Table I.
Kinetic Parameters for Hydrolysis of AHMMCE and 2-AG by hMGL in the Presence and Absence of Detergent or Phospholipid Bilayer Nanodiscs
| Substrate | ||||
|---|---|---|---|---|
| AHMMCE | 2-AG | |||
| Incubation conditions | Km (μM) | Vmax (μM/min/mg) | Km (μM) | Vmax (μM/min/μg) |
| Buffer | 18.3 ± 2.8 | 191.4 ± 10.5 | 41.9 ± 5.9 | 28.3 ± 1.1 |
| Buffer + POPC/POPG nanodiscs | 7.5 ±1.8a | 565.4 ± 52c | 11.9 ± 1.8c | 57.3 ± 3.2c |
| Buffer + POPC nanodiscs | 7.9 ± 2c | 590 ± 45a | n.d. | n.d. |
| Buffer + Triton X-100 | 18.3 ± 4 | 295 ± 37.2a | n.d. | n.d. |
For AHMMCE substrate, buffer was 50 mM Tris–HCl, pH 7.4. For 2-AG substrate, TME buffer (25 mM Tris base, 5 mM MgCl2, and 1 mM EDTA, pH 7.4) was used. Apparent Km and Vmax values are the mean ± SD for triplicate determinations across three independent enzyme preparations. Statistical significance of group-mean differences was evaluated by a two-sample independent t-test, the significance level set at P ≤ 0.05. n.d., not determined.
a
P ≤ 0.01 versus buffer.
bP ≤ 0.02 versus buffer + Triton X-100.
c
P ≤ 0.001 versus buffer.
dP ≤ 0.002 versus buffer + Triton X-100.