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

Darlene E. Berryman, Julio J. Mulero, L. Barry Hughes, D. L. Brasaemle, André Bensadoun

Research output: Contribution to journalArticle

10 Citations (Scopus)

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 either the myc or flag epitope and transfected them into 293 cells. The addition of the tag was shown not to alter enzyme secretion rate or specific activity of the lipase. Partially purified lipase From media of cotransfected cells was used to establish a dimer assay which employed a sandwich ELISA. This assay firmly established the presence of a rHL species which contained both the myc and flag tags, supporting an oligomeric subunit structure for rHL. Furthermore, the data using the epitope-tagged enzyme shows that this method could be a useful tool not only in identifying the region of the lipase responsible for dimer formation but also to study other protein-protein interactions.

Original languageEnglish (US)
Pages (from-to)217-229
Number of pages13
JournalBiochimica et Biophysica Acta - Protein Structure and Molecular Enzymology
Volume1382
Issue number2
DOIs
StatePublished - Feb 17 1998

Fingerprint

Lipase
Epitopes
Guanidine
Dimers
Liver
Enzymes
Ultracentrifugation
Assays
Proteins
Gels
Molecular weight
Gel Chromatography
Molecular Weight
Denaturation
Lipoprotein Lipase
Enzyme activity
Chromatography
Catalyst activity
Salts
Monomers

All Science Journal Classification (ASJC) codes

  • Biophysics
  • Structural Biology
  • Biochemistry
  • Molecular Biology

Keywords

  • Dimerization
  • Epitope tag
  • Hepatic lipase

Cite this

Berryman, Darlene E. ; Mulero, Julio J. ; Hughes, L. Barry ; Brasaemle, D. L. ; Bensadoun, André. / Oligomeric structure of hepatic lipase : Evidence from a novel epitope tag technique. In: Biochimica et Biophysica Acta - Protein Structure and Molecular Enzymology. 1998 ; Vol. 1382, No. 2. pp. 217-229.
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Oligomeric structure of hepatic lipase : Evidence from a novel epitope tag technique. / Berryman, Darlene E.; Mulero, Julio J.; Hughes, L. Barry; Brasaemle, D. L.; Bensadoun, André.

In: Biochimica et Biophysica Acta - Protein Structure and Molecular Enzymology, Vol. 1382, No. 2, 17.02.1998, p. 217-229.

Research output: Contribution to journalArticle

TY - JOUR

T1 - Oligomeric structure of hepatic lipase

T2 - Evidence from a novel epitope tag technique

AU - Berryman, Darlene E.

AU - Mulero, Julio J.

AU - Hughes, L. Barry

AU - Brasaemle, D. L.

AU - Bensadoun, André

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AB - 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 either the myc or flag epitope and transfected them into 293 cells. The addition of the tag was shown not to alter enzyme secretion rate or specific activity of the lipase. Partially purified lipase From media of cotransfected cells was used to establish a dimer assay which employed a sandwich ELISA. This assay firmly established the presence of a rHL species which contained both the myc and flag tags, supporting an oligomeric subunit structure for rHL. Furthermore, the data using the epitope-tagged enzyme shows that this method could be a useful tool not only in identifying the region of the lipase responsible for dimer formation but also to study other protein-protein interactions.

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