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

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