Ronald C. Coolbaugh

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 14 C 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 14 C 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.

Conversion of geranylgeranyl pyrophosphate to ent-kaurene in enzyme extracts of sonicated chloroplasts

Abstract Farnesyl pyrophosphate-[ 14 C] and geranylgeranyl pyrophosphate-[ 14 C] were biosynthesized from mevalonic acid-[2- 14 C] by cell-free enzyme extracts of pea ( Pisum sativum ) cotyledons containing MgCl 2 , MnCl 2 , ATP and AMO-1618. Maximum yields of farnesyl pyrophosphate were obtained after 30 min incubation while geranylgeranyl pyrophosphate was the primary product after 180 min. Biosynthesized geranylgeranyl pyrophosphate-[ 14 C] served as an efficient substrate for ent -kaurene biosynthesis in reaction mixtures containing cotyledon enzymes when AMO-1618 was omitted. Enzyme extracts from green pea shoot tips and chloroplasts also converted geranylgeranyl pyrophosphate to ent -kaurene in very low yields. Ent -kaurene production from mevalonic acid-[2- 14 C] in extracts of pea shoot tips was also enhanced by addition of chloroplast enzymes. This evidence indicates that kaurene synthetase is present in pea chloroplasts and adds to the possibility that some gibberellin biosynthesis may be compartmentalized in those organelles.

Biosynthesis of ent-kaurene in cell-free extracts of Pisum sativum shoot tips☆

Abstract Soluble enzyme preparations from pea shoot tips incorporated mevalonic acid-2- 14 C into ent -kaurene- 14 C, squalene- 14 C and other products. The assay for either ent -kaurene or squalene is quite direct; both products can be obtained apparently free of radioactive contaminants by TLC on silica gel G in hexane. The enzyme system is dependent upon added ATP and Mn 2+ or Mg 2+ , with Mn 2+ being a more effective activator than Mg 2+ under the experimental conditions. Reduced pyridine nucleotide had no effect on ent -kaurene production but stimulated squalene synthesis. The accumulation of both ent -kaurene and squalene was stimulated by dithiothreitol and carbon monoxide and was reduced by the addition of particulate cell components. AMO-1618 inhibited ent -kaurene production and had no effect on the synthesis of squalene. Enzyme extracts from shoot tips are much less active in ent -kaurene synthesis than extracts from the cotyledons of immature seeds on either a fresh weight or protein basis.

Localization of enzymes catalysing kaurene biosynthesis in immature pea seeds

Abstract Comparative assays of (–)-kaurene accumulation in cell-free extracts of whole pea ( Pisum sativum L. cv. Alaska) seeds, isolated seed coats, cotyledons, and embryonic shoot-root axes revealed that the enzymes catalysing the synthesis of (−)-kaurene from mevalonic acid-2- 14 C are apparently localized exclusively in the cotyledons. Differential centrifugation of extracts showed that the enzymes catalysing (−)-kaurene biosynthesis occur in the soluble fraction. The enzymes responsible for squalene (identification tentative) biosynthesis were present in both the seed coat and the cotyledon extracts.