Fucosterol epoxide lyase of insects: Synthesis of labeled substrates and development of a partition assay

Abstract

Fucosterol epoxide labeled with tritium in the C-29 methyl has been synthesized and employed in the development of a partition assay which allows the rapid determination of fucosterol epoxide lyase activity in vitro in homogenates of insect tissues. An independent synthesis of [24- 14C]fucosterol epoxide provided a control substrate to evaluate nondealkylative transfer of labeled steroid to the aqueous layer during the enzyme assay. The diastereomeric 24 R,28 R- and 24 S,28 S-[29- 3H]fucosterol epoxides were obtained via HPLC separation of their benzoate esters. Homogenates of the midgut tissue of larval tobacco hornworms ( Manduca sexta) were examined at pH 5 to 9 in several buffer systems, and at temperatures of 7 to 67°C in phosphate buffer. Optimal activity was found using pH 7.4, 76 m m phosphate buffer at 37°C. The 24 R,28 R diastereomer of fucosterol epoxide was metabolized at a rate at least 100 times that of the 24 S,28 S isomer by this enzyme system.