[Introduction of additional thiol groups into glucoamylase in Aspergillus awamori and their effect on the thermal stability and catalytic activity of the enzyme].

Five mutant forms of glucoamylase (GA) from the filamentous fungus Aspergillus awamori with artificial disulfide bonds (4D-G137A\A14C, 6D-A14C\Y419C\G137A, 10D-V13C\G396C, 11D-V13C\G396C\A14C\Y419C\G137A, and 20D-G137A\A246C\A14C) were constructed using computer simulation and experimentally tested for thermostability. The introduction of two additional disulfide bonds between its first and thirteenth alpha-helices and that of the loop located close to a catalytic residue--E400--made it possible to assess the effects of disulfide bridges on protein thermostability. The mutant proteins with combined amino acid substitutions G137A\A14C, V13C\G396C\A14C\Y419C\G137A, and G137A\A246C\A14C showed higher thermal stability as compared to the wild-type protein. At the same time, new disulfide bridges in the mutant A14C\Y419C\G137A and V13C\G396C proteins led to the destabilization of their structure and the loss of thermal stability.

[The effect of point amino acid substitutions in an internal alpha-helix on thermostability of Aspergillus awamori X100 glucoamylase].

Conformational flexibility of alpha-helices in glucoamylase of the fungus Aspergillus awamori was studied by molecular dynamics methods. Several amino acid substitutions (G127A, P128A, I136L, G137A, and G139A) optimizing intrinsic interactions in one of the alpha-helices (D) within the hydrophobic core of this protein were constructed and studied. It was found that these point mutations had different effects on the glucoamylase thermal inactivation constant. Unlike amino acid substitution P128A and substitutions G137A and A246C, I136L and G139A displayed a pronounced additive thermostabilizing effect.

[Destabilization and stabilization of poly(A).poly(U) structure by platinum(II) compounds].

On the basis of molecular biophysics, a methodology for the analysis of intramolecular structural order of the polynucleotide duplex poly(A).poly(U) has been developed. It was shown that the combination of circular dichroism spectroscopy with differential scanning calorimetry is an optimal approach, which ensures the screening of a wide set of substances and interaction conditions and the choice of compound(s) that can stabilize the structure and increase the biological activity of this duplex. The study is aimed at obtaining a new and highly active antiviral remedy.

Template-dependent biosynthesis of poly(G) x poly (C) and its antiviral activity in vitro and in vivo.

Experimental conditions for poly(G) synthesis from GTP on a poly(C) template with the aid of Escherichia coli DNA-dependent RNA polymerase were investigated. The reaction product was purified without the use of RNase. On the basis of spectral data, gel permeation chromatography, affinity adsorption and electron microscopic visualization, the poly(G) x poly(C) product was assumed to possess a high degree of structural regularity. Its in vitro and in vivo antiviral activities were compared with those of traditional poly(G) x poly(C) and poly(I) x poly(C). Template-dependent poly(G) x poly(C) was similar in its in vitro activity to poly(I) x poly(C) or even surpassed it, whereas the 'traditional' poly(G) x poly(C) was only slightly active in vitro. However, 'traditional' poly(G) x poly(C) and poly(I) x poly(C) had similar activity in vivo, whereas template-dependent poly(G) x poly(C) was much less active in vivo. The role of intramolecular structural regularity in the in vitro and in vivo antiviral activity of polyribonucleotide duplexes is discussed.

[Interrelationship between the chain length of poly-L-lysine and the degree of protection of polyribonucleotide interferon inducers from human blood nucleases]. / Vzaimosviaz' mezhdu dlinoi tsepi ékraniruiushchego poli-L-lizina i stepen'iu zashchity poliribonukleotidnykh induktorob interferona ot nukleas krovi cheloveka.

The action of the human total blood serum on polynucleotide interferon inducers, larifan and ridostin (natural double-stranded RNAs) and poly(I).poly(C) (a double-stranded complex of synthetic polyribonucleotides) used both in the free state and in the state shielded with poly-L-lysine was studied. The rate of the accumulation of the acid soluble products was compared with the residual interferon-inducing activity in mice. All the unshielded inducers were shown to completely loose their activity after a 4-hour contact with the serum. The protective activity of poly-L-lysine increased in parallel with the increase of its molecular weight and was maximal for the preparation with the molecular weight of 12300 +/- 1000 Da. Differences in the structure of the inducers and the mechanism of their biosynthesis and degradation must be taken into account.

[Resistance of natural and synthetic polyribonucleotide inducers of interferon to human blood ribonucleases]. / Ustoichivost' prirodnykh i sinteticheskikh poliribonukleotidnykh induktorov interferona k ribonukleazam chelovecheskoi krovi.

The resistance of polyribonucleotide inductors of interferon to blood ribonucleases was studied. Blood resistance of larifan and ridostin in the free and shielded state as well as that of the complexes of poly(I)-poly(C) and poly(G)-poly(C) were also investigated. A protective action of polylysine against the inductors was detected which, in case it had no effect on the biological activity of the drugs, could provide its recommendation as a compound for shielding the inductors.