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1.
Prikl Biokhim Mikrobiol ; 49(5): 513-21, 2013.
Artigo em Russo | MEDLINE | ID: mdl-25474875

RESUMO

Serine proteinases from three phytopathogenic microorganisms that belong to different fungal families and cause diseases in potatoes were studied and characterized. The oomycete Phytophthora infestans (Mont.) de Bary and the fungi Rhizoctonia solani and Fusarium culmorum were shown to secrete serine proteinases. An analysis of the substrate specificity of these enzymes and their sensitivity to synthetic and protein inhibitors allowed us to refer them to trypsin- and subtilisin-like proteinases. The correlation between the trypsin- and subtilisin-like proteinases depended on the composition of the culture medium, particularly on the form of the nitrogen source. A phylogenetic analysis was carried out. In contrast to basidiomycetes R. solani, ascomycetes F. culmorum and oomycetes P. infestans produced a similar set of exoproteinases, although they had more distant phylogenetic positions. This indicated that the secretion of serine proteinases by various phytopathogenic microorganisms also depended on their phylogenetic position. These results allowed us to suggest that exoproteinases from phytopathogenic fungi play a different role in pathogenesis. They may promote the adaptation of fungi if the range of hosts is enlarged. On the other hand, they may play an important role in the survival of microorganisms in hostile environements outside their hosts.


Assuntos
Proteínas Fúngicas/metabolismo , Fusarium/enzimologia , Peptídeo Hidrolases/metabolismo , Phytophthora infestans/enzimologia , Doenças das Plantas/microbiologia , Rhizoctonia/enzimologia
2.
Prikl Biokhim Mikrobiol ; 42(3): 338-44, 2006.
Artigo em Russo | MEDLINE | ID: mdl-16878552

RESUMO

The growth of Fusarium culmorum fungus on a medium containing thermostable proteins from potato tubers was accompanied by the production of proteinases, exhibiting activity over a broad pH range (from 6.0-10.0). When studied by SDS-PAGE in the presence of beta-mercaptoethanol, extracellular proteinases were represented by at least five species with a molecular weight of 30-60 kDa. Inhibitor analysis and studies of enzyme activities with synthetic substrates demonstrated that the culture liquid of Fusarium culmorum contained serine proteinases of various classes. The amount of subtilisin-like proteinases was the highest. A near-complete inhibition of the enzymes was caused by proteinaceous proteinase inhibitors from potato tubers. These data suggest that proteinases of the phytopathogen Fusarium culmorum serve as a metabolic target for natural inhibitors of potato proteinases.


Assuntos
Proteínas Fúngicas/metabolismo , Fusarium/enzimologia , Peptídeo Hidrolases/metabolismo , Proteínas Fúngicas/antagonistas & inibidores , Proteínas Fúngicas/química , Fusarium/crescimento & desenvolvimento , Peptídeo Hidrolases/química , Doenças das Plantas/microbiologia , Inibidores de Proteases/farmacologia , Solanum tuberosum/química , Solanum tuberosum/microbiologia
3.
Prikl Biokhim Mikrobiol ; 41(6): 612-5, 2005.
Artigo em Russo | MEDLINE | ID: mdl-16358748

RESUMO

The activity of trypsin-like proteinases and trypsin inhibitors was measured in fruiting bodies of various species of basidial fungi (Basidiomycetes). Fruiting bodies of all fungi contained these enzymes, with the exceptions of polypore (Coriolus versicolor (Fr.) Karst) and hedgehog fungus (Hericium erinaceus (Fr.) Quel), belonging to the families Polyporaceae and Hericiaceae, respectively, in which the enzyme activities were barely detectable. The activity of trypsin-like proteinases was the highest in fruiting bodies of Boletaceae and Agaricaceae. Fruiting bodies of all fungi contained trypsin inhibitors. The highest activity of trypsin inhibitors was detected in basidiomycetes of the families Boletaceae, Agaricaceae, and Pleurotaceae, including Boletus castanus (Fr.) Karst, orange-cap boletus (Leccinum aurantiacum (Fr.) Sing), and brown-cap boletus (Leccinum melanum (Fr.) Karst).


