A direct in vitro fatty acylation assay for hedgehog acyltransferase Journal Article
| Authors: | Schonbrun, A. R.; Resh, M. D. |
| Article Title: | A direct in vitro fatty acylation assay for hedgehog acyltransferase |
| Abstract: | Several assays have been developed to monitor the in vitro catalytic activity of Hedgehog acyltransferase (Hhat), an enzyme critical to the Hedgehog signaling pathway in cells. However, the majority of these previously reported assays involve radioactive fatty acyl donor substrates, multiple steps to achieve product readout, or specialized equipment. To increase safety, efficiency, and convenience, we developed a direct, fluorescent in vitro assay to monitor Hhat activity. Our assay utilizes purified Hhat, a fluorescently labeled fatty acyl-CoA donor substrate, and a Sonic hedgehog (Shh) peptide recipient substrate sufficient for fatty acylation. The protocol is a straightforward process that yields direct readout of fatty acylated Shh peptide via fluorescence detection of the transferred fatty acyl group. Copyright: © 2022 The Authors. |
| Keywords: | controlled study; unclassified drug; fluorescence; sonic hedgehog protein; in vitro study; enzyme activity; enzyme substrate; assay; enzyme purification; acyltransferase; acylation; hedgehog acyltransferase; acyl coenzyme a; fatty acyl-coa; article; hedgehog signaling; sonic hedgehog (shh); hedgehog (hh); hedgehog acyltransferase (hhat); nitrobenzoxadiazole (nbd) |
| Journal Title: | Bio-protocol |
| Volume: | 12 |
| Issue: | 24 |
| ISSN: | 2331-8325 |
| Publisher: | Bio-protocol |
| Date Published: | 2022-12-20 |
| Start Page: | e4573 |
| Language: | English |
| DOI: | 10.21769/BioProtoc.4573 |
| PROVIDER: | scopus |
| PMCID: | PMC9797357 |
| PUBMED: | 36618094 |
| DOI/URL: | |
| Notes: | Article -- Export Date: 1 May 2023 -- Source: Scopus |
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