Isolation and characterization of a fatty acyl esterase from rat lung.
In an effort to facilitate studies of the reaction involved in the removal of fatty acids from acyl proteins, we have synthesized an octanoic acid ester of doubly blocked serine, specifically octanoyl N-carbobenzoxy-L-serine-benzyl ester (octanoyl boc-serine), and used it as a substrate to guide the purification of an esterase from rat lung. The esterase was purified 228-fold by column chromatography on DE-52 cellulose, hydroxylapatite, octyl-Sepharose, and concanavalin A-Sepharose and by HPLC gel filtration. The final enzyme preparation ran as a single 77,000-Da band when subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis and exhibited a single symmetrical peak (sedimentation coefficient, 4.5 S) when centrifuged through a sucrose density gradient (empirical Mr, 63,000). The esterase is an acidic protein, pI 4.1, and is very active against p-nitrophenyl esters comprised of C4-C14 fatty acids; the highest specific activity (26.5 mumol/min/mg) was obtained using p-nitrophenyl caprylate as substrate. The pH optimum of the lung esterase is near 8.0 and the activity on octanoyl boc-serine is maximum when 0.3% (w/v) Myrj-52 is included in the assay medium. The activity of the esterase is not dependent on calcium ions. The enzyme does not remove acyl groups from the G-protein of vesicular stomatitis virus or the proteolipid of bovine brain. The possible role of the esterase in the metabolism of acylated proteins is considered.
Basu
A
A
Department of Microbiology, Biochemistry, and Molecular Biology, School of Medicine, University of Pittsburgh, Pennsylvania 15261.
Glew
R H
RH
Evans
R W
RW
Bandik
G
G
D000818:Animals; D002851:Chromatography, High Pressure Liquid; D004591:Electrophoresis, Polyacrylamide Gel; D004950:Esterases / Q000302:isolation & purification*; D005227:Fatty Acids / Q000378:metabolism*; D006358:Hot Temperature; D006801:Humans; D007231:Infant, Newborn; D007526:Isoelectric Point; D008168:Lung / Q000201:enzymology*; D051381:Rats; D013379:Substrate Specificity
D005227:Fatty Acids; D004950:Esterases