Phospholipid methyltransferase from Drosophila melanogaster: Purification and properties

Abstract

The phospholipid methyltransferase (PMTase) activity from Drosophila melanogaster has been purified ∼ 190,000-fold to give a preparation with a final sp.act. ∼4.3 μmol/min/mg protein. Gel filtration and HPLC methods show that this activity resides in a protein complex of M w = 140,000–150,000 dalton. Since the preparation gives several bands on SDS and native polyacrylamide gel electrophoresis, subunit composition has not been determined. The activity is sensitive to protein denaturing agents such as heat and proteases and shows inhibition by S-adenosyl homocysteine. Integrity of sulphydryl groups is essential for the stability of the enzyme. Zinc is a potent inhibitor, while manganese and calcium have no significant effect on activity. Micromolar concentrations of magnesium stimulate the activity, but millimolar concentrations inhibit the PMTase. There is no absolute requirement for exogenous lipid for activity, and evidence is presented that the enzyme is a lipoprotein and carries its own substrates. The incorporation of methyl groups into phosphatidylcholine and phosphatidyl- N, N-dimethylethanolamine was highest around pH 7.5. A high degree of methyl group incorporation into the monomethyl derivative also occurred at a lower pH. A Michaelis-Menten plot of methyl group incorporation into the total lipid fraction gives an average K m of 120 μM for S-adenosyl methionine. Methylation occurs on the base of the phospholipid. The ratio of the three methylated products formed is highly variable, with the monomethyl or the dimethyl products generally being the highest labelled under standard conditions. No separation of enzyme activities is observed during purification and on gel filtration a single peak is obtained, which shows all three methylating activities. Thus, while the variable ratios of the three products may indicate more than one enzyme, the single peak on gel filtration suggests these have almost identical molecular weights; it is possible they exist as a tight complex, or there is just one enzyme.