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

Add time:08/17/2019    Source:sciencedirect.com

The biochemical synthesis of labeled gibberellin A12-7-aldehyde (GA12ald) and gibberellin A12 (GA12) 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 GA12ald and GA12 in radiochemically pure form; (b) adjusting the pH of the reaction mix to pH 6.9 which increased the GA12aldGA12 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 GA12ald or GA12 displayed Michaelis-Menten kinetics with half-maximal synthesis at 0.4 mm MVA. Four-hour incubations afforded the highest yields from 0.25 mm MVA. Up to 15 and 7% of the MVA was incorporated into GA12ald and GA12, respectively. About one-quarter of the extracts incorporated at least 10% of the 0.25 mm MVA into GA12ald. One pumpkin fruit can provide sufficient endosperm to synthesize ca. 0.6 μmol of GA12ald.

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