PLANT PHYSIOLOGY , Vol 102, Issue 1 205-211, Copyright © 1993 by American Society of Plant Biologists
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METABOLISM AND ENZYMOLOGY |
Purification and Characterization of Geranyl Diphosphate Synthase from Vitis vinifera L. cv Muscat de Frontignan Cell Cultures
M. Clastre, B. Bantignies, G. Feron, E. Soler and C. Ambid
Ecole Nationale Superieure Agronomique, 145 Avenue de Muret, F-31076 Toulouse Cedex, France
A geranyl diphosphate synthase (EC 2.5.1.1), which catalyzes the formation
of geranyl diphosphate from dimethylallyl diphosphate and isopentenyl
diphosphate, was isolated from Vitis vinifera L. cv Muscat de Frontignan
cell cultures. Purification of the enzyme was achieved successively by
ammonium sulfate precipitation and chromatography on DEAE-Sephacel,
hydroxylapatite, Mono Q, Phenyl Superose, Superose 12, and preparative
nondenaturing polyacrylamide gels. The enzyme formed only geranyl
diphosphate as a product. In all cases, neither neryl diphosphate, the cis
isomer, nor farnesyl diphosphate was detected. The enzyme showed a native
molecular mass of 68 [plus or minus] 5 kD as determined by gel permeation.
On sodium dodecyl sulfate polyacrylamide gels, geranyl diphosphate synthase
purified to electrophoretic homogeneity migrated with a molecular mass of
66 [plus or minus] 2 kD. Michaelis constants for isopentenyl diphosphate
and dimethylallyl diphosphate were 8.5 and 56.8 [mu]M, respectively. The
enzyme required Mn2+ and Mg2+ as cofactors and its activity was enhanced by
Triton X-100. Inorganic pyrophosphate, aminophenylethyl diphosphate, and
geranyl diphosphate had inhibitory effects on the enzyme.
