[Study of intracellular localization of the proteolytic enzyme complex and its protein inhibitors in bombyx grain].

Intracellular localization of serine, cysteine and aspartate proteases, as well as their protein inhibitors, in bombyx grain in the postdiapause period of embryogenesis has been studied. Proteolytic activity of aspartate and cysteine proteases was found in lysosomal, mitochondrial, and nuclear fractions of grains. Serine protease activity was not observed in subcellular fractions of grains of the fourth day of postdiapause development. It has been shown that activities of protein inhibitors and certain peptide hydrolases in subcellular fractions provide consistent functioning and fine regulation of the proteolytic enzyme complex.

[Proteolytic enzymes as markers of productivity and heterosis of silkworm].

A positive correlation between the activity level of cysteine proteinases in developing eggs of common silkworm moth (Bombyx mori L.), on the one hand, and a set of commercial characteristics, on the other, was found. This allows the determination of cysteine proteinase activities (pH optima of 3.0, 3.6, and 8.6) to be recommended as a biochemical test for an early prediction of potential productivity of silkworm breeds. A positive correlation between the activity level of acid cysteine proteinases in eggs of parental breeds and a set of commercial characteristics of their hybrids was detected, indicating a principal possibility of predicting the degree of heterosis.

[Aminotransferases in early development of salmonid fish].

The activities of alanine and aspartate aminotransferases were assessed in the embryos and juveniles of the rainbow trout Parasalmo mukiss L. and chum salmon Oncorchyncus keta W. Changes in subcellular localization of these enzymes and their activities were found at different pH optima in each subcellular fraction of the rainbow trout during ontogenesis.

[Aspartate- and alanine-aminotransferase in early development of keta]. / Aspartat- i alaninaminotransferaza v rannem razitii kety.

We studied the activities of the marker enzymes of physiological state and adaptive reactions, aspartate and alanine aminotransferases, in early development of the keta Oncorhynchus keta. Aspartate aminotransferase with pH optima 6.8, 7.0, 7.6, and 8.0 and alanine aminotransferase with pH optima 7.0, 7.4, 7.6, 7.8, 8.0, and 8.2 were found in the eggs, larvae, and fry. The succession of enzymes with different pH takes place during ontogenesis, as well as stage specific changes in their activity. The maximum enzymatic activity was recorded in the larvae during their rise for "afloat." A correlation was established between the dynamics of enzymatic activity and soluble nitrogen and amine nitrogen contents.

[Specific interaction between esterases and 2-butylthio-2-thio-1,3,2-oxazaphosphorynane and its cyclic and acyclic analogs]. / Osobennosti vzaimodeistviia s ésterazami 2-butiltio-2-tio-1,3,2-oxsazafosforinana i nekotorykh ego tsiklicheskikh i atsiklicheskikh analogov.

We studied the anticholinesterase and anticarboxylesterase effects of 1,3,2-oxazaphosphorynane derivatives and certain cyclic and acyclic analogs on the two enzymes of homoiotherms (ACE from human erythrocytes and BuCE from horse serum) as well as the enzymes from insect tissues (the nerve cord of the American cock-roach and the cephalic region of the domestic fly). The differences in in vitro antiesterase activity of cyclic thionic and the corresponding oxo derivatives of phosphorinane were revealed. The mechanism of the esterase active center phosphorylation not only splitting off the outgoing group (in vivo) but also opening the cycle by P-O bond (in vitro and possibly in vivo) is usually proposed to explain the higher inhibiting activity of the thionic compounds compared to the oxonic ones. The possible involvement of this phosphorylation mechanism in the synergistic activity of the studied compounds is discussed.

[Ecdysterone induces the activity of multiple forms of acid phosphatase and malate dehydrogenase in fat bodies of Anterahea pernyi pupae]. / Ekdisteron indutsirue aktivnost' monzhestvennykh form kisloi fosfatazy i malatdegidrogenazy zhirovogo tela kukolok dubovogo shelkopriada.

The method of electrophoresis in PAAG has been used to study activity of multiple forms of acid phosphatase and malate dehydrogenase of the fat body of Anterahea pernyi pupae in norm and affected by ecdysterone. It is shown that an increase of total activity of acid phosphatase and malate dehydrogenase with administration of a steroid hormone is promoted by growth of activity of certain inducible multiple forms of the studied enzymes. Ecdysterone injection considerably elevates activity of the most electrophoretically mobile form of acid phosphatase and provokes new formation of malate dehydrogenase form. Ecdysterone-induced increase of activity of the studied enzyme forms in the fat body of Anterahea pernyi pupae is blocked by actinomycin D, an inhibitor of transcriptional processes, and by puromycin, a translation inhibitor. These data permit concluding that an increase in activity of the mentioned forms of enzymes observed with hormonal induction is a result of their synthesis de novo and is mediated by the DNA-dependent RNA-synthesis.

