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

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Abstract
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 °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 < 1.0). The inhibition properties of the enzyme can be fully restored by co-reconstituting pure 5′-nucleotidase with the remaining lymphocyte glycoproteins.