Purification and characterization of a porcine liver microsomal triacylglycerol hydrolase.
We have purified an enzyme from porcine liver microsomes which catalyzes hydrolysis of triacylglycerols. The enzyme was solubilized from the membranes by the zwitterionic detergent 3-[(3- cholamidopropyl)dimethylammonio]-l-propansulfonate (CHAPS) and was purified to apparent homogeneity by sequential chromatography on Q-Sepharose, hydroxyapatite, Affi-Gel heparin, and Mono-Q. The purified hydrolase migrated in SDS-polyacrylamide gel electrophoresis (PAGE) as a single polypeptide band of an apparent molecular mass of 60 kDa. The enzyme hydrolyzed long-, medium-, and short-chain triacylglycerols, as well as a chromogenic lipase substrate, 1,2-O-dilauryl-rac-glycero-3-glutaric acid resorufin ester. The highest specific activity was obtained with tributyroylglycerol (240 mumol.min-1.mg-1). The reaction rate was maximal at pH 8.5. Sulfhydryl-directed reagents, such as N-ethylmaleimide (NEM), 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB), and dodecyldithio-5-(2-nitrobenzoic acid) (C12-TNB) had no effect on the hydrolase activity; however, the enzyme was sensitive to HgCl2. Serine reagents, such as diethyl-p-nitrophenyl phosphate (E600) and diisopropyl fluorophosphate (DFP), used in 100-fold molar excess completely inhibited the activity, suggesting that it is a serine esterase. These results suggest that the enzyme may participate in the intracellular neutral lipid metabolism since the enzyme is located in the endoplasmic reticulum, an organelle where de novo triacylglycerol synthesis and assembly of lipoproteins take place.
Lehner
R
R
Laboratoire de Lipolyse Enzymatique, UPR 9025, CNRS, Marseille, France. rlehner@gpu.srv.ualberta.ca
Verger
R
R
D000595:Amino Acid Sequence; D000818:Animals; D006868:Hydrolysis; D008049:Lipase / Q000737:chemistry* / Q000276:immunology / Q000302:isolation & purification*; D008074:Lipoproteins / Q000378:metabolism; D008862:Microsomes, Liver / Q000201:enzymology* / Q000378:metabolism; D011233:Precipitin Tests; D011817:Rabbits; D017421:Sequence Analysis; D013379:Substrate Specificity; D013552:Swine; D014280:Triglycerides / Q000378:metabolism
D008074:Lipoproteins; D014280:Triglycerides; D008049:Lipase