An improved enzymatic synthesis of labeled gibberellin A12-aldehyde and gibberellin A12.
The biochemical synthesis of labeled gibberellin A12-7-aldehyde (GA12ald) and 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 developing a one-step HPLC procedure to isolate GA12ald and GA12 in radiochemically pure form; adjusting the pH of the reaction mix to pH 6.9 which increased the GA12ald/GA12 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 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 mumol of GA12ald.
D002474:Cell-Free System; D005875:Gibberellins / Q000096:biosynthesis*; D007700:Kinetics; D008798:Mevalonic Acid / Q000378:metabolism; D010944:Plants / Q000378:metabolism
D005875:Gibberellins; C048462:gibberellin A12; C048463:gibberellin A12 aldehyde; D008798:Mevalonic Acid