[Interaction of HIV-1 reverse transcriptase and bacteriophage T7 RNA polymerase with NTP phosphonate analogs and inorganic pyrophosphate]. / Vzaimodeistvie obratnoi transkriptazy VICh-1 i RNK-polimerazy bakteriofaga T7 s fosfonatnymi analogami NTP i neorganicheskogo pirofosfata.

We have examined the interaction of human immunodeficiency virus reverse transcriptase (HIV RT) and T7 RNA polymerase (T7 RNAP) with modified nucleoside triphosphates and inorganic pyrophosphate (PPi) analogs containing nonhydrolyzable bisphosphonate groups. We have synthesized a number of derivatives of bisphosphonic acid having different aromatic and nonaromatic side substituents, as well as the NTP derivatives whose incorporation into the growing nucleotide chain during the polymerization reaction results in formation of bisphosphonates as leaving groups. The competitive character of inhibition of both enzymes has been revealed for all the compounds under study, and the inhibition constants have been estimated. One of PPi analogs containing a bulky aromatic substituent is characterized by similar inhibition constants for both T7 RNAP and RT. The universal character of this inhibitor can serve as evidence for a similar structure of the NPT-binding sites in the two polymerases. It has been shown that nonsubstituted methylenebisphosphate is a better leaving group than that containing additional methyl and hydroxyl groups. The NTP analogs are very weak inhibitors of T7 RNAP, whereas HIV-1 RT is more sensitive to this type of compounds. On the basis of the X-ray crystallographic data on the T7 RNAP complex with a template and NTP, we have modeled the binding of some derivatives of bisphosphonic acid in the active center of the enzyme. The peculiarities observed in the model correlate well with the experimental data on inhibition.

[Structural-functional study of recombinant forms of onconase]. / Strukturno-funktsional'noe issledovanie rekombinantnykh form onkonazy.

A method for expression of an onconase gene leading to a soluble form of the protein was developed. The enzymatic and cytotoxic properties of the protein's recombinant forms were studied. Recombinant onconase with an additional N-terminal Met residue isolated in nondenaturing conditions did not substantially differ from the native enzyme in ribonucleolytic activity. The addition of a 33-mer peptide containing auxiliary elements for the simplification of isolation and detection of the recombinant protein did not affect the enzyme properties of onconase. The method proposed is useful for the onconase structure-function relation studies and enables construction of onconase-based fusion proteins for anticancer therapy.

[New acetylcholinesterase inhibitors--derivatives of vitamin B6]. / Novye ingibitory atsetilkholinésterazy--proizvodnye vitamina B6.

A new approach to design of reversible inhibitors of acetylcholinesterase (ACHE)--derivatives of natural compounds--has been worked out, as exemplified by vitamins of the B6 group. Analysis of the data obtained revealed main structural elements of the 3-hydroxypyridine molecule related to the inhibitory properties. Pyridoxamine derivatives are of interest in constructing new potent inhibitors of ACHE.

[Barnase mutant Ser57Ala: preparation and properties]. / Mutant barnazy Ser57Ala: poluchenie i svoistva.

Barnase, an extracellular ribonuclease produced by Bacillus amyloliquefaciens, belongs to a family of small microbial ribonucleases with similar structure and properties. These enzymes hydrolyze phosphodiester bonds on the 3' side of guanosine nucleotides in RNA. The guanylic specificity of barnase is more pronounced in the hydrolysis of dinucleotides or cyclonucleotide phosphates as substrates than in the hydrolysis of RNA or polynucleotides. To have an insight into the molecular basis of this phenomenon, we mutated amino acid residue Ser-57 in the "base recognition loop" of RNase Ba. The mutant protein was expressed in Escherichia coli producing system and purified for the study of the kinetic properties in the cleavage polynucleotide reactions. It was shown that the mutation of amino acid residue Ser-57 for Ala in the "recognition loop" of RNase Ba does not significantly influence the kinetic parametres of hydrolysis of polynucleotide substrates.

[Nonpolar interactions in phospholipase A2 molecules]. / Nepoliarnye vzaimodeistviia v molekule fosfolipazy A2.

Nonpolar regions (hydrophobic nuclei and microclusters) in porcine pancreatic phospholipase A2 have been investigated and described. Based on the atomic coordinates, we analyzed all nonpolar contacts for all nonpolar groups of each amino acid residue and identified three hydrophobic nuclei (gamma 1, gamma 2, gamma 3) and four microclusters (mu 4, mu 5, mu 6, mu 7) in the enzyme. These nonpolar regions include hydrophobic residues as well as residues with charged or polar side chain. Quantitative characteristics of hydrophobic nuclei show that the latter are condensations of nonpolar contacts in the protein. The stability of the nuclei is comparable to that of the secondary structure elements. Comparative analysis of nonpolar areas in native porcine pancreatic phospholipase A2, mutant enzyme, and its complex with a substrate analog demonstrated the role of nonpolar contacts in protein structure and function.

