Lignoceroyl-CoA ligase activity in rat brain microsomal fraction: Topographical localization and effect of detergents and α-cyclodextrin

Abstract

Lignoceroyl-CoA ligase activity has been detected in microsomal fractions prepared from rat brain. The synthesis of lignoceroyl-CoA from [1- 14C]lignoceric acid and CoASH by this enzyme had an absolute dependence on ATP and Mg 2+; ATP could not be replaced by GTP [ I. Singh, M. S. Kang, and L. Phillips (1982) Fed. Proc. 41, 1192]. The product has been characterized as lignoceroyl-CoA by the following criteria: R f on thin-layer chromatography; incorporation of [1- 14C]lignoceric acid and [ 3H]CoASH into the product; acid hydrolysis and identification of the radiolabel in lignoceric acid; and methanolysis and identification of the radiolabel in methyl lignocerate by thinlayer chromatography. The optimal concentrations for CoASH, ATP, and Mg 2+ were about 100 μ m, 10 m m, and 5 m m, respectively. Lignoceric acid, solubilized by α-cyclodextrin, Triton X-100, and deoxycholate, was utilized by the lignoceroyl-CoA ligase, but lignoceric acid solubilized by Triton WR-1339 was not. Topographical localization of lignoceroyl-CoA ligase in the plane of rat brain microsomal membranes was determined by the use of Triton X-100, trypsin, and mercury-Dextran, and was compared with the marker enzymes, ethanol acyltransferase and thiamine pyrophosphatase, which are known to be localized on the luminal (inner) surface of the microsomal vesicles. Mercury-Dextran (100 μ m) and trypsin (trypsin:microsomes, 1:56 w/w) treatment of the microsomes inhibited the lignoceroyl-CoA ligase activity by 70 and 90% without disrupting the microsomal vesicles. Disruption of the vesicles with Triton X-100 increased the activity of both ethanol acyltransferase and thiamine pyrophosphatase by 400% but there was no increase in lignoceroyl-CoA ligase activity. These results suggest that lignoceroyl-CoA ligase is localized on the cytoplasmic surface of the microsomal vesicles.