition of the photoreceptor cell from a photoexcited to a "dark" state was discussed in detail.

[An immunohistochemical study of localization of the calcium-binding protein recoverin in retina of the newt Pleurodeles waltl]. / Immunokhimicheskoe izuchenie lokalizatsii kal'tsii-sviazyvaiushchego belka rekoverina v setchatke tritona Pleurodeles waltl.

The presence and localization of the calcium-binding protein recoverin, initially found in photoreceptors of the bovine eye, were immunochemically studied in retina of the new Pleurodeles waltl. Polyclonal monospecific antibodies against recoverin were raised and the methods of immunoblotting and indirect immunofluorescence were used. A protein with an apparent molecular mass of 26 kDa was found in the retina extract, which was specifically stained by the antibodies against recoverin. Localization of recoverin was studied on the retina sections: an intense reaction was found in the inner segments and a weak reaction was found in the basal part of the outer segments of photoreceptors and in Landolt's clubs of displaced bipolars. The results we obtained suggest for the first time the presence of recoverin in the retina of a representative of the Urodeles and indicate to interspecific conservativeness of this protein and differences of its localization in the retina photoreceptors in different species. The data obtained open a possibility of using recoverin as a marker protein of photoreceptors and displaced bipolars in studied of retina regeneration in newts.

[The effect of proteolytic removal of the C-terminal fragment of rhodopsin on its ability to activate visual cascade]. / Vliianie proteoliticheskogo otshchepleniia C-kontsevogo fragmenta rodopsina na ego sposobnost' aktivirovat' zritel'nyi kaskad.

The role of the C-terminal domain of rhodopsin in the activation of transducin was studied. The treatment of photoreceptor membranes with trypsin, thermolysin, and Asp-N-endoprotease led to the respective rhodopsin species devoid of 9, 12-, or 19-aa C-terminal fragments. It was shown that the removal of 9-aa fragment by trypsin does not affect the catalytic activity of the receptor, whereas the thermolysin-induced truncation of the rhodopsin C-terminus by 12 aa about 1.5-fold enhances its activity. The Asp-N-endoprotease-assisted removal of 19 aa (i.e., the shortening by seven more C-terminal aa) virtually unchanges the rhodopsin catalytic activity compared to the preparation truncated with thermolysin. These results suggest that the part of the rhodopsin C-terminal fragment between the sites of its cleavage by trypsin and thermolysin (Val337-Ser338-Lys339) inhibits the signal transduction from rhodopsin to the next component of visual cascade. The English version of the paper.

[Point amino acid substitutions in Ca2+-binding centers of recoverin. III. Mutant with the fourth reconstructed Ca2+-binding site]. / Tochechnye aminokislotnye zameny v Ca2+-sviazyvaiushchikh tsentrakh rekoverina. III. Mutant s chetvertym rekonstruirovannym Ca2+-sviazyvaiushchim tsentrom.

Unlike wild type recoverin with only two (the second and the third) functioning Ca(2+)-binding sites out of four potential ones, the +EF4 mutant contains a third active Ca(2+)-binding site. This site was reconstructed from the fourth potential Ca(2+)-binding domain by the introduction of several amino acid substitutions in it by site-directed mutagenesis. The effect of these mutations in the fourth potential Ca(2+)-binding site of myristoylated recoverin on the structural features and conformational stability of the protein was studied by fluorimetry and circular dichroism. The apoform of the resulting mutant (free of Ca2+ ions) was shown to have a higher calcium capacity, significantly lower thermal stability, and noticeably different secondary and tertiary structures as compared with the apoform of wild type recoverin.

[Single amino acid substitutions in the Ca2+-binding site of recoverin.II.The unusual behavior of the protein upon the binding of calcium ions]. / Tochechnye aminokislotnye zameny v Ca2+-sviazyvaiushchikh tsentra rekoverina. II.Neobychnoe povedenie belka pri vzaimodeistvii s ionami kal'tsiia.

The structural properties of myristoylated forms of recombinant recoverin of the wild type and of its mutants with damaged second and/or third Ca(2+)-binding sites were studied by fluorimetry and circular dichroism. The interaction of wild-type recoverin with calcium ions was shown to induce unusual structural rearrangements in its molecule. In particular, protein binding with Ca2+ ions results in an increase in the mobility of the environment of Trp residues, in higher hydrophobicity, and in elevated thermal stability (its thermal transition shifts by 15 degrees C to higher temperatures) but has almost no effect on its secondary structure. Similar structural changes induced by Ca2+ are also characteristic of the -EF2 mutant of recoverin whose second Ca(2+)-binding site is modified and cannot bind calcium ions. The structural properties of the -EF3 and -EF2,3 mutants (whose third or simultaneously second and third Ca(2+)-binding sites, respectively, are modified and damaged) are practically indifferent to calcium ions.

[Point amino acid substitutions in the Ca2+-binding centers of recoverin. I. Mechanism of successive filling of Ca2+-binding centers]. / Tochechnye aminokislotnye zameny v Ca2+-sviazyvaiushchikh tsentrakh rekoverina. I. Mekhanizm posledovatel'nogo zapolneniia Ca2+-sviazyvaiushchikh tsentrov.

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.

[Preparation of the myristoylated and nonmyristoylated form of recombinant recoverin in E. coli cells and comparison of their functional activity]. / Poluchenie miristoilirovannoi i nemiristoilirovannoi form rekombinantnogo rekoverina v kletkakh E. coli i srvanenenie ikh funktsional'noi aktivnosti.

