Abstract
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).
Highlights
This resultsuggested that thioredoxin ias soluble cytoplasmic protein.Recentstudiesusing gentlylysedcells ( 5 ) suggest that this protein interacts with the cell membrane
Reduced thioredoxin was treated in the darkwith excess PAPA, iodoacetic acid (IAA), or PAPA and IAA
Reaction of thioredoxin with both PAPA and IAA caused the protein to migrate to a position characteristic of the PAPA
Summary
E. coli W3110 (thy+)was grown a t 37 “C in Tris minimal medium (TCG) containing 0.05% casamino acids (vitamin free, Difco), 0.1% glucose, and 0.3 mM phosphate [17]. Cells for experimentation were harvested a t a density of 3 X 10’ cells/ml by centrifugation a t 4 “C. T h e cells were harvested and resuspended to 1 X 10’’ cells/ ml in cold (6 “C) TCG medium containing 1 mM EDTA. This cell density corresponds to a thioredoxin concentration in the reaction mix of 0.2 p~ [18].PAPA, freshly prepared as a 10 X stock solution inmethanol, was addedtothe cellsuspension.Unlessotherwise. Thecross-linked cells were collected by centrifugation a t 4 "C
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Free) 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
