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An elicitor-inducible sesquiterpene cyclase, which catalyzes the conversion of
farnesyl diphosphate to 5-epi-aristolochene (IM Whitehead, DR Threlfall, DF
Ewing [1989] Phytochemistry 28:775-779) and representing a committed step in the
phytoalexin biosynthetic pathway in tobacco, was purified by a combination of
hydrophobic interaction, anion exchange, hydroxylapatite, and chromatofocusing
chromatography. From 2 kilograms of elicited tobacco (Nicotiana tabacum) cells,
approximately 500 micrograms of cyclase protein was purified, representing
greater than a 130-fold increase in the specific activity of the enzyme and a 4%
recovery of the starting activity. The purified enzyme resolved as two major
polypeptides of 60 and 62 kilodaltons by sodium dodecyl sulfate-polyacrylamide
gel electrophoresis (SDS-PAGE). Biochemical characterization of the enzyme
activity included an absolute requirement for magnesium, an isoelectric point of
4.5 to 4.9, and a K(m) for farnesyl diphosphate of 2 to 5 micromolar. The
purified cyclase protein was used to generate mouse polyclonal antibodies which
efficiently inhibited cyclase activity in an in vitro assay. Electrophoresis of
extracts from elicitor-treated cells or purified cyclase enzyme on native
polyacrylamide gels separated the cyclase enzyme into four polypeptides as shown
by immunoblot analysis using poly- and monoclonal antibodies. Proportionate
cyclase enzyme activity comigrated with those polypeptides. No cyclase
polypeptides were detectable in extracts of control cells by immunoblot
analysis. However, immunoblot analysis of proteins from elicitor-treated cells
using four independent monoclonal antibody lines and the polyclonal antibodies
detected the same polypeptides, regardless of whether the proteins were
separated by native or SDS-PAGE. The results suggest an induction of multiple
cyclase polypeptides in elicitor-treated cells resulting from either the
expression of multiple genes or multiple post-translational processing events.
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