Purification of P-glycoprotein from plasma membrane vesicles of Chinese hamster ovary cell mutants with reduced colchicine permeability.

Abstract

Plasma membrane vesicles were isolated from col- chicine-resistant mutant lines and sensitive wild type and revertant lines of Chinese hamster ovary cells after controlled cell disruptions. The pressures required to disrupt the mutant cells (150 p.s.i.) similarly were less than for the wild type (350 p.s.i.) or revertant (300 p.s.i.) cells indicating an increased fragility of the drug-re- sistant mutants. In all three cases, however, the final plasma membrane preparations consisted of homoge- neous populations of vesicles ranging between 0.1 and 0.7 pm in diameter and were enriched lo-fold in 5’- nucleotidase and (Na+ + K’)-Mti+ATPase. An integral membrane glycoprotein with a molecular weight of approximately 170,000 (“P-glycoprotein”) was present in vesicles from the drug-resistant cells but not the others. The amount of this protein was related to the degree of drug resistance. The protein was purified by the following procedure: removal of peripheral mem- brane proteins with 35 lll~ lithium diiodosalicylate, solubilization in 0.5% N-dodecylsarcosinate, and frac- tionation by lectin-agarose affinity chromatography using Ricinus communis-I which bound the P-glycopro- tein and Lens culinaris and Pisum sativum lectins which bound other membrane glycoproteins. The amount of purified P-glycoprotein thus obtained ac- counted for approximately 3 to 4% of the total plasma membrane protein in one colchicine-resistant mutant line.