Chemical cross-linking of thioredoxin to hybrid membrane fraction in Escherichia coli

Thioredoxin was cross-linked to a membrane fraction in vivo using the heterobifunctional photoreactive cross-linking reagent p-azidophenacyl bromide, chosen to couple thioredoxin via its highly reactive thiol. Under mild reaction conditions, a significant amount of thioredoxin (30%) was rapidly cros...

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Published inThe Journal of biological chemistry Vol. 261; no. 2; pp. 832 - 838
Main Authors Lunn, C A, Pigiet, V P
Format Journal Article
LanguageEnglish
Published Bethesda, MD Elsevier Inc 15.01.1986
American Society for Biochemistry and Molecular Biology
Subjects
Alkylation
Azides - pharmacology
Bacterial Proteins - metabolism
Bacteriology
Biological and medical sciences
Centrifugation
Escherichia coli - ultrastructure
Fundamental and applied biological sciences. Psychology
Glutathione - metabolism
Immunoelectrophoresis, Two-Dimensional
Kinetics
Membrane Proteins - metabolism
Microbiology
Models, Chemical
Permeability, membrane transport, intracellular transport
Thioredoxins - metabolism
In vivo
Escherichia coli
Bacteria
Membrane
Biosynthesis
Thioredoxin
Transport
Escherichieae
Enterobacteriaceae
Bound form
Immunological method
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Summary:Thioredoxin was cross-linked to a membrane fraction in vivo using the heterobifunctional photoreactive cross-linking reagent p-azidophenacyl bromide, chosen to couple thioredoxin via its highly reactive thiol. Under mild reaction conditions, a significant amount of thioredoxin (30%) was rapidly cross-linked to the crude membrane fraction. The cross-linking reaction was selective, with thioredoxin purified 15-fold in the cross-linked membrane fraction. Membrane fractionation studies showed that thioredoxin associated with the inner membrane and with a hybrid membrane fraction. This hybrid membrane fraction banded at a density between the inner and outer membranes. This result is consistent with the localization of thioredoxin in association with the bacterial membrane adhesion sites first described by Bayer (Bayer, M. (1968) J. Gen. Microbiol. 53, 395-404). Association of thioredoxin with the membrane adhesion sites defines a structure corresponding to the osmotically sensitive cytoplasmic compartment (Lunn, C. A., and Pigiet, V. (1982) J. Biol. Chem. 257, 11424-11430).
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ISSN:0021-9258
1083-351X
DOI:10.1016/S0021-9258(17)36171-9