Highly purified rat brain myelin was solubilized in Triton X-100 and myelin phospholipid N-methyltransferase was characterized. The enzyme activities were separated by isoelectric focusing and ion-exchange chromatography. The phospholipid methyl-transferase has shown at least four peaks of activity with pIapp. values of 4.5, 5.2, 6.2 and 8.4. After affinity purification each of these activities revealed a close set of bands of approx. 65 kDa on SDS/PAGE. These data together with those from preparative SDS/PAGE separations suggested that rat brain myelin contains three acidic and at least one basic phospholipid-methylating isoenzymes and that the major isoenzyme in each case is approx. 65 kDa in size. While the predominant product of the reaction catalysed by all detected isoforms was monomethylated phosphatidylethanolamine, the least acidic isoform (pIapp. 6.2) also formed about 20% phosphatidylcholine, suggesting that these isoenzymes may play different roles in vivo.

Myelin is a specialised extension of the plasma membrane of oligodendrocytes in the CNS which wraps around the axon and provides insulation for and facilitation of axonal conduction. Its composition is 70-75% lipid with six major proteins accounting for more than 95% of total protein. The earlier concept of myelin as an inert membrane has been revised and myelin is known to contain numerous enzymes, more than half of which metabolise lipids [ 13. Phospholipid methyltransferase (PLMTase) catalyses transfer of CH3-groups from the methyl donor Sadenosyl-methionine (SAM) to the amino head-group of phosphatidylethanolamine (PE) to yield monomethyl-PE (PME), dimethyl-PE (PDE) and phosphatidylcholine (PC). PLMTase was reported from rat liver [21 to be a single polypeptide, Mr 18.3 kDa, which possessed all three methylating activities. Others suggested PLMTase had two activities [3][4] [5]. The first one catalysed formation of PME and the second either completed PC formation from PME or catalysed all three steps [6]. Rat brain myelin PLMTase [7] was also reported to be a twoenzyme methylating system. Here we report that iso-electric focusing (IEF) of Triton X-100-solubilised myelin proteins resulted in separation of acidic (PI-5) and basic (PI-9) PLMTase activities. The acidic activity yielded three distinct peaks upon further re-focusing. Myelin was isolated according to the classical procedure [8] with the additional sucrose gradient [9] and solubilised in phosphate buffer (pH 8.0) containing 0.5% (w/v) Triton X-100. The solubilised proteins were subjected to IEF on Preparative IEF Cell (Bio-Rad) over pH gradient from 3 to 10. The harvested fractions were assayed for PLMTase activity after adjusting pH to 8 by incubating the aliquot from each IEF fraction for 30 min a t 4OoC with 200pM S-adenosyl-L-(3H-rnethyZ)methionine (12.5 mCi/mmol) and 1 0~1 of the mixture of PE (8mg/ml) and PME (lmg/ml). After the phospholipids were extracted and dried, the amount of (3H-methyl) incorporated into phospholipids was measured by liquid scintillation counting. The active fractions were pooled and subjected to re-focusing. The aliquots of each fraction were analysed by 12% SDS-PAGE and the gels silverstained. The results indicated that iso-electric focusing over the pH range 3-10 separated detergent-solubilised myelin PLMTase into two broad peaks of activity, one in the PI 4-7 and another in the basic region respectively. Upon refocusing (pH 3-7) the former revealed three distinct methylating activities with PI 4.5, 5.5 and 6.5 (Fig. 1). Refractionation of the basic activity (pH 7-10) did not result in tighter focusing but activity centred around PI-9. TLC separation and radiometric analysis of the products of PLMTase showed predominant formation of PME both from the PI 4.5 and 5.5 activities whilst the PI 6.5 activity yielded 30% PC and 260% PME. We cannot yet attribute these differences as evidence of separate monomethylating and di/tri-methylating activities. SDS-PAGE of each active acidic fraction unveiled the presence of one majo...

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