Abstract
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.
Highlights
We found that hormone-sensitive lipase (HSL) catalyzed the hydrolysis of water-soluble esters, which are useful in studies on the molecular dynamics of lipase, and we examined the relation between the lipase and esterase activities of purified HSL using triolein, cholesteryl oleate, and water-soluble p-nitrophenyl butyrate (PNPB) as substrates
Bovine serum albumin was obtained from Wako Pure Chemical Industries (Osaka, Japan) and Abbreviations: HSL, hormone-sensitive lipase; PNPB, p-nitrophenyl butyrate; CO, cholesteryl oleate; DFP, diisopropylfluorophosphate; FPLC, fast-protein liquid chromatography
The CO-hydrolyzing activity of adipose tissue homogenate was precipitated completely at pH 5.0. This CO-hydrolyzing activity of HSL was adsorbed to a column of phenyl-Sepharose, whereas 90% of the PNPB-hydrolyzing activity was not
Summary
One polypeptide of M , 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-hydrolyzingactivities were lost more rapidly than the PNPB-hydrolyzing activity. HSL is the key enzyme regulating fatty acid mobilization from fat cells (l), but its mechanism of action is not well understood. HSL shows less substrate specificity than pancreatic lipase, readily acting on triacylglycerols, diacylglycerols, monoacylglycerols, and cholesteryl esters [1, 5]. We found that HSL catalyzed the hydrolysis of water-soluble esters, which are useful in studies on the molecular dynamics of lipase, and we examined the relation between the lipase and esterase activities of purified HSL using triolein, cholesteryl oleate, and water-soluble PNPB as substrates
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