RESUMO
The study was carried out to evaluate the substrate specificity and activity of proteases secreted by strains of Klebsiella pneumoniae with various degree of virulence. The process included cultivation of the strains in semi-synthetic medium, after which the biomass was inactivated and the supernatant was separated from bacterial cells through centrifugation. Elastase-, trypsin-, and chymotrypsin-like proteolytic activity was measured in the supernatant and in all fractions obtained through gel-filtration, followed by DEAE-sepharose purification. Regardless of the degree of virulence, all the studied strains of K. pneumoniae secreted only one proteolitic enzyme, which was elastase with molecular weight of about 21 kDa. Addition of glycoprotein--the main structural component of eucaryotic cells--into the culture medium in the beginning of incubation, increased protein, polysaccharide, and lipopolysaccharide synthesis; proteolythic activity in the supernatant fluid increased from 7,476 to 15,731 mU/ml. The increase was associated with an elevation of polysaccharide synthesis from 173 to 349 mg dry weight. However, proteolythic activity per 1 gr of polysaccharide did not increase; it was 43.3 and 45.1 units, respectively. Thus, proteolytic activity increased in direct propotion to the increase of polysaccharide synthesis into the culture medium.
Assuntos
Klebsiella pneumoniae/metabolismo , Klebsiella pneumoniae/patogenicidade , Peptídeo Hidrolases/biossíntese , Polissacarídeos Bacterianos/biossíntese , Animais , Proteínas de Bactérias/biossíntese , Técnicas Bacteriológicas , Centrifugação , Cromatografia DEAE-Celulose , Cromatografia em Gel , Meios de Cultura , Interpretação Estatística de Dados , Modelos Animais de Doenças , Eletroforese em Gel de Poliacrilamida , Glicoproteínas/farmacologia , Infecções por Klebsiella/microbiologia , Klebsiella pneumoniae/citologia , Klebsiella pneumoniae/enzimologia , Klebsiella pneumoniae/crescimento & desenvolvimento , Klebsiella pneumoniae/isolamento & purificação , Lipopolissacarídeos/biossíntese , Camundongos , Peso Molecular , Elastase Pancreática/biossíntese , Peptídeo Hidrolases/metabolismo , Espectrofotometria , Especificidade por Substrato , Fatores de Tempo , VirulênciaRESUMO
The study of substrate specificity and activity of proteolytic enzymes secreted by K. pneumoniae strains with different virulence was carried out. The strains were cultivated in a liquid semi-synthetic medium. The biomass was inactivated, and the supernatant fluid was separated from microbial cells by centrifuging. In the supernatant thus obtained and in the fractions isolated by gel filtration with the subsequent purification on DEAE Sepharose elastase-like, trypsin-like and chemotrypsin-like proteolytic activity was determined. In K. pneumoniae strains with different virulence only a single proteolytic enzyme--elastase with a mol. wt. of 21 kD--was detected. The protease activity of the supernatant culture fluid did not depend on the virulence of the strain and was equal to 5,416-7,476 I.U./ml. The activity of the purified enzyme was 100% of the elastase-like activity of the supernatant culture fluid. The most virulent K. pneumoniae strain K2, whose LD50 for white mice was less than 10 microbial cells, was characterized by lower elastase-like activity. The absence of correlation between protease activity and K. pneumoniae virulence may be explained by the fact that surface glycoproteins of eukaryotic cells are glycosilated and thus slightly accessible for proteases.
Assuntos
Elastina/metabolismo , Klebsiella pneumoniae/enzimologia , Peptídeo Hidrolases/metabolismo , Animais , Modelos Animais de Doenças , Klebsiella pneumoniae/isolamento & purificação , Klebsiella pneumoniae/patogenicidade , Camundongos , Peso Molecular , Peptídeo Hidrolases/química , Especificidade por Substrato , VirulênciaRESUMO
Escherichia coli cells transformed with plasmids containing ricin B-chain coding sequences are shown to express this heterologous protein in inclusion bodies. After denaturation and renaturation of the product in the presence of glutathione and lactose, the recombinant ricin B-chain is soluble, biologically active and stable. Cytotoxicity of heterodimer containing this protein and ricin A-chain is found to be only ten times lower, than that of native ricin. Recombinant B-chain alone was nontoxic to cells (ID50 > 10(-6) M). Our data suggest that ricin B-chain oligosaccharides are essential for stability preserving protein from proteolytic degradation in cells.
Assuntos
Ricina/farmacologia , Sequência de Bases , Sobrevivência Celular/efeitos dos fármacos , Clonagem Molecular , Escherichia coli/genética , Hidrólise , Dados de Sequência Molecular , Oligodesoxirribonucleotídeos , Desnaturação Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/farmacologia , Ricina/química , Ricina/genéticaAssuntos
Ricina/metabolismo , Sobrevivência Celular , Células Cultivadas , Clonagem Molecular , Escherichia coli/genética , Glicosilação , Temperatura Alta , Conformação Proteica , Desnaturação Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/imunologia , Proteínas Recombinantes/metabolismo , Ricina/química , Ricina/imunologia , Espectrometria de Fluorescência , Espectrofotometria Ultravioleta , TermodinâmicaRESUMO
A chimeric toxic protein was prepared from the mistletoe lectin I A-chain and ricin B-chain by using the disulfide exchange reaction. Ricin and chimeric protein were indistinguishable in binding to immobilized asialofetuin in ELISA. The chimeric protein was more toxic for Jurkat cells than native mistletoe lectin I, but not as effective as native ricin. In the presence of NH4Cl, which enhances the toxicity of some toxins and immunotoxins, but does not influence ricin toxicity, both ricin and chimeric toxin had equal cytotoxic activity. The possibility is discussed that the ricin B-chain protects the ricin A-chain from degradation during delivery from the cell surface to the place where it is translocated into the cytosol.
Assuntos
Lectinas/química , Erva-de-Passarinho/química , Preparações de Plantas , Proteínas de Plantas , Plantas Medicinais , Proteínas Recombinantes de Fusão/síntese química , Ricina/química , Toxinas Biológicas/química , Assialoglicoproteínas/metabolismo , Transporte Biológico , Células Cultivadas , Citosol/metabolismo , Ensaio de Imunoadsorção Enzimática , Fetuínas , Lectinas de Plantas , Proteínas Recombinantes de Fusão/metabolismo , Proteínas Inativadoras de Ribossomos Tipo 2 , Ricina/metabolismo , alfa-Fetoproteínas/metabolismoRESUMO
By gene-engineering technique a chimeric protein made up of fragments of Staphylococcus aureus protein A and . Pseudomonas aeruginosa exotoxin A has been constructed. The chimeric protein was shown to preserve features characteristic of its both constituents--it ADP-ribosylates elongation factor 2 and binds to Ig. Cytotoxic properties of the chimeric protein were studied in two model systems. Treatment of target cells in both systems was performed successively with antibodies against corresponding antigens and after washing--with recombinant chimeric toxin which bound to antibodies on the surface of target cells. In the first model system human B-lymphoma cells (Daudi line) carrying Ig molecules on their surface were treated with polyclonal antibodies against human Ig L-chains. In the other system, human T-lymphoma cells (Jurkat line) were treated successively with monoclonal antibodies against cell surface CD5 antigen and further on--with polyclonal antibodies against mouse Ig. In both systems, only a slight inhibition of the target cells' growth was registered. The probable reasons of low cytotoxic activity of the chimeric protein and prospects of increasing it are discussed.