FIGURE 10.

Characterization of GOAT active site by photocross-linking. A, partial proteolysis of covalently photocross-linked mouse GOAT reveals a 15-kDa cross-linked fragment. GO-CoA-Tat F4BP (biotin-labeled) was photocross-linked to microsomal mouse GOAT-3xFLAG or to control microsomes (Ctrl; from empty-virus infected cells). Microsomes were then solubilized, digested with trypsin, and isolated using anti-FLAG beads (see “Experimental Procedures”). Samples were then immunoblotted using streptavidin or anti-FLAG. Note that although the biotin cannot be removed from GO-CoA-Tat F4BP during trypsin digest, the 3xFLAG tag contains multiple trypsin cleavage sites, and smaller tag-cleaved species may have been lost or may have fallen below the limit of detection. B, synthetic scheme to make ghrelin-28-Oct-diazirine, an acyl ghrelin product analog photocross-linker. As a complement to GO-CoA-Tat F4BP previously reported (8), we designed another set of photoaffinity labeling agents based on the fact that the amide analogs of the acyl ghrelin product have high affinity for GOAT (32). Photolabile diazirine was installed at the 7-position of the octanoic acid side chain, and biotinylated lysine was installed at the C terminus of ghrelin-28. Ghrelin-28-Oct-F4BP was made similarly, using unlabeled octanoic anhydride and replacing Phe-4 with benzoylphenylalanine. C–E, photocross-linking cross-competition binding assay. Each of three biotinylated photocross-linking compounds (GO-CoA-Tat F4BP (C), ghrelin-28-Oct-diazirine (D), and ghrelin-28-Oct-F4BP (E)) was cross-linked to purified, solubilized mouse GOAT in the presence of an excess of the indicated unlabeled competitors. Separate subpanels represent separate experiments. Coomassie Brilliant Blue (CBB) staining is used as a loading control.