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.
D015720:5'-Nucleotidase; D000818:Animals; D002462:Cell Membrane / Q000201:enzymology; D003208:Concanavalin A / Q000494:pharmacology*; D004789:Enzyme Activation; D007700:Kinetics; D008051:Lipid Bilayers; D008214:Lymphocytes / Q000201:enzymology*; D008954:Models, Biological; D009708:Nucleotidases / Q000302:isolation & purification / Q000378:metabolism*; D013552:Swine; D013816:Thermodynamics
D008051:Lipid Bilayers; D003208:Concanavalin A; D009708:Nucleotidases; D015720:5'-Nucleotidase