Identification and Biochemical Characterization of Ghrelin O-Acyltransferase (GOAT)
| dc.contributor.advisor | Goldstein, Joseph L. | en |
| dc.creator | Yang, Jing | en |
| dc.date.accessioned | 2010-07-12T17:23:26Z | |
| dc.date.available | 2010-07-12T17:23:26Z | |
| dc.date.issued | 2009-06-19 | |
| dc.description.abstract | Ghrelin 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.digitalOrigin | born digital | en |
| dc.format.medium | Electronic | en |
| dc.format.mimetype | application/pdf | en |
| dc.identifier.oclc | 754800058 | |
| dc.identifier.uri | https://hdl.handle.net/2152.5/271 | |
| dc.language.iso | en | en |
| dc.subject | Acyltransferases | en |
| dc.subject | Ghrelin | en |
| dc.subject | Octanoic Acids | en |
| dc.title | Identification and Biochemical Characterization of Ghrelin O-Acyltransferase (GOAT) | en |
| dc.type | Thesis | en |
| dc.type.genre | dissertation | en |
| dc.type.material | Text | en |
| thesis.date.available | 2009-06-19 | |
| thesis.degree.department | Graduate School of Biomedical Sciences | en |
| thesis.degree.discipline | Cell Regulation | en |
| thesis.degree.grantor | UT Southwestern Medical Center | en |
| thesis.degree.level | Doctoral | en |
| thesis.degree.name | Doctor of Philosophy | en |