[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.

Stereoisomeric specificity of the retinoid cycle in the vertebrate retina.

Understanding of the stereospecificity of enzymatic reactions that regenerate the universal chromophore required to sustain vision in vertebrates, 11-cis-retinal, is needed for an accurate molecular model of retinoid transformations. In rod outer segments (ROS), the redox reaction involves all-trans-retinal and pro-S-NADPH that results in the production of pro-R-all-trans-retinol. A recently identified all-trans-retinol dehydrogenase (photoreceptor retinol dehydrogenase) displays identical stereospecificity to that of the ROS enzyme(s). This result is unusual, because photoreceptor retinol dehydrogenase is a member of a short chain alcohol dehydrogenase family, which is often pro-S-specific toward their hydrophobic alcohol substrates. The second redox reaction occurring in retinal pigment epithelium, oxidation of 11-cis-retinol, which is largely catalyzed by abundantly expressed 11-cis-retinol dehydrogenase, is pro-S-specific to both 11-cis-retinol and NADH. However, there is notable presence of pro-R-specific activities. Therefore, multiple retinol dehydrogenases are involved in regeneration of 11-cis-retinal. Finally, the cellular retinaldehyde-binding protein-induced isomerization of all-trans-retinol to 11-cis-retinol proceeds with inversion of configuration at the C(15) carbon of retinol. Together, these results provide important additions to our understanding of retinoid transformations in the eye and a prelude for in vivo studies that ultimately may result in efficient pharmacological intervention to restore and prevent deterioration of vision in several inherited eye diseases.

[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.

Effects of mutations in the calcium-binding sites of recoverin on its calcium affinity: evidence for successive filling of the calcium binding sites.

A molecule of the photoreceptor Ca(2+)-binding protein recoverin contains four potential EF-hand Ca(2+)-binding sites, of which only two, the second and the third, are capable of binding calcium ions. We have studied the effects of substitutions in the second, third and fourth EF-hand sites of recoverin on its Ca(2+)-binding properties and some other characteristics, using intrinsic fluorescence, circular dichroism spectroscopy and differential scanning microcalorimetry. The interaction of the two operating binding sites of wild-type recoverin with calcium increases the protein's thermal stability, but makes the environment around the tryptophan residues more flexible. The amino acid substitution in the EF-hand 3 (E121Q) totally abolishes the high calcium affinity of recoverin, while the mutation in the EF-hand 2 (E85Q) causes only a moderate decrease in calcium binding. Based on this evidence, we suggest that the binding of calcium ions to recoverin is a sequential process with the EF-hand 3 being filled first. Estimation of Ca(2+)-binding constants according to the sequential binding scheme gave the values 3.7 x 10(6) and 3.1 x 10(5) M(-1) for third and second EF-hands, respectively. The substitutions in the EF-hand 2 or 3 (or in both the sites simultaneously) do not disturb significantly either tertiary or secondary structure of the apo-protein. Amino acid substitutions, which have been designed to restore the calcium affinity of the EF-hand 4 (G160D, K161E, K162N, D165G and K166Q), increase the calcium capacity and affinity of recoverin but also perturb the protein structure and decrease the thermostability of its apo-form.

[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.

Obtaining and characterization of EF-hand mutants of recoverin.

Several EF-hand recoverin mutants were obtained and their abilities to bind to photoreceptor membranes and to inhibit rhodopsin kinase were determined. The mutants with the 'spoiled' 2nd, 3rd or (2nd+3rd) EF-hand structures did not act upon the kinase activity in the microM range of Ca2+ concentrations. Mutations of the 4th EF hand, which 'repaired' its Ca2+-binding activity, resulted in recoverin with three 'working' Ca2+-binding sites. The latter mutant inhibited rhodopsin kinase even more effectively than the wild-type recoverin, containing two working Ca2+-binding structures.

[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.

N-myristoylation of recoverin enhances its efficiency as an inhibitor of rhodopsin kinase.

Recoverin, a recently identified member of the EF-hand superfamily of Ca(2+)-binding proteins, is capable to inhibit rhodopsin phosphorylation by rhodopsin kinase at high but not at low free [Ca2+]. The N-terminal glycine residue of retinal recoverin is heterogeneously acylated with myristoyl or related N-acyl group. To clarify the role of the N-terminal acylation of recoverin in its inhibitory action upon rhodopsin phosphorylation, we compared the efficiency of myristoylated and non-myristoylated forms of recombinant recoverin as inhibitors of rhodopsin kinase activity. We have found that rhodopsin phosphorylation by purified rhodopsin kinase, which does not depend on free [Ca2+] in the absence of recoverin, is regulated by Ca2+ in the presence of both forms of the recombinant protein. EC50 values for Ca2+ are the same (2 microM) for the myristoylated and non-myristoylated forms; the Hill coefficients of 1.7 and 0.9, respectively, indicate that the effect is cooperative with respect to Ca2+ only for myristoylated recoverin. In the presence of Ca2+, both forms of recoverin taken at saturated concentrations cause an almost equal inhibition of rhodopsin phosphorylation. However, the inhibitory action of the myristoylated form occurs at much lower its concentrations than that of the non-myristoylated form (EC50 are 0.9 and 6.5 microM, respectively).

