title: Lignoceroyl-CoA ligase activity in rat brain microsomal fraction: topographical localization and effect of detergents and alpha-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 Mg2+; 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: Rf 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 thin-layer chromatography. The optimal concentrations for CoASH, ATP, and Mg2+ were about 100 microM, 10 mM, and 5 mM, respectively. Lignoceric acid, solubilized by alpha-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 microM) 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.
authors.forename: I
authors.initials: I
authors.lastname: Singh
authors.forename: R
authors.initials: R
authors.lastname: Singh
authors.forename: A
authors.initials: A
authors.lastname: Bhushan
authors.forename: A K
authors.initials: AK
authors.lastname: Singh
mesh_terms: D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D001921:Brain / Q000201:enzymology*; D003065:Coenzyme A / Q000378:metabolism; D003066:Coenzyme A Ligases / Q000378:metabolism*; D003505:Cyclodextrins / Q000494:pharmacology; D003902:Detergents / Q000494:pharmacology; D003911:Dextrans; D005260:Female; D008274:Magnesium / Q000378:metabolism; D008297:Male; D008628:Mercury; D008861:Microsomes / Q000201:enzymology*; D017830:Octoxynol; D009941:Organomercury Compounds; D011092:Polyethylene Glycols; D051381:Rats; D011919:Rats, Inbred Strains; D013834:Thiamine Pyrophosphatase / Q000378:metabolism; D014357:Trypsin; D047391:alpha-Cyclodextrins
chemical_list: D003505:Cyclodextrins; D003902:Detergents; D003911:Dextrans; D009941:Organomercury Compounds; D047391:alpha-Cyclodextrins; C043248:mercury-dextran; D011092:Polyethylene Glycols; D000255:Adenosine Triphosphate; D017830:Octoxynol; D014357:Trypsin; D013834:Thiamine Pyrophosphatase; D003066:Coenzyme A Ligases; C044125:lignoceroyl-CoA ligase; D008628:Mercury; D008274:Magnesium; D003065:Coenzyme A; C016811:tyloxapol; C032613:alpha-cyclodextrin