Assuntos
Basidiomycota/enzimologia , Carpóforos/enzimologia , Peptídeo Hidrolases/metabolismo , Inibidores da Tripsina/metabolismo , Tripsina/metabolismo
4.
Prikl Biokhim Mikrobiol ; 40(2): 194-200, 2004.
Artigo em Russo | MEDLINE | ID: mdl-15125197

RESUMO

When grown in a medium containing heat-stable potato tuber proteins, the oomycete Phytophthora infestans (Mont.) de Bary produces a set of exoproteinases active at neutral and mildly basic pH values. These extracellular proteinases have been shown by SDS-PAGE with the presence of gelatin to include at least six components differing in molecular weight. Inhibitory analysis and study of the effects of the enzymes on various synthetic substrates show that the culture liquid of P. infestans contains mainly serine proteinases specific to trypsin and subtilisin and metalloproteinases. Their activity is suppressed by proteinase-inhibitor proteins from potato tubers. It is suggested that P. infestans exoproteinases may be the metabolic target for natural proteinase inhibitors from potato.


Assuntos
Exopeptidases/metabolismo , Phytophthora/enzimologia , Meios de Cultura , Exopeptidases/química , Concentração de Íons de Hidrogênio , Metaloproteases/análise , Phytophthora/crescimento & desenvolvimento , Proteínas de Plantas/metabolismo , Inibidores de Serina Proteinase/metabolismo , Solanum tuberosum/metabolismo , Especificidade por Substrato , Subtilisina/análise , Tripsina/análise
5.
Prikl Biokhim Mikrobiol ; 36(5): 541-4, 2000.
Artigo em Russo | MEDLINE | ID: mdl-11042876

RESUMO

A protein that inhibited the proteolytic activity of trypsin was isolated from amaranth leaves (Amaranthus cruentus) by affinity chromatography on trypsin-Sepharose. The inhibition was noncompetitive (with n-nitroanilide-N-alpha-benzoyl-DL-arginine as substrate) and had a Ki of 11.87 x 10(-7) 7 M. The protein caused a weaker inhibitory effect on chymotrypsin, had no effect on subtilisin, displayed a molecular weight of 8 kDa, and contained no cysteine residues.


Assuntos
Magnoliopsida , Inibidores da Tripsina/isolamento & purificação , Hidrólise , Cinética , Inibidores da Tripsina/análise , Inibidores da Tripsina/metabolismo
6.
Prikl Biokhim Mikrobiol ; 19(4): 521-7, 1983.
Artigo em Russo | MEDLINE | ID: mdl-6622441

RESUMO

A method of affinity chromatography of the inhibitor of cysteine proteinases from chick egg protein using immobilized ficin has been developed. This method yields a highly active inhibitor in an essentially homogeneous state. The molecular weight of the inhibitor is 14,000. The inhibitor suppresses the activity of ficin and papain but produces no effect on the proteolytic activity of trypsin, chymotrypsin, Asp. oryzae serine proteinase or subtilisine. Isoelectric focusing of the inhibitor has revealed the major band with pI 4.35.


Assuntos
Cistatinas , Proteínas do Ovo/análise , Inibidores de Proteases/isolamento & purificação , Proteínas/isolamento & purificação , Animais , Galinhas , Cromatografia de Afinidade/métodos , Eletroforese em Gel de Poliacrilamida , Ficina , Focalização Isoelétrica , Inibidores de Proteases/análise , Proteínas/análise
7.
Prikl Biokhim Mikrobiol ; 12(4): 572-7, 1976.
Artigo em Russo | MEDLINE | ID: mdl-1026941

RESUMO

Pancreatic Kallikrein was purified by affinity chromatography on BPTI-Sepharose. Immobilized BPTI was prepared via three stages: a) formation of the BPTI--acetylated trypsin complex; b) coupling of the resultant complex with CNBr-activated Sepharose; c) dissociation of the bound complex at pH 2.0 to yield immobilized BPTI and acetylated trypsin. The final product contained 59 X 10(-3) micronmoles insolubilized BPTI/ml Sepharose. Trypsin occurring in commercial Kallikrein preparations was separated by the batch procedure with ovomucoid-Sepharose. This method allows 100-fold purification of commercial Kallikrein.


Assuntos
Calicreínas/isolamento & purificação , Pâncreas/enzimologia , Animais , Cromatografia de Afinidade/métodos , Focalização Isoelétrica , Calicreínas/análise , Ovomucina/isolamento & purificação , Ovomucina/farmacologia , Tripsina/análise
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