Table 1.
Ghrelin substrate sequences and apparent Km measurements.
| Substrate | Sequence | Km (apparent), μM | Vmax (apparent) (nmol/min/mg) |
|---|---|---|---|
| Ghrelin27 | GSSFLSPEHQRVQQRKESKKPPAKLQPK (Biotin)G | 3.5 ± 0.60 | 230 ± 10 |
| Dap3-Ghrelin27 | GS(Dap)FLSPEHQRVQQRKESKKPPAKLQPK (Biotin)G | 3.4 ± 0.61 | 85 ± 5 |
| Ghrelin10 | GSSFLSPEHQ(Ahx)K(Biotin)G | 30.4 ± 5.1 | 160 ± 10 |
| Dap3-Ghrelin10 | GS(Dap)FLSPEHQ(Ahx)K(Biotin)G | 33.9 ± 9.6 | 74 ± 8 |
| Ghrelin10-Tat | GSSFLSPEHQ(Ahx) K(Biotin)YGRKKRRQRRR | 0.68 ± 0.09 | 120 ± 50 |
| Octanoyl-CoA | (vs 10 μM Ghrelin27) | 0.44 ± 0.04 | 250 ± 8 |
| Octanoyl-CoA | (vs 80 μM Ghrelin10) | 0.81 ± 0.04 | 190 ± 4 |
All reactions were carried out for 1 min at 30°C with 25 μg microsome protein (total 2 ng GOAT, quantified against a 50 kDa 3xFlag-tagged standard protein, not shown) and 50 μM palmitoyl-CoA. 1 μM octanoyl-CoA was used in the ghrelin measurements. Ahx=amino-hexanoic acid; K(Biotin) signifies that the biotin is attached to the epsilon amino group.