[Effect of the synthetic Leu enkephalin dalargin on the protein and nucleic acid content of the muscles in the rainbow trout]. / Vliianie sinteticheskogo analoga lei-énkefalina dalargina na soderzhanie belka i nukleinovykh kislot v myshtsakh raduzhnoi foreli.

After dalargin treatment of fish eggs (at the stage of swollen egg envelopes) and on juveniles (at the stage of early meiotic oocyte appearance in gonads) DNA content is observed to raise up to 85% and 66%, respectively in yearlings and up to 20% and 23% respectively in second-year fish as compared to the control ones. Dalargin also influences the exchange of muscle RNA in yearlings and second-year rainbow-trout. Dalargin effect is higher when peptide influences at the very onset of organism differentiation.

[Regularities of the content and organization of pyrimidine oligonucleotide sequences in insect DNA]. / Zakonomernosti v soderzhanii i organizatsii pirimidinovykh oligonukleotidnykh posledovatel'nostei v DNK nasekomykh.

Certain regularities in content and organization of pyrimidine oligonucleotide sequences of DNA from 15 insect species belonging to 4 orders were studied. The degree of nucleotide clusterization in insect DNA was found to be species-specific, being the highest in Hymenoptera and lowest in Lepidoptera; the Blattodea and Coleoptera occupy an intermediate position by this index between them. The changes in the DNA cluster structure during the evolution of insect species are not of vector type; the degree of clusterization of DNA nucleotide is either increased (Hymenoptera) or decreased (Lepidoptera as compared with Blattodea). In the DNA oligonucleotide fractions containing both pyrimidine nucleotides the percentage content of thymidyl nucleotides is much higher than that of cytidyl nucleotides, the thymine content being increased with the lengthening of oligopyrimidine clusters. The insect species with a higher degree of clusterization of DNA pyrimidine nucleotides contain more thymidyl nucleotide residues. These results agree well with the hypothesis suggesting that during the evolution of large taxons the accumulation of long pyrimidine sequences in animal DNA is accompanied by an increase of thymidyl nucleotide content in them. This can largely be due to the increase of matrix resistance during the evolution and is biologically significant for animals of any taxons, including insects.

[Separation and properties of multiple forms of acid phosphatase from the tissues and organs of the mulberry silkworm]. / Razdelenie i svoistva mnozhestvennykh form kisloi fosfatazy tkanei i organov tutovogo shelkopriada.

The multiple forms of acid phosphatase from the hemolymph, fat body, intestinal wall and silk gland of the silkworm larvae differing in their mobility during polyacrylamide gel electrophoresis were separated by isoelectrofocusing, gel filtration and ion-exchange chromatography and characterized in terms of their molecular weight, isoelectric points and substrate specificity. It was found that silkworm tissues contain several enzyme forms possessing broad substrate specificity (EC 3.1.3.2) as well as forms predominantly catalyzing the hydrolysis of glucose-1-phosphate (EC 3.1.3.10). The enzyme forms with a broad substrate specificity were detected in all tissues studied with the exception of hemolymph and were found to have the molecular weights above 60,000, pI greater than pH 6.0 and the highest activity within the pH range of 3.0-4.5. The enzyme forms causing predominant hydrolysis of glucose-1-phosphate detected in the hemolymph, fat body and intestinal wall are characterized by Mr = 30,000-100,000, pI below 6.0 and the highest activity within the pH range of 4.8-5.6. A possible role of the multiple forms of acid phosphatase in silkworm metabolism is discussed.

[Isolation and properties of carboxylesterase from hemolymph of the silkworm Bombyx mori L]. / Vydelenie i svoistva karboksilesterazy iz gemolimfy tutovogo shelkopriada Bombyx mori L.

