title: Purification and Properties of ent-Kaurene Synthase B from Immature Seeds of Pumpkin.
abstract: ent-Kaurene synthase B (KSB) was purified 291-fold from a crude enzyme preparation from endosperm of pumpkin (Cucurbita maxima L.). Separation of ent-kaurene synthase A and KSB was achieved by hydrophobic interaction chromatography. The fractions containing KSB activity were further purified by diethylaminoethyl, phenyl, and hydroxyapatite column chromatography. Using sodium dodecyl phosphate-polyacrylamide gel electrophoresis, the purest enzyme preparation showed a major band at an apparent molecular mass of 81 kD. The amount of protein in this band was correlated with KSB activity after diethylaminoethyl and hydroxyapatite chromatography. The N terminus of the 81-kD protein was blocked. Therefore, the protein was partially digested with protease and the amino acid sequences of the resulting major peptide fragments were analyzed. A polyclonal antibody was raised against a synthetic peptide based on the longest peptide fragment combined with a keyhole limpet hemocyanin. The antibody recognized only the 81-kD denatured protein and not the native KSB. The properties of KSB were examined using the phenyl-purified enzyme preparation. The Km value for copalyl pyrophosphate was 0.35 [mu]M, and the optimal pH was 6.8 to 7.5. The KSB activity required divalent cations such as Mg2+, Mn2+, and Co2+, whereas Cu2+, Ca2+, and Ba2+ inhibited the activity.
authors.affiliation: Frontier Research Program (T.S., N.T., Y.K.) and The Institute of Physical and Chemical Research (RIKEN) (H.A., A.S., K.T.), Wako-shi, Saitama, 351-01 Japan.
authors.forename: T.
authors.initials: T
authors.lastname: Saito
authors.forename: H.
authors.initials: H
authors.lastname: Abe
authors.forename: H.
authors.initials: H
authors.lastname: Yamane
authors.forename: A.
authors.initials: A
authors.lastname: Sakurai
authors.forename: N.
authors.initials: N
authors.lastname: Murofushi
authors.forename: K.
authors.initials: K
authors.lastname: Takio
authors.forename: N.
authors.initials: N
authors.lastname: Takahashi
authors.forename: Y.
authors.initials: Y
authors.lastname: Kamiya