Xanthine dehydrogenase in the fat body of Manduca sexta: Purification, characterization, subcellular localization and levels during the last larval instar

Abstract

Xanthine dehydrogenase was isolated and purified from the fat body of fifth instar tobacco hornworms, Manduca sexta (L.) by ammonium sulfate precipitation, Sephacryl S-400 gel filtration, QAE-Sephadex ion exchange chromatography and reactive blue-2 Sepharose affinity chromatography. The preparation appeared homogeneous following analysis by polyacrylamide disc gel electrophoresis, sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE), and rocket and crossed immunoelectrophoresis using rabbit antiserum against purified xanthine dehydrogenase. The molecular weight of native xanthine dehydrogenase was estimated at about 300,000 while SDS-PAGE indicated that the enzyme consisted of two 158,000 mol. wt subunits. Kinetic analyses revealed high affinities for xanthine and hypoxanthine as substrates. Electron acceptors (cofactors) included NAD +, as well as the dyes, p-iodonitrotetrazolium violet and nitro blue tetrazolium. A broad pH optimum was observed between pH 7 and 10, and xanthine dehydrogenase was competitively inhibited by allopurinol and irreversibly inhibited by the sulfhydryl inhibitor, p-hydroxymercuribenzoate. The levels of xanthine dehydrogenase in fat body extracts from fifth instar larvae, as determined by both enzyme assay and immunoelectrophoresis, decreased from days 1 to 5. Ultra-structural analysis of day 3 larval fat body cells by immunocytochemical techniques, using colloidal gold staining, revealed that xanthine dehydrogenase was specifically located either within urate storage vacuoles or near the basal lamina of the cells.