Isolation and characterization of membranes from oleic acid-induced peroxisomes of Candida tropicalis

Abstract

We report a methodology for the isolation of peroxisome membranes from the yeast Candida tropicalis pK233 grown on oleic acid, and the characterization of the polypeptide and lipid compositions of these membranes. Peroxisomes purified in either sucrose or Nycodenz gradients are treated with Tris-HCl (pH 8.5) and then with sodium carbonate (pH 11.5) to yield a final peroxisome membrane preparation (hereafter called 'peroxisome membranes'). Electron microscopy revealed peroxisome membranes that are approximately 8.1 nm thick, have a typical trilaminar appearance, and form either flattened sheets or whorled structures. Peroxisome membranes contain 3.1% and 2.2% of the total protein of sucrose- and Nycodenz-gradient-purified peroxisomes, respectively. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis showed three predominant polypeptide bands of 34 (PMP 34), 29 (PMP 29), and 24 (PMP 24) x 10(3) Mr in peroxisome membranes. Immunoblotting with an antiserum to PMP 24 showed that PMP 24 segregates with the peroxisome membrane fractions and is induced by growth of Candida tropicalis on oleic acid. Peroxisome membranes contain neutral lipids and phospholipids. The principal phospholipids are phosphatidyl choline and phosphatidyl ethanolamine. The phospholipid/protein ratio of peroxisome membranes is approximately 430 nmol mg-1.