CARDENOLIDE 16'-O-GLUCOHYDROLASE FROM DIGITALIS-LANATA - PURIFICATIONAND CHARACTERIZATION

A three-step chromatographic procedure was developed for purification of cardenolide 16'-O-glucohydrolase (CGH) from Digitalis lanata Ehrh, leaves, including Phenyl-Sepharose hydrophobic interaction chromatogra phy followed by SP-Sepharose cation exchange and Q-Sepharose anion-exc hange chromatography. Starting with acetone dry powder the purificatio n resulted in an 760-fold enrichment of CGH. Molecular weight, substra te specificity, pH optimum and temperature stability of CGH were deter mined. Antibodies against CGH were prepared in rabbits. The SDS gel el ectrophoresis of protein extracts from leaves of D. lanata and other D . species showed bands at 70 kDa and 36 kDa reacting with the antibodi es. The 70-kDa protein is the main protein stained with CGH antibodies in freshly prepared extracts of D. lanata. It may represent undegrade d CGH. The 36-kDa protein is enriched in aged CGH preparations. It is probably a degradation product. Proteins related to 70-kDa and 36-kDa bands also occur in crude protein preparations from leaves of D. heywo odii P. et M. Silva, D. mariana Boiss., D. purpurea L., and D. thapsi L. indicating that CGH is also present in these species. Purified CGH was digested with proteases V8 and Lys-C and the resulting fragments o btained were sequenced. One fragment had the typical amino-acid sequen ce of the catalytic center of family-1 glycosyl hydrolases (EC 3.2.1.x ). Cardenolide 16'-O-glucohydrolase, like the other members of this en zyme family, appeared to have a glutamic acid residue directly involve d in glycosidic bond cleavage as a nucleophile.