PANCREATIC CHOLESTEROL ESTERASES .2. PURIFICATION AND CHARACTERIZATION OF HUMAN PANCREATIC FATTY-ACID ETHYL-ESTER SYNTHASE

被引:66
作者
RILEY, DJS [1 ]
KYGER, EM [1 ]
SPILBURG, CA [1 ]
LANGE, LG [1 ]
机构
[1] WASHINGTON UNIV,JEWISH HOSP ST LOUIS,MED CTR,DIV CARDIOL,YALEM 305,216 S KINGSHIGHWAY,ST LOUIS,MO 63110
关键词
D O I
10.1021/bi00468a007
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
Human pancreatic fatty acid ethyl ester synthase has been isolated and purified 1200-fold to homogeneity, and its activities, binding properties, and N-terminal amino acid sequence indicate that it is a member of the lipase family. This 52-kDa monomeric protein is present at 0.6–1.2 mg/g of pancreas, and it catalyzes the synthesis and hydrolysis of ethyl oleate at rates of 2400 nmo1 mg−1 h−1 and 30 nmo1 mg−1 h−1, respectively. Kinetic analyses reveal a pronounced substrate specificity for unsaturated octadecanoic fatty acids, with ethyl ester synthetic rates of 2400 nmol mg−1 h−1 (linoleic), 2400 nmol mg−1 h−1 (oleic), 400 nmol mg−1 h−1 (arachidonic), 300 nmol mg−1 h−1 (palmitic), and 100 nmol mg−1 h−1 (stearic). Like cholesterol esterase, the enzyme binds to immobilized heparin, and this property was critical for its purification to homogeneity. Its N-terminal amino acid sequence is virtually identical with that reported for human triglyceride lipase, NH2-X-Glu-Val-Cys-5Tyr-Glu-Arg-Leu-Gly-10Cys-Phe-Ser-Asp-Asp-15Ser-Pro-Trp-Ser-Gly-20Ile, and it differs by only four residues from that reported for porcine pancreatic lipase. The synthase purified here also cleaves triglycerides, hydrolyzing triolein at a rate of 30 nmo1 mg−1 h−1, and this activity is stimulated by colipase and inhibited by sodium chloride. Conversely, commercially available porcine triglyceride lipase exhibits fatty acid ethyl ester synthase activity (1530 nmo1 mg−1 h−1) and hydrolyzes triolein at a rate of 23 nmol mg−1 h−1. Thus, the predominant source of pancreatic fatty acid ethyl ester synthase activity is the exocrine product triglyceride lipase. These data indicate that triglyceride lipase and the cholesterol esterases belong to a family of enzymes whose members recognize both a fatty acid and an alcohol and bind specifically to heparin. © 1990, American Chemical Society. All rights reserved.
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页码:3848 / 3852
页数:5
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