Table 1.
Effect of respiratory substrates and ATP/Mg2+ on the methylation of mitochondrial [3H]-methyl-PMME in the in vitro reconstituted system
| Additive(s) | Relative methylation of [3H]-methyl-PMME |
|---|---|
| None | 1.0 |
| Ethanol (17 mM) | 1.24 ± 0.05 |
| Ethanol (17 mM) + FCCP (10 μM) | 1.04 ± 0.05 |
| NADH (2 mM) | 1.0 ± 0.05 |
| NADH (2 mM) + FCCP (10 μM) | 1.0 ± 0.05 |
| ATP (2 mM) | 1.15 ± 0.07 |
| ATP (2 mM) + MgCl2 (5 mM) | 0.7 ± 0.2 |
| MgCl2 (5 mM) | 0.6 ± 0.2 |
Mitochondria from the opi3Δ strain and wild-type microsomes were incubated for 2 h at 1 mg/ml each in the presence of AdoMet and the additives indicated, as detailed in MATERIALS AND METHODS. When ethanol or NADH was added, the buffer also contained 5 mM Pi to maintain mitochondrial intactness (Janssen et al., 2002b). FCCP was added from a 10 mM stock solution in ethanol. Methylation was quantified as the increase in the percentage of [3H]-methyl label present in PDME and PC upon 2 h of incubation and was normalized to that in the absence of additives (32 ± 5% of the lipid-incorporated [3H]-methyl label). Results are mean ± SD (n = 3).