An improved enzymatic synthesis of labeled gibberellin A12-aldehyde and gibberellin A12

Abstract The biochemical synthesis of labeled gibberellin A 12 -7-aldehyde (GA 12 ald) and gibberellin A 12 (GA 12 ) from labeled R,S -mevalonic acid (MVA) using cell-free extracts from pumpkin ( Cucurbita maxima ) endosperm has been improved over the previously reported procedure. Three major improvements were (a) developing a one-step HPLC procedure to isolate GA 12 ald and GA 12 in radiochemically pure form; (b) adjusting the pH of the reaction mix to pH 6.9 which increased the GA 12 ald GA 12 ratio over that at pH 7.8 by ca. 18-fold while reducing the combined yield of these two compounds by less than 17%; and (c) developing a technique that permitted sampling of the fruits without interrupting growth; this doubled the fraction of extracts with “high activity.” Conversion of MVA into GA 12 ald or GA 12 displayed Michaelis-Menten kinetics with half-maximal synthesis at 0.4 m m MVA. Four-hour incubations afforded the highest yields from 0.25 m m MVA. Up to 15 and 7% of the MVA was incorporated into GA 12 ald and GA 12 , respectively. About one-quarter of the extracts incorporated at least 10% of the 0.25 m m MVA into GA 12 ald. One pumpkin fruit can provide sufficient endosperm to synthesize ca. 0.6 μmol of GA 12 ald.