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

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Title: Identification and Biochemical Characterization of Ghrelin O-Acyltransferase (GOAT)
Author: Yang, Jing
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 .
URI: http : / /hdl .handle .net /2152 .5 /271
Date: 2009-06-19

Citation

Identification and Biochemical Characterization of Ghrelin O-Acyltransferase (GOAT). Graduate School of Biomedical Sciences. Available electronically from http : / /hdl .handle .net /2152 .5 /271 .

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