Interaction of Cholera Toxin B-subunit with Human T-lymphocytes.

In this work, 125I-labeled cholera toxin B-subunit (CT-B) (specific activity 98 Ci/mmol) was prepared, and its high-affinity binding to human blood T-lymphocytes (Kd = 3.3 nM) was determined. The binding of the 125I-labeled CT-B was inhibited by unlabeled interferon-α2 (IFN-α2), thymosin-α1 (TM-α1), and by the synthetic peptide LKEKK, which corresponds to sequences 16-20 of human TM-α1 and 131-135 of IFN-α2 (Ki 0.8, 1.2, and 1.6 nM, respectively), but was not inhibited by the unlabeled synthetic peptide KKEKL with inverted sequence (Ki > 1 µM). In the concentration range of 10-1000 nM, both CT-B and peptide LKEKK dose-dependently increased the activity of soluble guanylate cyclase (sGC) but did not affect the activity of membrane-bound guanylate cyclase. The KKEKL peptide tested in parallel did not affect sGC activity. Thus, the CT-B and peptide LKEKK binding to a common receptor on the surface of T-lymphocytes leads to an increase in sGC activity.

Binding of Synthetic LKEKK Peptide to Human T-Lymphocytes.

The synthetic peptide LKEKK corresponding to sequence 16-20 of human thymosin-α1 and 131-135 of human interferon-α2 was labeled with tritium to specific activity 28 Ci/mol. The [3H]LKEKK bound with high affinity (Kd = 3.7 ± 0.3 nM) to donor blood T-lymphocytes. Treatment of cells with trypsin or proteinase K did not abolish [3H]LKEKK binding, suggesting the non-protein nature of the peptide receptor. The binding was inhibited by thymosin-α1, interferon-α2, and cholera toxin B subunit (Ki = 2.0 ± 0.3, 2.2 ± 0.2, and 3.6 ± 0.3 nM, respectively). Using [3H]LKEKK, we demonstrated the existence of a non-protein receptor common for thymosin-α1, interferon-α2, and cholera toxin B-subunit on donor blood T-lymphocytes.

Amino acid sequences of two immune-dominant epitopes of recoverin are involved in Ca2+/recoverin-dependent inhibition of phosphorylation of rhodopsin.

Antibodies AB(60-72) and AB(80-92) against two immune-dominant epitopes of photoreceptor Ca(2+)-binding protein recoverin, 60-DPKAYAQHVFRSF-72 and 80-LDFKEYVIALHMT-92, which can be exposed in a Ca(2+)-dependent manner, were obtained. The presence of AB(60-72) or AB(80-92) results in a slight increase in Ca(2+)-affinity of recoverin and does not affect significantly a Ca(2+)-myristoyl switch mechanism of the protein. However in the presence of AB(60-72) or AB(80-92) recoverin loses its ability to interact with rhodopsin kinase and consequently to perform a function of Ca(2+)-sensitive inhibitor of rhodopsin phosphorylation in photoreceptor cells.

[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.

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.

[The effect of pesticides on health indices and the test indicators for their determination]. / Vliiane pestitsidov na pokazateli zdorov'ia i testy-indikatory ikh opredeleniia.

It is suggested that causal-sequel relationships of the levels of morbidity and functional changes in the health condition of the population reflect the intensity in the use of pesticides and are locally manifested in conditions of extreme loads of pesticides. The authors distinguish priority unspecific tests-indicators according to nosological entities of pathology and indices of the functional state of the body that may be recommended for systemic evaluation of links with territorial loads of pesticides and for providing information in the monitoring of the health of the population in regions of intensive use of agrochemical agents.

[Determination of the activity of neurotoxic esterase in the peripheral blood lymphocytes for assessing the action of organophosphorus compounds]. / Opredelenie aktivnosti neirotoksicheskoi ésterazy v limfotsitakh perifericheskoi krovi dlia otsenki deistviia fosfororganicheskikh soedinenii.

It was shown on a model of intoxication of hens with aphos possessing a selective neuroparalytic action that the target-enzyme neurotoxic esterase changed its activity in the brain, spleen and lymphocytes of the peripheral blood. The authors describe a method of determination of the activity of neurotoxic esterase in human peripheral blood lymphocytes that may be used for biomonitoring of the effect of phosphorus organic compounds possessing a delayed neurotoxic action.