PURIFICATION AND CHARACTERIZATION OF LANATOSIDE 15'-O-ACETYLESTERASE FROM DIGITALIS-LANATA EHRH

Lanatoside 15'-O-acetylesterase (LAE) from in-vitro-cultivated cells o f Digitalis lanata Ehrh. was isolated and partially sequenced. The enz yme was extracted with citrate buffer from acetone dry powder. It was purified in a two-step chromatographical procedure including Phenyl Se pharose hydrophobic interaction chromatography followed by CM Sepharos e cation-exchange chromatography to more than 330 mu mol.s(-1).(g prot ein)(-1). Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (S DS-PAGE) of the purified protein showed a major band at 39 kDa. The pr otein was identified by correlation of band intensity on SDS-PAGE and enzyme activity of CM Sepharose column fractions. Size-exclusion chrom atography on Sephacryl 200 revealed a single activity peak with an app arent molecular mass of about 85 kDa. Electrophoresis under nondenatur ating conditions of purified LAE showed only one band with esterase ac tivity. The intensity of this band was correlated with that of the 39- kDa band after SDS-PACE. About 30% of the protein, including the N-ter minus and several fragments obtained by Lys-C protease digestion, was sequenced. A fragment obtained by Lys-C digestion showed partial homol ogy to other hydrolases and apoplasmic proteins. It included the proba ble location of an active-site histidine. The activity of LAE was high in non-morphogenic D. lanata cell strains selected for high activitie s in the chemical transformation of cardenolides, but rather low in th e proembryogenic masses of the embryogenic cell strain VIII. It increa sed during the development of somatic embryos. The LAE activity in lea ves of D. lanata plants was in the range 4-24 nmol.s(-1).(g protein)(- 1).