Inhibition of hepatic lipase by m-aminophenylboronate. Application of phenylboronate affinity chromatography for purification of human postheparin plasma lipases.
Phenylboronates are competitive inhibitors of serine. hydrolases including lipases. We studied the effect of m-aminophenylboronate on triglyceride-hydrolyzing activity of hepatic lipase (EC 3.1,1.3). m-Aminophenylboronate inhibited hepatic lipase activity with a Ki value of 55 microM. Furthermore, m-aminophenylboronate protected hepatic lipase activity from inhibition by di-isopropyl fluorophosphate, an irreversible active site inhibitor of serine hydrolases. Inhibition of hepatic lipase activity by m-aminophenylboronate was pH-dependent. The inhibition was maximal at pH 7.5, while at pH 10 it was almost non-existent. These data were used to develop a purification procedure for postheparin plasma hepatic lipase and lipoprotein lipase. The method is a combination of m-aminophenylboronate and heparin-Sepharose affinity chromatographies. Hepatic lipase was purified to homogeneity as analyzed on sodium dodecyl sulfate polyacrylamide gel electrophoresis. The specific activity of purified hepatic lipase was 5.46 mmol free fatty acids h-1 mg-1 protein with a total purification factor of 14,400 and a final recovery of approximately 20%. The recovery of hepatic lipase activity in m-aminophenylboronate affinity chromatography step was 95%. The purified lipoprotein lipase was a homogeneous protein with a specific activity of 8.27 mmol free fatty acids h-1 mg-1. The purification factor was 23,400 and the final recovery approximately 20%. The recovery of lipoprotein lipase activity in the m-aminophenylboronate affinity chromatography step was 87%. The phenylboronate affinity chromatography step can be used for purification of serine hydrolases which interact with boronates.
Uusi-Oukari
M
M
Department of Biochemistry, National Public Health Institute, Helsinki, Finland.
Ehnholm
C
C
Jauhiainen
M
M
D001897:Boronic Acids / Q000737:chemistry / Q000494:pharmacology*; D002846:Chromatography, Affinity / Q000379:methods*; D004591:Electrophoresis, Polyacrylamide Gel; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D006801:Humans; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D007531:Isoflurophate / Q000494:pharmacology; D008049:Lipase / Q000037:antagonists & inhibitors* / Q000097:blood; D008071:Lipoprotein Lipase / Q000302:isolation & purification*; D008099:Liver / Q000201:enzymology*; D014280:Triglycerides / Q000378:metabolism
D001897:Boronic Acids; D004791:Enzyme Inhibitors; D014280:Triglycerides; D007531:Isoflurophate; D008049:Lipase; D008071:Lipoprotein Lipase; C010686:benzeneboronic acid