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. 2010 Dec 10;49(50):9630-4.
doi: 10.1002/anie.201003387.

cat-ELCCA: a robust method to monitor the fatty acid acyltransferase activity of ghrelin O-acyltransferase (GOAT)

Amanda L Garner  1 , Kim D Janda
Affiliations

cat-ELCCA: a robust method to monitor the fatty acid acyltransferase activity of ghrelin O-acyltransferase (GOAT)

Amanda L Garner et al. Angew Chem Int Ed Engl. .

Abstract

Assays armed with catalytic signal amplification have arisen as superior systems for ultrasensitive detection of analytes. Here we describe a conceptually new enzyme assay based on cat-ELISA, catalytic assay using enzyme-linked click chemistry assay (cat-ELCCA), where an enzyme-linked azide is utilized to arm the assay with catalytic fluorescence signal amplification. Using this assay technology, we have developed the first potentially high-throughput screen for the recently disclosed acyltransferase, ghrelin O-acyltransferase (GOAT).

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Figures

Figure 1
Figure 1
Comparison of A) cat-ELISA and B) catalytic assay using enzyme-linked click chemistry assay (cat-ELCCA). E = enzyme. S = substrate.
Figure 2
Figure 2
GOAT-catalyzed n-octyanoyl transfer to ghrelin.
Figure 3
Figure 3
cat-ELCCA for screening GOAT activity. A) Assay design. B) Proof-of-concept data. ++ = Sample treated with both GOAT-containing membranes and n-octynoyl-CoA. RFU = normalized relative fluorescence units at 585 nm.
Figure 3
Figure 3
cat-ELCCA for screening GOAT activity. A) Assay design. B) Proof-of-concept data. ++ = Sample treated with both GOAT-containing membranes and n-octynoyl-CoA. RFU = normalized relative fluorescence units at 585 nm.
Figure 4
Figure 4
Characterization of GOAT acyltransferase activity using cat-ELCCA. A) Time dependence. B) Dependence on the amount of GOAT membranes. C) Dependence on n-octynoyl-CoA concentration. D) Dependence on immobilized ghrelin(1–5)pentapeptide concentration. RFU = normalized relative fluorescence units at 585 nm.
Figure 4
Figure 4
Characterization of GOAT acyltransferase activity using cat-ELCCA. A) Time dependence. B) Dependence on the amount of GOAT membranes. C) Dependence on n-octynoyl-CoA concentration. D) Dependence on immobilized ghrelin(1–5)pentapeptide concentration. RFU = normalized relative fluorescence units at 585 nm.
Figure 4
Figure 4
Characterization of GOAT acyltransferase activity using cat-ELCCA. A) Time dependence. B) Dependence on the amount of GOAT membranes. C) Dependence on n-octynoyl-CoA concentration. D) Dependence on immobilized ghrelin(1–5)pentapeptide concentration. RFU = normalized relative fluorescence units at 585 nm.
Figure 4
Figure 4
Characterization of GOAT acyltransferase activity using cat-ELCCA. A) Time dependence. B) Dependence on the amount of GOAT membranes. C) Dependence on n-octynoyl-CoA concentration. D) Dependence on immobilized ghrelin(1–5)pentapeptide concentration. RFU = normalized relative fluorescence units at 585 nm.
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