A high-performance liquid chromatography-based radiometric assay for acyl-CoA:alcohol transacylase from jojoba.
Acyl-CoA:alcohol transacylase catalyzes the final step in the biosynthesis of storage liquid wax esters from acyl-CoA fatty acids and fatty alcohols in a limited number of microbes, algae, and Simmondsia chinensis Link (jojoba). An improved and automated method of enzyme assay for this catalyst from cotyledons of jojoba is described. The assay method uses reversed-phase C18 high performance liquid chromatography (HPLC) to separate the labeled C30:1 liquid wax product, [14C]-dodecanyl-octadecenoate, from the unreacted substrate, [14C]octadecenoyl-CoA (oleyl-CoA), and other components produced from enzymes present in the crude homogenate of jojoba cotyledons, including [14C]-octadecenoic acid (oleic acid) and [14C]octadecenol (oleyol). Methods are also described for microscale chemical synthesis in one vessel of 14C-radiolabeled substrates and products for the transacylase. These labeled reagents are required to confirm the HPLC separations of reaction products. The radioactive components are quantitated using an on-line flow-through scintillation detector enabling sensitive and precise analysis of the reaction products.
Garver
W S
WS
Plant Genetic Engineering Laboratory, New Mexico State University, Department 3C, Las Cruces 88003-0001.
Kemp
J D
JD
Kuehn
G D
GD
D000214:Acyl Coenzyme A / Q000302:isolation & purification / Q000378:metabolism; D000217:Acyltransferases / Q000032:analysis* / Q000378:metabolism; D002250:Carbon Radioisotopes; D002851:Chromatography, High Pressure Liquid / Q000379:methods; D007553:Isotope Labeling / Q000379:methods; D007700:Kinetics; D009829:Oleic Acids / Q000302:isolation & purification*; D010944:Plants / Q000201:enzymology*; D011865:Radioisotope Dilution Technique; D013816:Thermodynamics
D000214:Acyl Coenzyme A; D002250:Carbon Radioisotopes; D009829:Oleic Acids; C017585:oleoyl-coenzyme A; C079166:dodecyl oleate; D000217:Acyltransferases; C079165:long-chain-alcohol O-fatty-acyltransferase