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Behavior of cysteine mutants of human lysozyme in de novo synthesis and in vivo secretion



Behavior of cysteine mutants of human lysozyme in de novo synthesis and in vivo secretion



European Journal of Biochemistry 198(2): 477-484



To investigate the mechanism of disulfide-bond-coupled de novo folding of human lysozyme, we have constructed 23 mutant enzymes in which cysteine residue(s) were replaced by alanine(s). The mutant genes were translated in vitro in a system composed of rabbit reticulocyte lysate, canine pancreatic microsomal vesicles and oxidized glutathione. This system allows the formaton of intramolecular disulfide bonds in translation products translocated into the microsomal lumen. The mobilities of the translation products were analyzed by SDS/PAGE in non-reducing conditions. Some mutant lysozymes were found to form a compact of conformation with native-like mobidity in the presence of SDS. The de novo formation of the SDS-resistant compact conformation of each mutant correlated well with its efficiency of secretion by Saccharomyces cerevisiae. Our results suggest that the de novo synthesized products reflect the conformational states in vivo to some extent, and that theformation of SDS-resistant compact conformation can be regarded as a necessary condition for allowing lysozyme to be secreted. In addition, the analysis of a mutant C116A (Cys116 .fwdarw. Ala) under different oxidative conditions suggests two distinct pathways for the disulfide-bond-coupled formation of the compact conformation.

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Accession: 007051523

Download citation: RISBibTeXText

PMID: 2040307

DOI: 10.1111/j.1432-1033.1991.tb16038.x


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