Role of cholesterol in the structure and function of gastric microsomal vesicles

Digitonin was used as a tool to investigate the organization and function of cholesterol in gastric microsomes. Microsomal vesicles were treated with digitonin for different time at 0-4 degrees C under isotonic conditions. The effects of digitonin treatment of the vesicles on removal of cholesterol, ultrastructural changes, (H+ + K+)-ATPase activity, and gastric ATPase-dependent H+ uptake ability were investigated. Microsomal cholesterol was extracted in an exponential manner with a t1/2 of 32 min. There was no release of microsomal phospholipids by digitonin treatment during the same period. Digitonin treatment (30 min) produced visible "holes" in the vesicles; at the same time (H+ + K+)-ATPase-dependent H+ uptake was abolished. Under the same conditions the K+-stimulated ATPase activity, however, was moderately (about 35%) reduced, although the response of K+ stimulation to valinomycin was obliterated. Longer digitonin treatment resulted in gradual diffusion and eventual disappearance of the "holes" with the generation of distorted cup-shaped microsomes. The data strongly suggest that membrane lipids are freely mobile and that there is a certain degree of specialization in the organization of gastric microsomal cholesterol for the proper maintenance of the membrane structure and function.