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Properties and Partial Purification of 1-Aminocyclopropane-1-carboxylate Synthase ¹

MSU-DOE Plant Research Laboratory, Michigan State University, East Lansing, Michigan 48824

We studied the regulation of 1-aminocyclopropane-1-carboxylate (ACC) synthase activity in tomato (Lycopersicon esculentum Mill.) fruit tissue and attempted the purification of this enzyme. The increase of ACC synthase activity in wounded tomato pericarp was inhibited by cordycepin and cycloheximide. Density labeling studies showed a 0.75% increase in the buoyant density of ACC synthase isolated from tomato pericarp tissue that had been incubated on ²H₂O as compared to ACC synthase from H₂O-treated tissue. These data are consistent with the hypothesis that ACC synthase is synthesized de novo following wounding of tomato pericarp tissue. SDS-gel electrophoresis and fluorography showed that the pattern of incorporation of L-[³⁵S]methionine into protein changed with time after wounding of the tissue. Radioactive protein bands that were not detected 1 hour after wounding, became apparent 2 to 3 hours after wounding.

Gel filtration on Sephadex G-100 gave a molecular weight estimate for ACC synthase of 57,000 ± 1,500 daltons. Hydrophobic interaction chromatography on phenyl-Sepharose yielded a 60- to 70-fold purification of the enzyme. SDS-gel electrophoresis of this preparation indicated the presence of one intense band at 57,000 daltons and several less intense bands. Affinity chromatography was of limited usefulness in the purification of ACC synthase since the enzyme could not be eluted specifically from any of the affinity gels tried. Purification methods that involved pH changes led to a rapid loss of ACC synthase activity. ACC synthase was estimated to comprise less than 1% of the total protein in tomato pericarp tissue.

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