[Structure-activity analysis of ribonuclease A and related proteins]. / Strukturno-funktsional'nyi analiz ribonukleazy A i rodstvennykh belkov.

A detailed analysis of noncovalent interactions in the RNase A molecule was carried out. For this purpose, contact maps based on structural data were constructed. Three hydrophobic nuclei and five microclusters were identified, and their quantitative parameters were calculated. The contacts between amino acid residues of the active center and the hydrophobic nuclei were established. The distribution of the charged amino acid residues on the of RNase A surface was shown. The data obtained was discussed in the context of the ribonuclease A family. The substitutions seen from the alignment of amino acid sequences in the family might have influence on the spatial structure of different parts of RNase A and their mutual orientation and, consequently on the biological activity of family members.

[Formation and properties of S-protein complexes with S-peptide-containing hybrid polypeptides]. / Obrazovanie i svoistva kompleksov S-belka s S-peptidsoderzhashchimi gibridnymi polipeptidami.

The fused polypeptides of human epidermal growth factor and one or two S-peptide of RNase A was shown to form stoichiometric (1:1) strong noncovalent and enzymatically active complexes with S-protein of RNase A. The dissociation constants for these complexes were found to be 5.0 x 10(-7) M and 1.1 x 10(-7) M. The complexes of polypeptides with S-protein were capable to hydrolyze ribopolynucleotides, and pyrimidine-2',3'-cyclophosphates specifically, like RNase S'. A possibility was shown of effective purification of the S-peptide-containing polypeptides by affinity chromatography in which S-protein is immobilized on solid supports.

[Extracellular alkaline ribonuclease of Bacillus thuringiensis var. subtoxicus]. / Vnekletochnaia shchelochnaia ribonukleaza Bacillus thuringiensis var. subtoxicus.

Intraspecific selection of Bacillus thuringiensis strains producing extracellular alkaline ribonucleases was carried out. Subtoxicus subspecies with increased expression of the enzyme was detected. A method was developed to isolate preparative amounts of homogeneous extracellular RNase of B. thuringiensis var. subtoxicus. The physico-chemical and catalytic properties of the enzyme was studied and compared with extracellular RNases of others Bacillus species. The conclusion about the structural and evolutional conservation of Bacillus extracellular RNases was drawn.

[Conservativeness of residues in nonpolar nuclei of microbial ribonucleases]. / Konservativnost' ostatkov v nepoliarnykh iadrakh mikrobnykh ribonukleaz.

We present the application of a new method for analysis of nonpolar structure of proteins. A detailed analysis of the composition and properties of nonpolar nuclei and microclusters of microbial ribonucleases with known sequence have been carried out on the basis of 3D-structure of RNase Pbi and that of RNase Ti. It has been shown that all residues in nonpolar nuclei have high homology, about 95% for proteins with an identical scheme of S-S bridges and about 75% for nonidentical scheme of S-S bridges. The stability of nonpolar nuclei, conservation of their composition and their position in the protein globule allows one to assume that they play an important functional role in protein structure and possibly can be considered as independent structural elements of 3D-structure of a protein.

[Directed synthesis of 2',5'-oligoadenylates with increased stability towards phosphodiesterases]. / Napravlennoe sozdanie 2',5'-oligoadenilatov s povyshennoi ustoichivost'iu k fosfodiésterazam.

Phosphodiesterase stability of synthetic analogs of 2',5'-oligoadenylates, the mediators of antiviral and antiproliferative action of interferons was analysed. The analogs with a 3'-terminal acyclic nucleoside residue were prepared. These analogs were treated with NIH3T3 cell lysate, mice liver homogenate and snake venom phosphodiesterase. All analogs have demonstrated a high stability as compared with the natural 2',5'-oligoadenylate and its 3'-deoxyderivative. The possible biological activity of these stable analogs of 2',5'-oligoadenylates is discussed.

[Extracellular ribonuclease from Bacillus thuringiensis]. / Vnekletochnaia ribonukleaza iz Bacillus thuringiensis.

The ability of the strain Bacillus thuringiensis var. subtoxicus to produce extracellular ribonuclease (ribonuclease Bt) was studied. It was found that the culture medium possesses a RNA-depolymerizing activity whose maximum is observed 4-5 hours after the beginning of the linear growth phase. A three-step chromatography of the culture extract on phosphocellulose resulted in a homogeneous enzyme with a molecular mass of 12000 Da. The enzyme showed the maximum activity towards RNA at pH 8.5, catalyzed the hydrolysis of polyribonucleotides and guanosine-2',3'-cyclophosphate. Hence, the enzyme can be related to base-nonspecific cyclizing ribonucleases showing the guanylic specificity towards nucleoside-2',3'-cyclophosphates.

[5'-C-methylnucleoside triphosphates in the reaction of RNA synthesis catalyzed by Escherichia coli RNA-polymerase]. / 5'-C-methylnukleozidtrifosfaty v reaktsii sinteza RNK, kataliziruemoi RNK-polimerazoi Escherichia coli.

