Alkaline density gradient floatation of membranes: polypeptide composition of the mammalian peroxisomal membrane.
A method for purification of the peroxisomal membrane from rat liver is described. The procedure consists of floating the (contaminated) peroxisomal membranes through an alkaline sucrose density gradient. A good resolution between the peroxisomal membrane and other membrane systems is achieved. Using 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 and Western blotting analysis.
Gouveia
A M
AM
Instituto de Biologia Molecular e Celular, Universidade do Porto, Porto, Portugal.
Reguenga
C
C
Oliveira
M E
ME
Eckerskorn
C
C
Sá-Miranda
C
C
Azevedo
J E
JE
D000595:Amino Acid Sequence; D000818:Animals; D002458:Cell Fractionation / Q000379:methods; D002499:Centrifugation, Density Gradient / Q000379:methods*; D004591:Electrophoresis, Polyacrylamide Gel; D007425:Intracellular Membranes / Q000737:chemistry*; D008297:Male; D008969:Molecular Sequence Data; D010455:Peptides / Q000737:chemistry*; D020675:Peroxisomes / Q000737:chemistry*; D051381:Rats; D017208:Rats, Wistar
D010455:Peptides