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

Soluble enzyme preparations from pea shoot tips incorporated mevalonic acid-2-¹⁴C into ent-kaurene-¹⁴C, squalene-¹⁴C 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 Mn²⁺ or Mg²⁺, with Mn²⁺ being a more effective activator than Mg²⁺ 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.