It was shown that RNA-polymerase is able to discriminate diastereoisomers of 5'-methyl-substituted analogs of ribonucleoside triphosphates (rNTP). Under conditions of soil substrate reactions when the analog is added to the presynthesized ternary complexes, D-allo- and L-talo-stereoisomers incorporate into RNA 100 and 1000 times, respectively, less effectively, then the natural rNTP. The effectivities of incorporation of other 2'- and 3'-substituted analogs of rNTP were measured under the same conditions and compared with that for 5'-Me-rNTP. It was shown also that RNA-polymerase does not support long-chain RNA synthesis from 5'-Me-rNTP in the absence of natural rNTP. No more then two analog residues can be attached to the 3'-end of the presynthesized RNA under such conditions. Addition of one natural rNTP to this reaction mixture results in the synthesis of long alternating RNA containing D-allo-stereoisomer and natural rNTP residues. In the case of L-talo-stereoisomer RNA elongation is not inhibited, if the distance between the analog residues in the RNA chain is not shorter then five nucleotide residues. The rate of pyrophosphorolysis from the RNA of the analogs studied was the same as for the natural rNTP residues.

[Metabolism of oligoadenylates in cell nuclei and regulation of protein ADP-ribosylation]. / Metabolizm oligoadenilatov v kletochnykh iadrakh i reguliatsiia ADP-ribozilirovaniia belkov.

2',5'-Oligo(A)synthetase (2-5A) and 2-phosphodiesterase were found in the L cells nuclei. In the cell nuclei 2-5A is 10-30 times higher, than in the cytoplasm. It is induced by interferon and depends on the cell growth state. 2-Phosphodiesterase activity has two pH optima of hydrolysis of 2-5A, namely 7.1, and 7.9 and decreases after interferon treatment of cells. Thus, interferon treatment of cells leads to an increase of the 2-5A level in cell nuclei. One of the possible pathways for 2-5A action in cell nuclei is the regulation of (ADP-ribose)transferase activity. Treatment of L cells with 2-5A (A2pA2pA) leads to activation of ADP-ribosylation of proteins by a factor of 1.5 in a concentration range of 10(-9)-10(-7) M, but more higher concentrations of 2-5A inhibit this process up to 60%. Treatment of cells with actinomycin D has no influence on 2-5A induced changes in protein ADP-ribosylation. This result is indicative for a new pathway of interferon action and 2-5A mediated regulation of cell metabolism.

[1-(3-C-methyl-beta-D-ribofuranosyl)uracil-5'-phosphate--a terminator of RNA synthesis catalyzed by RNA polymerase from E. coli]. / 1-(3'-C-metil-beta-D-ribofuranozil)uratsil-5'-trifosfat--terminator sinteza RNK, kataliziruemogo RNK-polimerazoi E. coli.

It is shown that 1-(3'-C-methyl-beta-D-ribofuranosyl)uracil 5'-triphosphate is a terminator of RNA synthesis and may be used for nucleic acid sequencing with DNA-dependent RNA polymerase from E. coli.

[Regulation of the 2',5'-oligoadenylate level in mouse L cells]. / Reguliatsiia urovnia 2',5'-oligoadenilatov v L-kletkakh myshei.

A study of pH dependence for ppp5'A2'p5'A2'p5'A hydrolysis in interferon treated and untreated mouse L-cells extracts led to the detection of two types of the 2'-phosphodiesterase activities: interferon dependent and interferon resistant. Several pH-optima were observed for hydrolysis of ppp5'A2'p5'A2'p5'A in cell extracts after their treatment with non-ionic detergent NP-40 or their differential centrifugation. The 2'-phosphodiesterase activity was found in the membrane fraction as well as in the cytoplasmic one. The presence of several pH-optima for 2'-phosphodiesterase activity in L-cells and changes of the level of this activity depending on the growth stage of cells and time of their interferon treatment indicate the complicated character of the regulation of 2'-5'-oligoadenylate's concentration and localization. The results obtained suggest that in mouse L-cells several 2'-phosphodiesterases or one enzyme in different forms may be present.

[Topochemical aspects of pyrimidine specificity of ribonuclease A]. / Topokhimicheskie aspekty pirimidinovoi spetsifichnosti ribonukleazy A.

To get insight into the origin of pyrimidine specificity of ribonuclease A, a study of the enzyme interaction with the substrate analogs having a modified nucleobase was undertaken. Pyridine and pyrimidine cyclophosphates were obtained by phosphorylation of 2'(3')-O-dibutyl stannylidene derivatives of nonprotected nucleosides in high yields. The results of kinetic and NMR studies suggested that a substrate should be locked in anti-conformation in the productive enzyme-substrate complex as it was shown for the crystalline complexes of the enzyme with pyrimidine nucleotides by X-ray analysis. The interaction between carbonyl group in position 2 of substrate nucleobase and proton accepting group of the protein (NH of Thr45) was found to be a prerequisite for the specific recognition of a substrate by the enzyme. The rate constants for transformation of lactam form (slow) and lactim form (fast) of pyrimidine substrates were estimated.