Metabolism of kaurene in cell-free extracts of immature pea seeds

Abstract

Geranylgeraniol, (—)-kaurene, (—)-kaurenol, (—)-kaurenal and (—)-kaurenoic acid were tentatively identified as products synthesized from mevalonic acid-2 14C in cell-free reaction mixtures containing 10,000 g supernatant of extracts of cotyledons of immature pea ( Pisum sativum L. cv. Alaska) seeds. The identity of (—)-kaurenol was confirmed by co-crystallization of the radioactive product with authentic kaurenol to constant specific radioactivity. Repeated efforts to observe cell-free metabolism of exogenous (—)-kaurene in a variety of enzyme extracts consistently resulted in failure. Only when (—)-kaurene was synthesized in situ from mevalonic acid in reaction mixtures containing 10,000 g supernatant from cotyledon extracts could the metabolism of (—)-kaurene be observed. Thus, the procedure employed most extensively was to allow (—)-kaurene to be formed from mevalonic acid-2 14C for 60 min, then to inhibit its further synthesis by the addition of AMO-1618, and finally to assay the carbon monoxide-sensitive oxidation of (—)-kaurene which occurred during a subsequent 60-min incubation. By this procedure it was demonstrated that the oxidation of (—)-kaurene which was formed in situ was enzymic, or at least heat labile; markedly sensitive to inhibition by carbon monoxide; and greater in extracts prepared with insoluble polyvinyl-pyrrolidone (PVP) than in extracts prepared without PVP. Possible reasons for the failure of exogenous (—)-kaurene to be oxidized are discussed.