PURIFICATION AND CHARACTERIZATION OF THE CARDENOLIDE-SPECIFIC BETA-GLUCOHYDROLASE CGH-I FROM DIGITALIS-LANATA LEAVES

A cardenolide-hydrolysing beta-D-glucosidase (CGH I) was solubilised f rom young leaves of Digitalis lanata Ehrh. The specific enzyme activit y reached about 4 mu kat mg(-1) protein in buffered leaf extracts. Opt imal enzyme activities occurred at around pH 4.5 and 55 degrees C. CGH I was purified approximately 600-fold by hydrophobic interaction chro matography, cation exchange chromatography, anion exchange chromatogra phy and chromatofocusing. The apparent molecular mass of CGH I was 154 kDa, as determined by non-denaturating PAGE. Fragments of about 27, 3 7 and 76 kDa were obtained in SDS-PAGE. Kinetic constants of the enzym e for a variety of cardenolide and non-cardenolide substrates were det ermined (e.g., lanatoside A: K-m 21 mu m, V-max 225 nkat mg(-1) protei n; lanatoside C: K-m 146 mu M, V-max 225 nkat mg(-1) protein; purpurea glycoside A: K-m 38 mu M, V-max 120 nkat mg(-1) protein; deacetyllanat oside C: K-m 154 mu M, V-max 82 nkat mg(-1) protein; p-nitro-phenyl-be ta-beta-D-glucoside: K-m 10.5 mM, V-max 5 nkat mg(-1) protein).