This Article Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via CrossRef Citing Articles via Google Scholar Google Scholar Articles by Dueber, M. T. Articles by West, C. A. Search for Related Content PubMed PubMed Citation Articles by Dueber, M. T. Articles by West, C. A. Agricola Articles by Dueber, M. T. Articles by West, C. A.

Articles

Biosynthesis of the Diterpene Phytoalexin Casbene

Partial Purification and Characterization of Casbene Synthetase from Ricinis communis ¹

Division of Biochemistry, Department of Chemistry, University of California, Los Angeles, California 90024

Casbene synthetase from 67-hour seedlings of Ricinis communis L. which had been treated with Rhizopus stolonifer spores was purified 700-fold by a combination of ammonium sulfate fractionation, QAE A-50 Sephadex chromatography, and G-100 Sephadex chromatography. Polyacrylamide disc gel electrophoresis revealed that the purified fraction was heterogeneous. No casbene synthetase was detected in extracts of seedlings which had not been exposed to the fungal spores; maximum activity was obtained from seedlings 14 hours after exposure to spores.

The partially purified enzyme exhibited a broad pH optimum from pH 7.5 to 9.0 with half-maximal activity at pH 6.0 and 9.8. Chromatography on a calibrated Sephadex G-100 column indicated a molecular weight of 53,000 ± 3,000 for casbene synthetase. Concentrations of Mg²⁺ above 5 mM gave maximal stimulation of the activity. Mn²⁺ was much less effective and was inhibitory at concentrations above 0.2 mM. The K_m for geranyl-geranyl pyrophosphate was estimated as 1.9 µM. The activity was inhibited 50% by 2.5 mM N-ethylmaleimide; 10 mM iodoacetamide was not inhibitory. N,N-Dimethylaminoethyl-2,2-diphenylpentanoate (SKF-525A) and the growth retardant 2'isopropyl-4'-(trimethylammonium chloride)-5'-methylphenyl piperidine-1-carboxylate (Amo-1618) were ineffective inhibitors of casbene synthetase, but the growth retardant tributyl-2, 4-dichlorobenzyl-phosphonium chloride (Phosphon D) at a concentration of 5 µM inhibited the activity by 55%.

¹ This work was supported by National Institutes of Health Grant GM-07065 and National Science Foundation Grant PCM 77-14027.

This article has been cited by other articles:

				J. Bohlmann, G. Meyer-Gauen, and R. Croteau Plant terpenoid synthases: Molecular biology and phylogenetic analysis PNAS, April 14, 1998; 95(8): 4126 - 4133. [Abstract] [Full Text] [PDF]