Purification of an acyl-CoA hydrolase from rat intestinal microsomes. A candidate acyl-enzyme intermediate in glycerolipid acylation.
We have purified to apparent homogeneity an acyl-CoA hydrolase activity from rat intestinal villus cell microsomes by heparin and anion exchange and affinity chromatography. The purified 54-kDa acyl-CoA hydrolase along with several microsomal proteins form a covalent acyl-protein bond upon incubation with an activated fatty acid (acyl-CoA). The acyl moiety of the acylated acyl-CoA hydrolase is stable to denaturation and extraction with organic solvents, but is displaced by neutral hydroxylamine or mercaptoethanol, indicating a labile high energy (thio)ester linkage. The enzyme activity is inhibited by thiol-directed reagents and activated by the presence of dithiothreitol suggesting the presence of a cysteine residue(s) at or near the active site. Common serine-esterase inhibitors (NaF, phenylmethylsulfonyl fluoride) and activators (Mg2+, Ca2+) had no effect on the hydrolase activity. The enzyme hydrolyzed (transferred to water) 14-20 carbon acyl-CoA with similar efficiencies and did not utilize glycerophospholipids or mono- and diacylglycerols as potential acyl donors/acceptors. Phospholipids and mono- and diradylglycerols at concentrations below 100 microM or polyclonal antibodies raised against the purified hydrolase did not inhibit the enzyme activity. However, the acyl-CoA hydrolase activity could be immunoprecipitated from solubilized microsomes or purified enzyme preparations with corresponding decrease of the hydrolase activity in the supernatant of the immunoprecipitate. Immunoblotting studies show cross-reactivity with a protein of an identical molecular mass in other rat or human tissues. It is concluded that the microsomal acyl-CoA hydrolase deserves consideration as a candidate acyl-enzyme intermediate in glycerolipid synthesis when associated with appropriate acyltransferases.
Lehner
R
R
Banting and Best Department of Medical Research, C. H. Best Institute, University of Toronto, Ontario, Canada.
Kuksis
A
A
D000215:Acylation; D000818:Animals; D002846:Chromatography, Affinity; D002852:Chromatography, Ion Exchange; D004591:Electrophoresis, Polyacrylamide Gel; D007413:Intestinal Mucosa / Q000201:enzymology*; D007421:Intestine, Small / Q000201:enzymology; D050356:Lipid Metabolism; D008297:Male; D008861:Microsomes / Q000201:enzymology*; D010170:Palmitoyl-CoA Hydrolase / Q000302:isolation & purification* / Q000378:metabolism; D051381:Rats; D017208:Rats, Wistar
D010170:Palmitoyl-CoA Hydrolase