A recombinant plasmid was constructed for expressing a gene for bovine recoverin under the control of the lac promoter. Coexpression of the recoverin and N-myristoyl transferase genes was performed to prepare recombinant myristoylated recoverin. The obtained systems provide high levels of biosynthesis of the recombinant recoverins in the E. coli cells. Using a reconstructed system, containing urea-washed rod outer segment membranes, purified rhodopsin kinase (RK), and a recoverin, it was shown that the three recoverin forms (natural, recombinant nonmyristoylated, and recombinant myristoylated ones) perform the calcium-dependent regulation of the activity of RK with half a maximum effect at a free calcium concentration of 2 microM. Interestingly, the N-terminal myristoylation of recoverin increased substantially its functional activity.

[Function of the calcium-binding protein p26 (recoverin) in bovine retinal rods]. / Izuchenie funktsii kal'tsiisviazyvaiushchego belka p26 (rekoverina) v palochkakh setchatki byka.

The bovine retina rod cell protein with an apparent M(r) 26 kDa (p26 [1] or recoverin [2]) was suggested to be a calcium-sensitive regulator of photoreceptor guanylate cyclase. The data obtained show that highly purified p26 is not capable of restoring guanylate cyclase in washed ROS membranes up to the level of a ROS suspension. At the same time we found that a Ca(2+)-sensitive complex of p26 and a protein with apparent M(r) 67 kDa, presumably rhodopsin kinase, is present in the ROS extract. We suggest that rhodopsin kinase can be a functional target for p26 in retina rod cells.

[New 26 kDa protein specific for photoreceptor cells, capable of binding with immobilized delipidized rhodopsin]. / Novyy spetsifichnyi dlia fotoretseptornykh kletok belok s molekuliarnoi massoi 26 kDa, sposobnyi sviazyvat'sia s immobilizvannym delipidizirovannym rodopsinom.

A protein p26 with molecular weight 26 kDa capable of binding to delipidated rhodopsin immobilized on Concanavalin A-Sepharose was found in photoreceptor cells of bovine retina. Mono specific antibodies against this protein were used to demonstrate this protein to be located in a layer of photoreceptor cells, both in their inner and outer segments. On the basis of its antigenic properties p26 is different from any other known photoreceptor cells-specific proteins.

[A study of the interaction of immobilized delipidized rhodopsin with G-proteins]. / Izuchenie vzaimodeistviia immobilizovannogo delipidizirovannogo rodopsina s G-belkami.

It was shown that delipidated rhodopsin immobilized on concanavalin A-Sepharose (Rimm) binds with high selectivity transducin from total extracts of rod outer segment protein as well as the regulatory GTP-binding Gi- and Go-like proteins from solubilized membranes of bovine brain. The Rimm-bound proteins are eluted in the presence of the nonionic detergent, octyl glucoside, and GTP. This suggests that Rimm can be used as an affinity adsorbent for the isolation and purification of G-proteins.

[The enzymology of visual reception: phosphodiesterase cascade of signal amplification]. / Enzimologiia zritel'noi retseptsii: fosfodiésteraznyi kaskad usileniia signala.

According to present-day concepts an important and, presumably, a key role in signal transmission in photoreceptor cells is ascribed to a system containing the photosensitive protein rhodopsin, GTP-binding protein transducin and cyclic GMP phosphodiesterase which in many features is similar to the adenylate cyclase system from other eukaryotic cells. The experimental and literary data concerning the already established and hypothetical mechanisms of transmission, enhancement and switch-off of the signal in the rhodopsin----transducin----phosphodiesterase chain are reviewed.

[The nature of binding between the coenzyme and protein in transketolase from the swine liver]. / O kharaktere sviazi mezhdu kofermentom i belkom v transketolaze pecheni svin'i.

An analysis of a proteolytic hydrolysate of pig liver transketolase by thin-layer chromatography revealed the presence of a coenzyme-containing material which differed from free thiamine pyrophosphate in chromatographic behaviour. This coenzyme-containing material is distinct from the free coenzyme in terms of other properties as well, e.g., stability, pH dependence of thiochrome fluorescence, etc. It was demonstrated that incubation of enzyme preparations possessing a high specific activity (on the average, 2 E/mg) in acidic acetate buffer caused no or little detachment of the coenzyme, mainly in the composition of the heterogeneous material which, at least partly, was not represented by thiamine pyrophosphate.

[Pig liver transketolase: covalent binding between the coenzyme and protein]. / Transketolaza iz pecheni svin'i: kovalentnaia sviaz' mezhdu kofermentom i belkom.

Transketolase from pig liver contains a tightly bound coenzyme, which is not split off after severe treatment (e.g. boiling for 12 hrs in 1 n. HCl, 30 min boiling in 2 n HCl, boiling in 1% DS-Na, standing in 6 M solution of quanidine chloride, etc.). The proteolysis of the enzyme results in a coenzyme-containing material differing in some of its properties from free thiamine by pyrophosphate. The data obtained suggest that pig liver transketolase contains covalently bound thiamine pyrophosphate.

[Properties of pig liver transketolase]. / Svoistva transketolazy iz pecheni svinyi.

Some properties of homogeneous transketolase from pig liver were studied. It was shown that the pH optimum of the transketolase reaction lies within the range of 7.8--8.2. The isoelectric point is at pH 7.6--7.8. The molecular weight of transketolase is 138,000 +/- 3,000 as determined by the sedimentation equilibrium method and about 152,000 according to the data from gel filtration through Sephadex G-200. The enzyme molecule is a tetramer of the alpha 2 beta 2 type. The molecular weights of the alpha- and beta- subunits determined by polyacrylamide gel in the presence of sodium dodecyl sulfate are 52,000--56,000 and 27,000--29,000, respectively. Transketolase contains about two moles of TPP per mole of protein and does not require metal ions for its catalytic activity.