Reconstitution of lymphocyte 5'-nucleotidase in lipid bilayers: behaviour and interaction with concanavalin A.

Pure 5'-nucleotidase (EC 3.1.3.5) and a membrane glycoprotein fraction (partially purified 5'-nucleotidase) were isolated from pig lymphocyte plasma membrane by affinity chromatography techniques, using the cationic detergent dodecyltrimethylammonium bromide as a solubilizing agent. A detergent-dialysis technique was used to reconstitute both partially purified and pure enzyme into large unilamellar phospholipid vesicles, where it remains functional. 5'-Nucleotidase is relatively unstable in detergent solutions, but is highly stable once reconstituted into lipid vesicles. Arrhenius plots of the enzyme in bilayers of dimyristoyl phosphatidylcholine show a break point at 22-23 degrees C, with a different activation energy above and below the phospholipid gel-to-liquid crystalline phase transition. 5'-Nucleotidase in intact plasma membrane is inhibited more than 95% by concanavalin A in a positively cooperative fashion (Hill coefficient = 2.1), as is partially purified reconstituted enzyme. Purification of the enzyme before reconstitution results in less than 50% inhibition by concanavalin A and a complete loss of positive cooperativity (Hill coefficient less than 1.0). The inhibition properties of the enzyme can be fully restored by co-reconstituting pure 5'-nucleotidase with the remaining lymphocyte glycoproteins.