Portal de Periódicos da CAPES

Thiroedoxin from Escherichia coli. Radioimmunological and enzymatic determinations in wild type cells and mutants defective in phage T7 DNA replication.

1978; Elsevier BV; Volume: 253; Issue: 2 Linguagem: Inglês

10.1016/s0021-9258(17)38227-3

1083-351X

Arne Holmgren, Ingrid Ohlsson, M.L. Grankvist,

bioluminescence and chemiluminescence research

A competition radioimmunoassay for Escherichia coli thioredoxin using Y-labeled thioredoxin-S, and a double antibody technique was developed.The method permits determination of picomole amounts of thioredoxin in crude cell extracts and was used to study the localization of thioredoxin cell fractions.E. coli B was calculated to have approximately 10,000 copies of thioredoxin per cell mainly located in the soluble fraction after separation of the membrane and soluble fractions by gentle lysis and centrifugation.E. coli B tsnC mutants which are defective in the replication of phage T7 DNA in uiuo and in vitro (Chamberlin, M. (1974) J. Viral.14, 509-516) were examined for their content of thioredoxin.E. coli B tsnC 7004 contained no detectable level of thioredoxin in cell-free extracts examined under a variety of conditions with the following methods: radioimmunqassay, immunoprecipitation, and immunodiffusion, enzymatic assays employing excess thioredoxin reductase, and NADPH in reduction of 5,5'-dithiobis(Z-nitrobenzoic acid), or disulfides in bovine insulin.The results strongly suggest that tsnC 7004 is a nonsense or deletion mutant.The growth properties of E. coti B tsnC 7004 in rich medium containing glucose were little affected compared with its parent strain E. coli B/l.Growth in minimal medium was slow but addition of L-cystine normalized the growth behavior, suggesting that a function of thioredoxin in E. coli is to participate in the biosynthesis of L-cystine through enzymatic reduction of sulfate, as has been proposed by others.Two other E. coli tsnC mutants, 7007 and 7008, contained detectable levels of thioredoxin in crude extracts as measured by thioredoxin reductase and gave similar immunoprecipitation reactions as the parent strain B/l.By radioimmunoassay incompletely cross-reacting material was present in both strains.These results show that tsnC 7007 and 7008 belong to a type of thioredoxin mutants with missence mutations in the thioredoxin gene affecting the function of thioredoxin as subunit in phage T7 DNA polymerase.