Comparative characterization of a wild type and transmembrane domain- deleted fatty acid amide hydrolase

Identification of the transmembrane domain as a site for oligomerization

Matthew P. Patricelli, Hilal A. Lashuel, Dan K. Giang, Jeffery W. Kelly, Benjamin F. Cravatt

Research output: Contribution to journalArticle

124 Citations (Scopus)

Abstract

Fatty acid amide hydrolase (FAAH) is an integral membrane protein responsible for the hydrolysis of a number of primary and secondary fatty acid amides, including the neuromodulatory compounds anandamide and oleamide. Analysis of FAAH's primary sequence reveals the presence of a single predicted transmembrane domain at the extreme N-terminus of the enzyme. A mutant form of the rat FAAH protein lacking this N-terminal transmembrane domain (ΔTM-FAAH) was generated and, like wild type FAAH (WT-FAAH), was found to be tightly associated with membranes when expressed in COS-7 cells. Recombinant forms of WT- and ATM-FAAH expressed and purified from Escherichia coli exhibited essentially identical enzymatic properties which were also similar to those of the native enzyme from rat liver. Analysis of the oligomerization states of WT- and ΔTM-FAAH by chemical cross-linking, sedimentation velocity analytical ultracentrifugation, and size exclusion chromatography indicated that both enzymes were oligomeric when membrane- bound and after solubilization. However, WT-FAAH consistently behaved as a larger oligomer than ΔTM-FAAH. Additionally, SDS-PAGE analysis of the recombinant proteins identified the presence of SDS-resistant oligomers for WT-FAAH, but not for ΔTM-FAAH. Self-association through FAAH's transmembrane domain was further demonstrated by a FAAH transmembrane domain - GST fusion protein which formed SDS-resistant dimers and large oligomeric assemblies in solution.

Original languageEnglish
Pages (from-to)15177-15187
Number of pages11
JournalBiochemistry
Volume37
Issue number43
DOIs
Publication statusPublished - 27 Oct 1998
Externally publishedYes

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Oligomerization
Oligomers
Enzymes
Membranes
fatty-acid amide hydrolase
Size exclusion chromatography
COS Cells
Ultracentrifugation
Automatic teller machines
Sedimentation
Recombinant Proteins
Amides
Dimers
Liver
Escherichia coli
Gel Chromatography
Rats
Polyacrylamide Gel Electrophoresis
Hydrolysis
Membrane Proteins

ASJC Scopus subject areas

  • Biochemistry

Cite this

Comparative characterization of a wild type and transmembrane domain- deleted fatty acid amide hydrolase : Identification of the transmembrane domain as a site for oligomerization. / Patricelli, Matthew P.; Lashuel, Hilal A.; Giang, Dan K.; Kelly, Jeffery W.; Cravatt, Benjamin F.

In: Biochemistry, Vol. 37, No. 43, 27.10.1998, p. 15177-15187.

Research output: Contribution to journalArticle

Patricelli, Matthew P. ; Lashuel, Hilal A. ; Giang, Dan K. ; Kelly, Jeffery W. ; Cravatt, Benjamin F. / Comparative characterization of a wild type and transmembrane domain- deleted fatty acid amide hydrolase : Identification of the transmembrane domain as a site for oligomerization. In: Biochemistry. 1998 ; Vol. 37, No. 43. pp. 15177-15187.
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