The isolation by self forming gradients of Percoll of plasma membrane enriched fractions from bovine retinal pigment epithelium

A method is described for the preparation of plasma membrane enriched fractions from bovine retinal pigment epithelium (RPE) by means of differential centrifugation followed by the use of self forming gradients of Percoll. A detailed analysis of the distribution of organelle specific markers (nuclei, mitochondria, lysosomes, endoplasmic reticulum, cytosol) in the different fractions is presented. Comparison of 125I-wheat germ agglutinin (WGA) binding with more conventional plasma membrane enzyme markers demonstrates that also in RPE radiolabeled lectin is a specific and extremely sensitive marker to follow quantitatively the distribution of outer cell membranes. Results of 125I-WGA displacement experiments indicate that plasma membranes are mostly (90%) composed of right side out vesicles or sheets. On the basis of 125I-WGA radioactivity the overall recovery of plasma membranes was about 10% and purification over 15 fold. NADH cytochrome c reductase activity, which is shown to be a specific marker for endoplasmic reticulum in retinal pigment epithelium, has been utilized to evaluate microsomal contamination of the plasma membrane preparation.