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

Many hydrolytic enzymes are attached to the extracellular face of the plasma membrane of eukaryotic cells by a glycosylphosphatidylinositol (GPI) anchor. Little is currently known about the consequences for enzyme function of anchor cleavage by phosphatidylinositol-specific phospholipase C. We have examined this question for the GPI-anchored protein 5´-nucleotidase (5´-ribonucleotide phosphohydrolase; EC 3.1.3.5), both in the native lymphocyte plasma membrane, and following purification and reconstitution into defined lipid bilayer vesicles, using Bacillus thuringiensis phosphatidylinositol-specific phospholipase C (PI-PLC). Membrane-bound, detergent-solubilized and cleaved 5´-nucleotidase all obeyed Michaelis–Menten kinetics, with a K_m for 5´-AMP in the range 11–16 µM. The GPI anchor was removed from essentially all 5´-nucleotidase molecules, indicating that there is no phospholipase-resistant pool of enzyme. However, the phospholipase was much less efficient at cleaving the GPI anchor when 5´-nucleotidase was present in detergent solution, dimyristoyl phosphatidylcholine, egg phosphatidylethanolamine and sphingomyelin, compared with the native plasma membrane, egg phosphatidylcholine and a sphingolipid/cholesterol-rich mixture. Lipid molecular properties and bilayer packing may affect the ability of PI-PLC to gain access to the GPI anchor. Catalytic activation, characterized by an increase in V_max, was observed following PI-PLC cleavage of reconstituted 5´-nucleotidase from vesicles of several different lipids. The highest degree of activation was noted for 5´-nucleotidase in egg phosphatidylethanolamine. An increase in V_max was also noted for a sphingolipid/cholesterol-rich mixture, the native plasma membrane and egg phosphatidylcholine, whereas vesicles of sphingomyelin and dimyristoyl phosphatidylcholine showed little activation. K_m generally remained unchanged following cleavage, except in the case of the sphingolipid/cholesterol-rich mixture. Insertion of the GPI anchor into a lipid bilayer appears to reduce the catalytic efficiency of 5´-nucleotidase, possibly via a conformational change in the enzyme, and activity is restored on release from the membrane.