Abstract
lac permease mutated at each of the 8 cysteinyl residues in the molecule was solubilized from the membrane, purified, and reconstituted into proteoliposomes. The transport activity of proteoliposomes reconstituted with each mutant permease relative to the wild-type is virtually identical with that reported for intact cells and/or right-side-out membrane vesicles. Moreover, a double mutant containing Ser in place of both Cys148 and Cys154 exhibits significant ability to catalyze active lactose transport. The results provide strong confirmation for the contention that cysteinyl residues in lac permease do not play an important role in the transport mechanism. The effect of sulfhydryl oxidant 5-hydroxy-2-methyl-1,4-naphthoquinone on lactose transport in proteoliposomes reconstituted with wild-type or mutant permeases was also investigated, and the results indicate that inactivation is probably due to formation of a covalent adduct with Cys148 and/or Cys154 rather than disulfide formation. Thus, it seems unlikely that sulfhydryl-disulfide interconversion functions to regulate permease activity.
Highlights
The transport activity of proteoliposomes reconstituted with each mutant permease relative to the wild-type is virtually identical with that reported for intact cells and/or right-side-out membrane vesicles
The results provide strong confirmation for the contention that cysteinyl residues in lac permease do not play an important role in the transport mechanism
The effect of sulfhydryl oxidant 6-hydroxy-2-methyl-1,4-maphthoquinonoen lactose transport in proteoliposomesreconstitutedwith wild-type or mutant permeases was investigated, and the results indicate that inactivation is probably due to formation of a covalent adduct with CYS'~'and/ or CYS''~ rather than disulfide formation
Summary
Characterization of Purified, Reconstituted Site-directed Cysteine Mutants of the Lactose Permease of Escherichia coli Iwaarden, Pierre R. van; Driessen, Arnold J.M.; Menick, Donald R.; Kaback, H. Characterization of Purified, Reconstituted Site-directed Cysteine Mutants of the Lactose Permease of Escherichia coli. More information can be found on the University of Groningen website: https://www.rug.nl/library/open-access/self-archiving-pure/taverneamendment. Take-down policy If you believe that this document breaches copyright please contact us providing details, and we will remove access to the work immediately and investigate your claim. For technical reasons the number of authors shown on this cover page is limited to 10 maximum
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