PLANT PHYSIOLOGY , Vol 109, Issue 4 1239-1245, Copyright © 1995 by American Society of Plant Biologists
|
BIOCHEMISTRY AND ENZYMOLOGY |
Purification and Properties of ent-Kaurene Synthase B from Immature Seeds of Pumpkin
T. Saito, H. Abe, H. Yamane, A. Sakurai, N. Murofushi, K. Takio, N. Takahashi and Y. Kamiya
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
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.
