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

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 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.

Cell Surface Display and Intracellular Trafficking of Free Glycosylphosphatidylinositols in Mammalian Cells

“…The amount of the ER marker ribophorin I and the mitochondrial marker Hsp70 was determined by Western blot analysis as described above. 5Ј-Nucleotidase activity (PM marker) was assayed as described previously (37).…”

Release of the glycosylphosphatidylinositol-anchored enzyme ecto-5′-nucleotidase by phospholipase C: catalytic activation and modulation by the lipid bilayer

“…Briefly, homogenized porcine mesenteric lymph nodes (30 g) were layered over a sucrose cushion (41 %, w\v), which was followed by ultracentrifugation at 95 000 g. Lymphocyte 5h-NTase was purified as described previously [15,17], by solubilizing plasma membrane in 50 mM CHAPS and performing two sequential affinitychromatography steps, the first using lentil lectin-Sepharose 4B (Pharmacia Canada, Baie D'Urfe! , QC, Canada), the second using 5h-AMP-Sepharose (Sigma).…”

“…Purified 5h-NTase was reconstituted into lipid bilayer vesicles using a modification of the detergent-dialysis technique described previously [15,17,18]. A mixture of the desired lipids (1-3 mg) in methanol\CHCl $…”

“…5h-NTase plays an important role in purine salvage in lymphocytes, where it is known as CD73, and has also been implicated in transmembrane signalling in itro in T-lymphocytes [12]. The enzyme is N-glycosylated, and binding of a variety of lectins to N-linked carbohydrates on 5h-NTase is known to inhibit activity [15,16], suggesting that some communication exists between the glycan chain(s) and the catalytic site.…”

“…Previous work in our laboratory led to the isolation and reconstitution of porcine-lymphocyte 5h-NTase into lipid bilayers, where it retains enzymic activity that is inhibited by lectin binding [15,17]. Reconstitution of purified GPI-anchored proteins into bilayers of defined phospholipids provides a powerful tool to delineate the effects of membrane properties on the behaviour and interactions of this class of proteins.…”