PMID: 9540793Apr 16, 1998Paper

Oligomeric structure of hepatic lipase: evidence from a novel epitope tag technique

Biochimica Et Biophysica Acta
D E BerrymanA Bensadoun

Abstract

The subunit structure of purified rHL (rHL) was determined by gel filtration chromatography, density gradient ultracentrifugation studies and a novel approach using epitope-tagged rHL. By gel filtration studies, native rHL had an apparent molecular weight of 179 kDa whereas enzyme treated with 6 M guanidine hydrochloride (GuHCl) for 22 h at room temperature gave a protein peak at 76 kDa. Using milder conditions for denaturation of rHL, such as 1 M GuHCl for 2 h, rHL eluted in two distinct peaks, one at 179 kDa and the other at 76 kDa. In addition, both protein peaks produced under mild denaturing conditions possessed detectable catalytic activity. Consistent with studies on lipoprotein lipase, the denatured rHL eluted from heparin-Sepharose at a lower salt concentration of 0.42 M NaCl than the native rHL which eluted at 0.72 M NaCl. By density gradient ultracentrifugation studies, the estimated molecular weight of native rHL was determined to be 113 kDa. Together, the data suggest that native rHL exists as a dimer that can be denatured into monomers by GuHCl and that a fraction of the denatured enzyme has detectable enzyme activity. To confirm these results, we designed two different rHL constructs that were epitope-tagged with...Continue Reading

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Citations

Jul 2, 2009·The Journal of Biological Chemistry·Nathalie GriffonDaniel J Rader
Feb 3, 1999·Annual Review of Genetics·J W Jarvik, C A Telmer
Sep 1, 2007·Arteriosclerosis, Thrombosis, and Vascular Biology·Laeticia LichtensteinSander Kersten
Apr 29, 2000·Current Opinion in Lipidology·D J Rader, M Jaye
Nov 8, 2005·Biochemistry. Biokhimii︠a︡·V G ArtyukhovO D Trofimova

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