p-nitrophenyl butyrate hydrolyzing activity of hormone-sensitive lipase from bovine adipose tissue.
The "esterase" activity of hormone-sensitive lipase (HSL) was studied using water-soluble p-nitrophenyl butyrate (PNPB) as a substrate. Bovine adipose tissue HSL was purified to near homogeneity by precipitation at pH 5.0, followed by chromatography on DEAE-cellulose, phenyl-Sepharose, and high performance ion-exchange columns on Mono Q and Mono S. The purified preparation hydrolyzed emulsified triolein and cholesteryl oleate (CO), and water-soluble PNPB. In the two last steps of purification, the elution profile of the CO-hydrolyzing activity coincided with that of PNPB-hydrolyzing activity. The HSL was adsorbed to heparin-Sepharose and the CO- and PNPB-hydrolyzing activities were eluted together in the same peak. Diisopropylfluorophosphate (DFP) strongly inhibited the HSL activities and the inhibition profiles of the triolein-; CO-, and PNPB-hydrolyzing activities were essentially identical. Only one polypeptide of Mr 84,000 in partial purified HSL fraction was labeled by affinity labeling with [3H]DFP. On digestion of the enzyme with trypsin, the triolein- and CO-hydrolyzing activities were lost more rapidly than the PNPB-hydrolyzing activity. Phosphorylation increased the triolein-hydrolyzing activity to 40% more than that of the control, but did not affect the CO- and PNPB-hydrolyzing activities.
Tsujita
T
T
Department of Medical Biochemistry, School of Medicine, Ehime University, Japan.
Ninomiya
H
H
Okuda
H
H
D000273:Adipose Tissue / Q000201:enzymology*; D000818:Animals; D002087:Butyrates / Q000378:metabolism*; D002265:Carboxylic Ester Hydrolases / Q000378:metabolism*; D002417:Cattle; D002788:Cholesterol Esters / Q000378:metabolism; D002847:Chromatography, Agarose; D004591:Electrophoresis, Polyacrylamide Gel; D006868:Hydrolysis; D002787:Sterol Esterase / Q000037:antagonists & inhibitors / Q000302:isolation & purification / Q000378:metabolism*; D013379:Substrate Specificity; D014304:Triolein / Q000378:metabolism; D014357:Trypsin
D002087:Butyrates; D002788:Cholesterol Esters; D014304:Triolein; C033592:4-nitrophenyl butyrate; C014409:cholesteryl oleate; D002265:Carboxylic Ester Hydrolases; D002787:Sterol Esterase; D014357:Trypsin