[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.

[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.

[Dependence of the antiviral activity of the poly(G).poly(C) complex on the size of the continuous poly(C)segments]. / Zavisimost' protivovirusnoi aktivnosti kompleksa poli(G).poli(Ts) ot razmera nepreryvnykh uchastkov poli(Ts).

Modification of poly(C) by various frequency treatment with adenosine non-complementary to guanosine has produced poly(G) X poly (C.A) complexes with continuous double-stranded areas the length of which is determined by C/A ratio. Studies of the antiviral activity of poly(G).poly(C,A) complexes with C/A from 10:1 to 90:1 and poly(G).poly(C) in vesicular stomatitis virus-infected chick embryo cell cultures and in experimental tick-borne encephalitis of mice demonstrated that the maximum activity is achieved at an average lengths of double-stranded areas of 90 nucleotide pairs. At the same time, a low but statistically significant antiviral activity is observed at a length of double-stranded areas of 10-30 nucleotide pairs.

[Operon of riboflavin biosynthesis in Bacillus subtilis. XVII. A study of the regulatory functions of the intermediate products and their derivatives]. / Issledovanie operona biosinteza riboflavina u Bacillus subtiis. Soobshchenie XVII. Izuchenie reguliatornykh funktsii promezhutochnykh produktov i ikh proizvodnykh.

Repression of synthesis of GTP-cyclohydrolase and riboflavinsynthetase was studied in different regulatory mutants of Bacillus subtilis. The results of experiments with some riboflavin precursors and their derivatives revealed that 5-amino-2,6-dioxo-4-ribitylaminopyrimidine and 6-methyl-7-(1',2'-dioxyethyl)-8-ribityllumazine can serve as effectors in riboflavin biosynthesis.

[Riboflavin biosynthesis operon of Bacillus subtilis. XIV. Operator-constitutive mutants]. / Issledovanie operona biosinteza riboflavina u Bacillus subtilis. Soobshchenie XIV. Izuchenie operator-konstitutivnykh mutantov.

Numerous operator-constitutive mutants of riboflavin biosynthesis were selected. All of them map in a short region of the Bacillus subtilis chromosome. The yield of riboflavin synthetase from this mutant is different, but in most cases much lower than the maximal yield from a repressor minus strain. Our tentative explanation is a partial overlap of the sites for the adsorption of repressor and RNA-polymerase. Therefore the affinity to the transcribing enzyme is diminished in the operator constitutive strains. The affinity of the repressor-effector complex to the operator depends on the effector structure.

[Analgos of riboflavin, lumiflavin and alloxazine derivatives. II. Effect of roseoflavin on 6,7-dimethyl-8-ribityllumazine and riboflavin synthetase synthesis and growth of Bacillus subtilis]. / Analogi riboflavina, liumiflavina i proizvodnye alloksazina. Soobshchenie II. Vliianie rozeoflavina na sintez 6,7-dimetil-8-ribitillumazina, riboflavinsintetazy i rost Bacillus subtilis.

The replacement of 8-CH3 group in the riboflavin molecule results in the formation of specific antimetabolites. They are rozeoflavin, 7-desmethylrozeoflavin, 8-amino (nor) riboflavin, 8-ribitylamino (nor) riboflavin. Effect of rozeoflavin and other riboflavin analogues on the growth and regulatory characteristics of Bacillus subtilis strains with different genetic state of riboflavin operon is studied. Roseoflavin at a concentration of 0.05 mkg/ml inhibits DRL synthesis in rib-b110 strain. An analogue inhibits the growth of auxotrophic and prototrophic strains at concentrations of 0.5 mkg/ml and 50 mkg/ml respectively. Riboflavin (1 mkg/ml) recovers the growth of bacteria. The curve of rozeoflavin regulation of DRL and riboflavin synthetase synthesis is shifted in 100 times in the direction of lesser concentrations as compared with riboflavin and 8 amino (nor) riboflavin. 180 mutants resistant to 100 mkg/ml of rozeoflavin were selected. 150 mutants over-synthetize riboflavin.

[Operon study of riboflavin biosynthesis in Bacillus subtilis. XII. The determination of the ATP:riboflavin-5'-phosphotransferase and riboflavinsynthetase content in the cells with varying genotypes]. / Issledovanie operona biosinteza riboflavina u Bacillus subtilis. Soobshchenie XII. Opredelenie soderzhaniia ATF:riboflavin-5'-fosfotransferazy i riboflavinsintetazy v kletakh s razlichnym genotipom.

Activities of riboflavinkinase and riboflavinsynthetase were measured in 15 strains of Bacillus subtilis with different genotype. The increased level of riboflavinkinase was observed in strains, resistant to lumiflavin or lumichrome. Specific activity of riboflavinkinase was found to be about 100 times lower than that of riboflavinsynthetase. The regulation of biosynthesis of these enzymes seems to proceed non-coordinately. This phenomenon can be the sequence of the existence of many operators, controlling the flavinogenesis in Bac. subtilis.

[Riboflavin biosynthesis operon of Bacillus subtilis. XIII. Genetic and biochemical study of mutants with regard to intermediate stages of biosynthesis]. / Issledovanie operona biosinteza riboflavina u Bacillus subtilis. Soobshchemie XIII. Genetichekoe i biokhimicheskoe issledovanie mutantov, otnosiashchikhsia k promezhutochnym stadiiam biosinteza.

New riboflavin dependent mutants of Bacillus subtilis accumulating different pteridines were studied. The data obtained show that the formation of ribityl side chain proceeds in a few steps at least on a part of riboflavin precursors. The oxidation of connected ribosyl into ribulose with subsequent restoration of it into ribityl proceeds at first. The corresponding genes are located on terminal part of riboflavin operon, as show the results of two-factor transformational crosses with different donors and recipients.

[Operon of riboflavin synthesis in Bacillus subtilis. IX. Preparation and properties of lumiflavin- or lumichrome-resistant mutants]. / Issledovanie operona biosinteza riboflavina u Bacillus subtilis. Soobshchenie IX. Poluchenie i svoistva mutantov, ustoichivykh k liumiflavinu ili liumikhromu

Lumiflavin and lumichrome both inhibit the growth of different strains of Bacillus subtilis including riboflavin-constitutive mutant. Lumiflavin has no regulating effect, but lumichrome is an effector and it participates in the repression of riboflavin precursors synthesis and the synthesis of riboflavinsynthetase. Mutants resistant to lumiflavin or lumichrome accumulate a mixture of riboflavin, FMN and FAD. Their regulatory characteristics are substantially altered. They are repressed by higher concentrations of all effectors comparing with the wild type. The mapping of resistant mutants showed that the most of them are located near to ribC gene, probably inside of it. The simultaneous presence of all three flavins in the cultural medium shows that the regulatory protein is universal to the biosynthesis of all flavins.