Identification and Biochemical Characterization of Ghrelin O-Acyltransferase (GOAT)

dc.contributor.advisorGoldstein, Joseph L.en
dc.creatorYang, Jingen
dc.date.accessioned2010-07-12T17:23:26Zen
dc.date.accessioned2014-02-19T22:01:21Z
dc.date.available2010-07-12T17:23:26Zen
dc.date.available2014-02-19T22:01:21Z
dc.date.issued2009-06-19en
dc.description.abstractGhrelin is a 28-amino acid, appetite-stimulating hormone secreted by the food-deprived stomach. Ser-3 of ghrelin is acylated with an eight-carbon fatty acid, octanoate, which is critically required for its endocrine actions. However, the octanoylating enzyme had remained elusive for nearly a decade. By expression cloning, I have identified GOAT (Ghrelin O-Acyltransferase), an enzyme belonging to a family of 16 polytopic membrane-bound O-acyltransferases. GOAT activity requires catalytic Asn and His residues, which are conserved through vertebrates. Consistent with its function, GOAT mRNA is largely restricted to stomach and intestine, the major ghrelin-secreting tissues. To further characterize GOAT function biochemically, I have developed a robust in vitro assay using membranes from insect cells infected with baculovirus encoding recombinant mouse GOAT. GOAT-containing membranes catalyze the transfer of [3H]octanoyl from [3H]octanoyl CoA to recombinant proghrelin in vitro. 50 microM palmitoyl CoA is necessary in the assays to prevent the deacylation of [3H]octanoyl CoA by crude membrane preparations. Maximal GOAT activity is observed at pH 7.0, and there is no apparent requirement for metals as determined by a lack of inhibition by 1 mM EDTA. The apparent Km for proghrelin is 6 microM and for [3H]octanoyl CoA is 0.6 microM. The octanoylation reaction strictly depends on the GOAT recognition site comprising three of the four N-terminal amino acids of proghrelin: Gly-1, Ser-3, and Phe-4. A pentapeptide containing only the N-terminal five amino acids of ghrelin is octanoylated by the enzyme. Moreover, I have demonstrated that the activity of GOAT is subjected to end-product inhibition. Together, the insights provided by my research may facilitate the design of useful inhibitors of GOAT.en
dc.format.digitalOriginborn digitalen
dc.format.mediumElectronicen
dc.format.mimetypeapplication/pdfen
dc.identifier.other754800058en
dc.identifier.urihttp://hdl.handle.net/2152.5/271en
dc.language.isoenen
dc.subjectAcyltransferasesen
dc.subjectGhrelinen
dc.subjectOctanoic Acidsen
dc.titleIdentification and Biochemical Characterization of Ghrelin O-Acyltransferase (GOAT)en
dc.type.genredissertationen
dc.type.materialTexten
thesis.date.available2011-06-19en
thesis.degree.departmenten
thesis.degree.disciplineCell Regulationen
thesis.degree.grantorGraduate School of Biomedical Sciencesen
thesis.degree.levelPh.Den
thesis.degree.nameDoctor of Philosophyen

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