Presence of endogenous ent-kaurene in a microsomal preparation from Cucurbita maxima L. endosperm and implications for kinetic studies of ent-kaurene oxidase.
1993
Hazebroek J.P. | Schechter S.E. | Metzger J.D. | Coolbaugh R.C.
Microsomal and soluble cell-free extracts prepared from liquid endosperm of Cucurbita maxima L. were found to contain high concentrations of endogenous ent-kaurene and ent-kaurenol by gas chromatography-mass spectrometry-chemical ionization with deuterated internal standards. Increases in the levels of ent-kaurenol, ent-kaurenoic acid, and ent-7 alpha-hydroxykaurenoic acid are correlated with a decline in the amount of endogenous ent-kaurene following a 10 min incubation of microsomes with NADPH and FAD. The rate of oxidation of radiolabeled ent-kaurene by the microsomal fraction was determined, and the need to account for endogenous substrate is shown. Endogenous ent-kaurene present in soluble extracts had the effect of diluting the [14C]ent-kaurene synthesized from [14C]mevalonic acid, resulting in reduced specific radioactivity of the product. The dilution of [14C]ent-kaurene was more pronounced in extracts with higher endogenous ent-kaurene levels or when the reactions were run in the presence of O2 and NADPH. Evidence is presented that suggests differential metabolism of endogenous ent-kaurene and radiolabeled ent-kaurene in both microsomal and soluble extracts.
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