Bilayer orientation of membrane-bound NH 2 groups across microsomal vesicles and their role in the function of gastric K +-stimulated adenosine triphosphatase

Abstract

The transversal distribution of the free NH 2 groups associated with phosphatidyl ethanolamine and the intrinsic membrane proteins of the purified pig gastric microsomes was quantitated and their relations to the function of the gastric K +-stimulated ATPase was investigated. Three different chemical probes such as 2,4,6-trinitrobenzene sulfonic acid (TNBS), 1-fluoro-2,4-dinitrobenzene (FDNB), and 2-methoxy-2,4-diphenyl-3(2H)-furanone (MDPF) were used for the study. The structure-function relationship of the membrane NH 2 groups was studied after modification with the probes under various conditions and relating the inhibition of the K +-stimulated ATPase to the ATPase-dependent H + accumulation by the gastric microsomal vesicles. TNBS (2 m m) inhibits nearly completely the K +-stimulated ATPase and the vesicular dye accumulation, both in presence and absence of valinomycin plus K +. Both the K +-ATPase and dye uptake were largely (about 50%) protected against TNBS inhibition if the treatment with TNBS was carried out in presence of 2 m m ATP. TNBS and FDNB labeled 70% of the total microsomal PE; the intra- and extravesicular orientation being 48 and 22%, respectively. The presence or absence of ATP did not have any effect on the TNBS labeling of microsomal PE. ATP, however, significantly ( P < 0.05) reduced the labeling of protein-bound NH 2 groups of gastric microsomes by TNBS. The intra- and extravesicular orientation of the protein NH 2 groups were 60 and 40%, respectively. Eighteen percent of the total protein-NH 2 appeared to be associated with the K +-stimulated ATPase; the rest being associated with non-ATPase proteins of the microsomes. About half (50%) of the total free NH 2 groups of the K +-stimulated ATPase were exposed to the vesicle exterior and were found to play critical roles in gastric ATPase function. The generation of florescence after MDPF conjugation of gastric microsomes was largely (50%) inhibited by ATP. ATP also protected completely the MDPF inhibition of gastric K +-stimulated ATPase and dye uptake.