Two NH2-reactive probes (2,4,6-trinitrobenzesulphonic acid and 1-fluoro-2,4-dinitrobenzene) were used to study the vectorial orientation of the membrane-associated free NH2 groups across pig gastric microsomal vesicles. Unlike 1-fluoro-2,4-dinitrobenzene, 2,4,6-trinitrobenzenesulphonic acid is ordinarily an impermeant probe that becomes permeant in the presence of K+ and valinomycin. Although 2,4,6-trinitrobenzenesulphonic acid alone reacts with about 28% of the total microsomal phosphatidylethanolamine, 2,4,6-trinitrobenzenesulphonic acid in the presence of valinomycin plus K+ or 1-fluoro-2,4-dinitrobenzene alone reacted with 75% of the phosphatidyl- ethanolamine. Under similar conditions the free NH2 groups associated with the microsomal proteins also exhibited an asymmetric labeling pattern, the intra- and extravesicular orientation being 74 and 26% respectively.
