Alkaline density gradient floatation of membranes: Polypeptide compositionof the mammalian peroxisomal membrane

A method for purification of the peroxisomal membrane from rat liver is des cribed. The procedure consists of floating the (contaminated) peroxisomal m embranes through an alkaline sucrose density gradient. A good resolution be tween the peroxisomal membrane and other membrane systems is achieved. Usin g these floated peroxisomal membranes we have determined that only 7.8 +/- 0.9% of the total peroxisomal protein is alkali resistant. The polypeptide composition of these highly pure peroxisomal membranes was analyzed by SDS- PAGE. Bands corresponding to polypeptides with apparent molecular masses of 15, 18, 22, 24, 26, 29, 35, 36, 38, 40, 52, 55, 70, 74-77, and 88 kDa are detected upon Coomassie blue staining of polyacrylamide gels. The identity of several of these polypeptides was determined by N-terminal sequencing an d Western blotting analysis. (C) 1999 Academic Press.