Lectin affinity chromatography and electrophoretic properties of human platelet gamma-glutamyl transferase

Abstract

The sialoglycoprotein, gamma-glutamyl transferase (GGT, gamma-GT, EC 2.3.2.2) is a membrane enzyme found in many cells including platelets and leukocytes. In platelets GGT converts leukotriene C-4 (LTC4) to leukotriene D-4 (LTD4) and is involved in glutathione metabolism. In this study, human platelet GGT was solubilized with Triton X-100 and purified by lectin affinity chromatography on Con A Sepharose 4B to determine its electrophoretic properties. The specific activity of purified GGT was 236 mU/mg protein; 73.7% of human platelet GGT activity was found bound to Con A and 50% of the bound activity was released with 0.3 mol/l methyl alpha-D-mannopyranoside. We observed that human platelet GGT has only one isoenzyme band showing a carbohydrate stained band near the origin on polyacrylamide gel electrophoresis (PAGE). The electrophoretic mobility of papain-solubilized GGT was higher than that of Triton X-100-solubilized GGT at PAGE. Also GGT activities were determined on neuraminidase, trypsin or n-butanol-DIPE (diisopropyl ether)-treated Triton X-100-solubilized membrane fractions. This characterization may be useful when trying to establish the contribution of platelet GGT to serum GGT activity. This marker may reflect the extent of platelet activation.