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 includ ing lipases. We studied the effect of m-aminophenylboronate on triglyc eride-hydrolyzing activity of hepatic lipase (EC 3.1.1.3). m-Aminophen ylboronate inhibited hepatic lipase activity with a K-i value of 55 mu M. Furthermore, m-aminophenylboronate protected hepatic lipase activi ty from inhibition by di-isopropyl fluorophosphate, an irreversible ac tive site inhibitor of serine hydrolases. Inhibition of hepatic lipase activity by m-aminophenylboronate was pH-dependent. The inhibition wa s 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 pla sma hepatic lipase and lipoprotein lipase, The method is a combination of m-aminophenylboronate and heparin-Sepharose affinity chromatograph ies. Hepatic lipase was purified to homogeneity as analyzed on sodium dodecyl sulfate polyacrylamide gel electrophoresis. The specific activ ity 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 re covery of approximately 20%. The recovery of hepatic lipase activity i n m-aminophenylboronate affinity chromatography step was 95%. The puri fied lipoprotein lipase was a homogeneous protein with a specific acti vity of 8.27 mmol free fatty acids h(-1) mg(-1). The purification fact or was 23 400 and the final recovery approximately 20%, The recovery o f lipoprotein lipase activity in the m-aminophenylboronate affinity ch romatography step was 87%. The phenylboronate affinity chromatography step can be used for purification of serine hydrolases which interact with boronates.