Fucosterol epoxide lyase of insects: synthesis of labeled substrates and development of a partition assay.
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 24R,28R- and 24S,28S-[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 degrees C in phosphate buffer. Optimal activity was found using pH 7.4, 76 mM phosphate buffer at 37 degrees C. The 24R,28R diastereomer of fucosterol epoxide was metabolized at a rate at least 100 times that of the 24S,28S isomer by this enzyme system.
D000446:Aldehyde-Lyases / Q000378:metabolism*; D002250:Carbon Radioisotopes; D002851:Chromatography, High Pressure Liquid; D007700:Kinetics; D007915:Lepidoptera / Q000201:enzymology*; D009036:Moths / Q000201:enzymology*; D010840:Phytosterols / Q000138:chemical synthesis*; D011865:Radioisotope Dilution Technique; D013265:Stigmasterol / Q000031:analogs & derivatives / Q000138:chemical synthesis*; D013379:Substrate Specificity; D014316:Tritium
D002250:Carbon Radioisotopes; D010840:Phytosterols; D014316:Tritium; C024932:fucosterol epoxide; D013265:Stigmasterol; D000446:Aldehyde-Lyases; C047957:fucosterol epoxide lyase