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Hoffmann, R. A wiki for the life sciences where authorship matters. Nature Genetics (2008)
 
 
 
 
 

Phosphatidylethanolamine N-methyltransferase activity in isolated rod outer segments from bovine retina.

Phosphatidylcholine (PC) can be synthesized in isolated rod outer segments from bovine retina by successive transfer of methyl groups from S-adenosyl-L-methionine (SAM) to phosphatidylethanolamine (PE), with the intermediate formation of phosphatidyl-N-monomethylethanolamine (PMME) and phosphatidyl-N,N-dimethylethanolamine (PDME). This reaction is time-protein-and SAM concentration-dependent. Phosphatidylethanolamine N-methyltransferase (PE N-MTase) has two pH optima, 8.5 and 10, at low (10 microM) and high (200 microM) SAM concentrations and requires magnesium ions for full activity. When ROS membranes were incubated at 5 to 200 microM SAM concentrations at pH 8.5 or pH 10, the major methylated product was PMME, followed by PC and PDME. The apparent Kms for SAM at pH 8.5 and at pH 10 were similar (37 and 38 microM, respectively). The Vmax was 13 pmol h-1 (mg protein)-1 at pH 8.5 and 12.50 pmol h-1 (mg protein)-1 at pH 10. Pulse-chase experiments demonstrated a precursor-product relationship with [3H]PC as the end product. The level of PE N-Mtase activity in the purified ROS preparation obtained from crude ROS fractions by discontinuous sucrose gradient centrifugation, was as high as 65% of the level found in the microsomal fraction obtained from the remainder of the retinas. The presence of microsomal and mitochondrial marker enzymes, however, was minimal in the ROS preparation. The radioactivity incorporated into ROS PC was measured in an upper and lower band of PC obtained by two-dimensional TLC. We found that the amount of [methyl-3H] groups incorporated into the upper PC band was 2.5-fold greater than that incorporated into the lower one. The fatty acid composition of the upper band was very different from that of the lower band, the former being enriched in very long-chain polyunsaturated fatty acids and the latter in saturated fatty acids. Phosphatidyl-ethanolamine N-methyltransferase activity increased in the presence of exogenous phospholipid substrates. PDME being augmented ten-fold and PC eight-fold when the incubations were carried out in the presence of PMME and PDME, respectively. At a 2 mM concentration, S-adenosyl-L-homocysteine (SAH) inhibited the methyl groups' incorporation into the endogenous phospholipids by 40%. When ROS membranes were selectively depleted of soluble or peripheral and soluble proteins, the PE N-MTase activity remained mainly associated to the membrane, suggesting that this enzyme (s) is an intrinsic membrane protein.(ABSTRACT TRUNCATED AT 400 WORDS)[1]

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