Purification and characterization of a fatty acyl-CoA hydrolase from the uropygial glands of Peking ducks (Anas domesticus).
In a previous communication the occurrence of a medium-chain acyl-CoA hydrolase designated thioesterase-B in the uropygial gland of mallard ducks was reported [L. Rogers, P. E. Kolattukudy, and M. J. de Renobales (1982) J. Biol. Chem. 257, 880-886]. In the present study, thioesterase-B was purified from the postmicrosomal supernatant of homogenized uropygial glands from Peking ducks (Anas domesticus). Most of the contaminating thioesterase activities were removed by ammonium sulfate fractionation. The 55% ammonium sulfate supernatant, containing thioesterase-B, was chromatographed on hydroxylapatite followed by gel filtration on Sephadex G-100. The remaining contaminants were removed by chromatofocusing followed by desalting on Sephadex G-75. This procedure gave a 26% yield with a nearly 200-fold purification. Gel filtration of the purified enzyme showed that the molecular weight of the native enzyme was 56,300, whereas sodium dodecyl sulfate-gel electrophoresis of components separated by chromatofocusing showed that the purified enzyme contained enzymatically active proteins of molecular weights 59,400, 58,300, 56,000, and 55,800. The four species differed slightly in pI (4.9, 4.7, 4.45, and 4.40) but they were kinetically and immunologically indistinguishable. All four had the same N-terminal sequence. The purified thioesterase preparation showed a pH optimum of 9.3 with C12-CoA but the pH optimum was dependent on the chain length of the acyl group. At pH 8.0, C10 was the preferred substrate with less activity on C12, C8, and C14. The enzymatic activity was stimulated by bovine serum albumin and was inhibited by p-hydroxymercuribenzoate. Involvement of active serine in catalysis was suggested by inhibition of the enzyme by diethylpyrocarbonate, diisopropylfluorophosphate and phenylmethylsulfonyl fluoride.
D000596:Amino Acids / Q000032:analysis; D000818:Animals; D055598:Chemical Phenomena; D002621:Chemistry; D002845:Chromatography / Q000379:methods; D004372:Ducks / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D005088:Exocrine Glands / Q000201:enzymology*; D006120:Grooming; D006863:Hydrogen-Ion Concentration; D013379:Substrate Specificity; D013869:Thiolester Hydrolases / Q000302:isolation & purification* / Q000378:metabolism
D000596:Amino Acids; D013869:Thiolester Hydrolases; C044320:thioesterase B