Fatty Acyl-CoA elongation in Blatella germanica integumental microsomes

Insect cuticular hydrocarbons are synthesized de novo in integumental tissue through the concerted action of fatty acid synthases (FASs), fatty acyl-CoA elongases, a reductose, and a decarboxylase to produce hydrocarbons and CO2. Elongation of fatty acyl-CoAs to very long chain fatty acids was studied in the integumental microsomes of the German cockroach, Blatella germanica. Incubation of [1-C-14] palmitoyl-CoA, malonyl-CoA, and NADPH resulted in the production of 18-CoA with minor amounts of C20, C22, C24, GO, and C32 labeled acyl-CoA moieties. Similar experiments with [1-C-14] stearoyl-CoA rendered C20-CoA as the major product, and lesser amounts of C22 and C24-CoAs were also detected. After solubilization of the microsomal FAS, kinetic parameters were determined radiochemically or by measuring NADPH consumption. The reaction velocity was linear for up to 3 min incubation time, and with a protein concentration up to 0.025 mug/mud The effect of the chain length on the reaction velocity was compared for paimiioyl-CoA, stearoyl-CoA, and eicosanoyl-CoA. The optimal substrate concentration was 10 muM for C16-CEA, between 8 and 12 muM for C18-CoA, and close to 3 muM for C20-CoA. In vivo hydrocarbon biosynthesis was inhibited from 55.5 to 72.5% in the presence of 1 mM trichloroacetic acid, a known inhibitor of elongation reactions. Arch. Insect Biochem. (C) 2004 Wiley-Liss, Inc.