The β-glucan synthase from Lolium multiflorum. Detergent solubilization, purification using monoclonal antibodies, and photoaffinity labeling with a novel photoreactive pyrimidine analogue of uridine 5'-diphosphoglucose

The membrane-bound β-glucan synthase from Italian ryegrass (Lolium multiflorum L.) endosperm cells has been solubilized by both non-ionic and zwitterionic detergents. A complex relationship exists between the ratio of (1→3)-, (1→4)-, and (1→3, 1→4)-β-glucan products of the solubilized enzyme, the cations present, and the concentration of the uridine 5'-diphosphoglucose substrate. Monoclonal antibodies directed against the β-glucan synthase complex were generated by immunization of mice with an unfractionated microsomal preparation. Hybridoma cell lines were screened using a combination of indirect enzyme-linked immunosorbent assay followed by an enzyme-capture assay. The purified monoclonal antibodies were used with Pansorbin (stabilized protein A-bearing staphylococcal cells) to immunoprecipitate an active β-glucan synthase complex which had been solubilized from a microsomal preparation with 0.6% CHAPS. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of the immunoprecipitated synthase complex revealed four major polypeptides of apparent molecular mass 30, 31, 54, and 58 kDa together with several minor components. The immunoprecipitated β-glucan synthase complex was capable of synthesizing both (1→ 3)- and (1→4)-β-glucans. A new photoreactive pyrimidine analogue of uridine 5'-diphosphoglucose, 5-[3-(p-azidosalicylamide)]allyl-uridine 5'-diphosphoglucose was synthesized in a three-step reaction sequence involving mercuration of UDP-Glc, alkylation of 5-Hg-UDP-Glc, and acylation of 5-(3-amino)allyl-UDP-Glc and characterized by chemical and spectroscopic analysis. The analogue inhibits (K(i)(app) 16 μM) and, upon UV irradiation, irreversibly inactivates the β-glucan synthase. The analogue was iodinated with Na¹²⁵I to give a radiolabeled, photoreactive compound, and was used in photoaffinity labeling of UDP-Glc pyrophosphorylase, UDP-Glc dehydrogenase, and several putative UDP-Glc-binding proteins from L. multiflorum. The radiolabeled analogue specifically labeled the 31-kDa polypeptide in the immunoprecipitated synthase complex. The photolabeling of this polypeptide is strictly dependent on UV irradiation, is blocked by uridine 5'-diphosphoglucose and uridine 5'-diphosphate, and reaches saturation at analogue concentrations above 300 μM. These results indicate that the 31-kDa polypeptide in the β-glucan synthase complex bears a uridine 5'-diphosphoglucose-binding site and is involved in the catalysis of β-glucan synthesis.