RESUMO
Ten monoclonal antibodies were produced against a k-1-type crystal protein of Bacillus thuringiensis subsp. kurstaki. Eight of the antibodies belong to the immunoglobulin G1 (IgG1) subclass, with pI values ranging from 5.5 to 8.6, one could be assigned to the IgG2b subclass, and one could be assigned to the IgM class. Competitive antibody-binding assays and analysis of antibody specificity indicated that the 10 antibodies recognized at least nine distinct antigenic determinants. Eight antibodies bound to both protoxin and toxin, whereas the other two interacted with protoxin only. One antibody completely inhibited the biological activity of the delta-endotoxin, five antibodies reduced it by 15 to 82%, and four antibodies did not affect it at all. Based on cross-reaction studies, homologies and differences in the crystal protein structures of different B. thuringiensis subspecies were revealed. All of the monoclonal antibodies strongly cross-reacted with crystal proteins from strains of B. thuringiensis subsp. tolworthi, B. thuringiensis subsp. galleriae, B. thuringiensis subsp. dendrolimus, B. thuringiensis subsp. sotto, and B. thuringiensis subsp. subtoxicus. Some antibodies interacted only weakly with crystal proteins from strains of B. thuringiensis subsp. morrisoni and B. thuringiensis subsp. entomocidus, and some of these did not interact with B. thuringiensis subsp. kenyae and B. thuringiensis subsp. darmstadiensis. No cross-reaction was found with the parasporal inclusion protein of B. thuringiensis subsp. israelensis. Studies with the monoclonal antibodies also disclosed that crystal proteins from strains of the same subspecies can exhibit substantial differences in antigenic structure. In particular, striking strain-specific differences in the protoxins of B. thuringiensis subsp. kurstaki and B. thuringiensis subsp. thuringiensis were observed.
Assuntos
Anticorpos Monoclonais/imunologia , Bacillus thuringiensis/imunologia , Proteínas de Bactérias/imunologia , Toxinas Bacterianas/imunologia , Endotoxinas , Afinidade de Anticorpos , Toxinas de Bacillus thuringiensis , Ligação Competitiva , Proteínas Hemolisinas , Testes de Neutralização , Precursores de Proteínas/imunologia , Especificidade da EspécieRESUMO
The indole-3-glycerolphosphate synthase/anthranilate synthase complex from Saccharomyces cerevisiae was purified to apparent homogeneity. The native complex with Mr approximately equal to 130 000 consists of two different subunits, the TRP2 gene product with Mr = 64 000 and the TRP3 gene product with Mr = 58 000. The larger polypeptide was identified as anthranilate synthase and is active in vitro with ammonia as cosubstrate without need of complex formation. The smaller polypeptide carries both glutamine amidotransferase activity and indole-3-glycerolphosphate synthase activity. Various steady-state kinetic parameters as well as the amino acid composition of the two polypeptides were determined.
Assuntos
Antranilato Sintase/isolamento & purificação , Carboxiliases/isolamento & purificação , Indol-3-Glicerolfosfato Sintase/isolamento & purificação , Saccharomyces cerevisiae/enzimologia , Aminoácidos/análise , Antranilato Sintase/genética , Fenômenos Químicos , Precipitação Química , Química , Cromatografia/métodos , Indol-3-Glicerolfosfato Sintase/genética , Saccharomyces cerevisiae/genéticaRESUMO
The free tryptophan pool and the levels of two enzymes of tryptophan biosynthesis (anthranilate synthase and indoleglycerolphosphate synthase)have been determined in a wild type strain of Saccharomyces cerevisiae and in mutants with altered regulatory properties.