[Oligonucleotide-directed mutagenesis of inhibitory gamma-subunits of cGMP phosphodiesterase from bovine outer rod segments. A new hypothesis on mechanisms for inhibiting catalytic subunits by gamma-subunits and activation of a holoenzyme by transducin]. / Oligonukleotidnapravlennyi mutagenez ingibitornoi gamma-sub''edinitsy fosfodiésterazy cGMP iz naruzhnykh segmentov palochek setchatki byka. Novaia gipoteza o mekhanizmakh ingibirovaniia kataliticheskikh sub''edinitz gamma-sub''edinitsei ii aktivatsii kholofermenta transdutsinom.

Two mutants of the phosphodiesterase (PDE) gamma subunit (PDE gamma) from bovine retinal rods were synthesized by sequential transcription and translation in vitro. PDE gamma mutants R24E and H79L exhibited inhibitory properties similar to those of the wild-type PDE gamma (wtPDE gamma). At the same time, affinity to the rod outer segment (ROS) membranes is lower for R24E and higher for H79L in comparison with wtPDE gamma. The transducin alpha subunit (in a complex with the GTP non-hydrolyzable analogue, GTP gamma S) activates the trypsin-treated PDE (tPDE) inhibited by wtPDE gamma weaker than tPDE inhibited by R24E and stronger than tPDE inhibited by H79L. To explain the properties of these and earlier studied PDE gamma mutants, a new hypothesis on the mechanisms of inhibition of the PDE catalytic subunit dimer (PDE alpha beta) by PDE gamma and mechanism of the PDE holoenzyme (PDE alpha beta gamma 2) activation by the transducin alpha subunit in a complex with GTP (T alpha.GTP) is proposed: 1) two sites on PDE alpha beta for the PDE gamma binding (A- and the B-site) are different in structure. Sites on PDE gamma interacting with A- and the B-sites on PDE alpha beta are also different in structure. The site on PDE gamma interacting with the B-site partially overlaps with the T alpha.GTP binding site; 2) PDE gamma bound to the B-site provides the main contribution to inhibition of the enzyme catalytic activity; 3) T alpha.GTP first interacts with the PDE gamma bound to the A-site in the PDE holoenzyme and removes this PDE gamma in a PDE gamma.(T alpha.GTP) complex. This results in a slight increase of the catalytic activity of the PDE alpha beta gamma complex remaining bound to the ROS membranes; 4) after removal of PDE gamma from the A-site, another T alpha.GTP molecule is enabled to interact with both PDE alpha beta and PDE gamma bound to the B-site on PDE alpha beta. This interaction results in the formation of a ROS membrane-bound fully catalytically active triple complex PDE alpha beta.PDE gamma.(T alpha.GTP).

[The effect of the phage lambda ral gene on the level of synthesis of the EcoK restriction endonuclease beta-subunit]. / Vliianie gena ral faga lambda na uroven' sinteza beta-sub''edinitsy éndonukleazy restriktsii EcoK.

E. coli hsd genes were subcloned from lambda 642 (ral+) into lambda L47.1 vector (ral-after replacement). The influence of bacteriophage lambda ral gene on the expression efficiency of hsdS kappa, hsdM kappa genes was investigated. It was shown, that its presence in vitro enhanced the synthesis of beta-subunit, hsdM gene product, and the increase of modification in vivo was observed. It is proposed that the increase of modification rate of lambda phage fully unmodified DNA is connected with the appearance of E. coli DNA methylase consisting of beta- and gamma-subunits but lacking alpha-subunit.

[Effect of gentamicin and pentoxyl on the immunological reactivity of the body in the treatment of sepsis caused by Ps. aeruginosa]. / Vliianie gentamitsina i pentoksila na immunologicheskuiu reaktivnost' organizma pri lechenii sepsisa, vyzvannogo Ps. aeruginosa

The state of immunological reactivity was studied on 250 albino mice treated with gentamycin and pentoxyl used alone or in combination because of sepsis caused by Ps. aeruginosa. Morphological changes in the spleen and lymph nodes, as well as the dynamics of the agglutinins accumulation in the blood serum were investigated. Gentamycin had no significant effect on the plasmocytal reaction in the lymph nodes and at the same time lowered the number of the immunocompetent cells in the spleen and the titer of agglutinins in the blood serum. The use of gentamycin in combination with pentoxyl had a stimulating effect on transformation of the plasmic cells in the lymphoid organs and resulted in increased titers of agglutinins in the blood serum of the animals treated.