An original procedure for isolation and purification of carboxylesterase from the hemolymph of stage V larvae of one of Bombyx mori strains including precipitation with 10% polyethyleneglycol, ion-exchange chromatography on Sephadex G-200 and chromatography on DEAE-Sephadex A-50, has been developed. The specific activity of the enzyme after purification makes up to 1250 units per mg of protein with a 59% yield. Some physicochemical properties of the enzyme (Mr = 69 000, pI congruent to 4.9, temperature optimum = 40 degrees, pH optimum = 7.2 Km for alpha-naphthyl- and beta-naphthylacetate = 0.11 X 10(-3) and 0.52 X 10(-3) M, respectively) have been determined. Using immunodiffusion in agar gel, the antigenic identity of the enzymes isolated from the hemolymph of two silkworm species has been established.

[Subcellular localization of some enzymes in silkworm eggs]. / Issledovanie subkletochnoi lokalizatsii nekotorykh fermentov v iaitsakh tutovogo shelkopriada.

Differential centrifugation of the silkworm (Bombyx mori L.) egg homogenates resulted in nuclear, mitochondrial, lysosomal and cytosol infarctions, which were analyzed for the activities of alanine, aspartate- and tyrosine aminotransferases, 3-glycerophosphate- and lactate dehydrogenases and acid and alkaline DNAases. Alanine- and aspartate aminotransferases as well as 3-glycerophosphate dehydrogenase are localized mainly in the cytosol, where their activities made up to 86.2, 95.4 and 98.4% of their total activity, respectively. The activities of lactate dehydrogenase and acid DNAase are distributed between the nuclear and mitochondrial fractions; this distribution is even in the case of the former enzyme, whereas in the case of the latter the bulk (90.6%) of total enzyme activity is found in the nuclei. In contrast to the other enzymes whose activity is distributed between different cell fractions, tyrosine aminotransferase is localized exclusively in mitochondria, while alkaline DNAase--exclusively in the nuclei. No correlation between the level of enzyme specific activity and its total content in the fractions was established. The role of the enzymes under study in silkworm metabolism is discussed.

[Isolation, purification and properties of acid deoxyribonuclease from silkworm Bombyx mori eggs]. / Vydelenie, ochistka i svoistva kisloi dezoksiribonuckleazy greny tutovogo shelkopriada Bombyx mori.

The acid deoxyribonuclease was isolated from Bombyx mori eggs and its physico-chemical properties were investigated. The enzyme purified 160-fold did not contain admixtures of phosphomono- and phosphodiesterases or ribonuclease. The molecular weight of the enzyme is 40 000 +/- 1000, isoelectric point lies at 6.5. The maximum activity is revealed at pH 5.2, 50 degrees. The DNAase is insignificantly activated by Mg2+ and is inhibited by Cu2+ and Zn2+. The enzyme preferentially hydrolyzes native DNA and is an endonuclease splitting DNA down to 5'-oligonucleotides.

[Metabolism of 14C-soluble proteins from silkworm tissues during silk formation]. / Metabolizm of 14C-rastvorimykh belkov tkanei tutovogo shelkopriada na zakliuchitel'nom etape ego lichinochnogo razvitiia.

The functional role of individual labelled fractions of haemolymph, fat body, carcass, gut and anterior and posterior divisions of the silk gland of the silkworm Bombyx mori was studied, using polyacrylamide gel electrophoresis. It was found that the protein fractions with Rf = 0.02, 0.05, 0.09 and 0.24 are formed in the gut, carcass and fat body during active feeding and are utilized by the silk gland in the spinning period by means of haemolymph. The contents of amino acids in several electrophoretic protein patterns (Rf = 0.02, 0.05, 0.09 and 0.24) in the haemolymph were determined. These protein fractions are sources of nitrogen-containing material used for increasing the level of free amino acids in the silk gland and for biosynthesis of major silk amino acids.

[Isolation, purification and properties of acid ribonuclease from the lysosomal fraction of silkworm eggs]. / Vydelenie, ochistka i svoistva kisloi ribonuckleazy lizosomal'noi fraktsii greny tutovogo shelkopriada.

Acid ribonuclease (ribonucleate-3'-oligonucleotide hydrolase, EC 3.1.4.23) has been isolated from the lysosomal fraction of Bombyx mori eggs. The enzyme has a pH optimum of 4,7 and a molecular weight of 17 000 +/- 1000; the isoelectric point of the enzyme lies around 6,0. The enzyme splits RNA and poly(U) down to nucleoside-3'-phosphates to form intermediates--nucleoside-2',3'-cyclophosphates. Polyadenylic acid is hydrolyzed in the presence of the enzyme down to oligonucleotides. Mg2+ suppress the enzyme activity.