Enzymes of Phosphatidylcholine Synthesis in Lemna, Soybean, and Carrot

Abstract

ABSTRACICell-free extracts from Lemna and suspension cultured carrot (Daucus Carota L.) catalyze S-adenosylmethionine-dependent N-methylations of phosphoethanolamine, phosphomethylethanolamine, and phosphodimethylethanolamine; extracts of suspension cultured soybean (Glycine max), of phosphoethanolamine only. Material pelleted from each tissue between 15,000 and 100,000g catalyzes S-adenosylmethionine-dependent N-methylations of phosphatidylmethylethanolamine and phosphatidyldimethylethanolamine, but not phosphatidylethanolamine. Extracts from each tissue catalyze CTP-dependent cytidylyltransfers to each of the three methylated phosphoethanolamine derivatives, forming the corresponding CDP derivatives. Some of the properties of the activities investigated are reported. On the basis of in vivo labeling experiments, we have proposed (AH Datko, SH Mudd 1988 Plant Physiol 88: 854-861) differing pathways for phosphatidylcholine synthesis in which, after a common committing step, N-methylation of phosphoethanolamine, subsequent methylations occur in Lemna almost exclusively at the phospho-base level; in soybean, at the phosphatidyl-base level; and in carrot, at both levels. Thus, among the activities investigated, at least those required for the operation of the proposed pathways have been positively demonstrated. The extent to which the present results explain the differences between these pathways is discussed, and a speculation offered as to how these differences may have arisen phylogenetically.We have recently reported the results of studies of the kinetics of entry of the radiolabeled methyl group of L-methionine into the network of methylated derivatives of EA2 which lead ultimately to PtdCho. The patterns for each of the three plants studied, Lemna (19), soybean and carrot (4), were qualitatively different. The working models we suggested based on the results ' Reprint requests should be addressed to the authors at Building 36, Room 3D06, National Institute of Mental Health, Bethesda, MD 20892.2 Abbreviations: EA, ethanolamine; MEA, N-methylethanolamine; DMEA, N, N-dimethylethanolamine; Cho, choline. The phosphate esters of these compounds are designated by the prefix, P-(e.g. P-EA; P-Cho); the corresponding phosphatidyl, glycerylphospho-base, or CDP derivatives, by the prefixes, Ptd, GP-, or CDP-(e.g. PtdEA and PtdCho; GP-EA and GP-Cho; or CDP-EA and CDP-Cho). The methylations involved in PtdCho biosynthesis are all N-methylations. For brevity in the text Nmethylation has generally been replaced by methylation (unless special emphasis is desired). The of these studies include a committing step common to the three plants: namely an initial N-methylation of P-EA to form P-MEA. Subsequent methylations occur either virtually exclusively at the phospho-base level (Lemna) (19), at the phosphatidyl-base level (soybean), or at both of these levels (carrot) (4). These suggested pathways require the activity of certain phospho-base N-methyltransferases which have not hitherto been described, and of additional cytidy...

cDNA Cloning of PhosphoethanolamineN-Methyltransferase from Spinach by Complementation inSchizosaccharomyces pombe and Characterization of the Recombinant Enzyme

“…that of the 25 complemented S. pombe strains recovered, 24 harbored a plasmid encoding PEAMT, for this implies that N-methyltransferases acting on Ptd-EA or EA are either absent or expressed at far lower levels. This evidence reinforces the conclusion from in vivo radiolabeling studies (2-5) and enzyme assays (1,4,17) that in plant leaves the first methylation in the synthesis of Cho moieties takes place exclusively at the phosphobase level.…”

“…Enzyme Assays-PEAMT activity was measured by a modification of published methods (1,4), under conditions in which product formation was proportional to enzyme concentration and time. To compensate for H 2 SO 4 present in the [methyl- 14 C]AdoMet solution, the assay buffer was titrated to a pH such that, after adding [methyl- 14 C]AdoMet and other assay components, the mixture had the desired pH.…”

“…Nerve tissues have a phosphobase methylation route as well as a phosphatidylbase route, but the first methylation appears not to be restricted to the phosphobase level (9,10). The initial methylation of P-EA in plants is catalyzed by S-adenosyl-L-methionine:phosphoethanolamine N-methyltransferase (PEAMT), which has been detected in all species tested (1,4,11). PEAMT was recently purified 5,400-fold from spinach leaves, giving a preparation containing several polypeptides (12).…”

“…Flowering plants are unusual in how they synthesize choline (Cho) 1 moieties. In leaves and other vegetative tissues, the first and committing step is N-methylation of phosphoethanolamine (P-EA) to give phosphomonomethylethanolamine (P-MME), and the subsequent N-methylations occur at the phosphobase level, the phosphatidyl base level, or both, depending on the species (1, 2) (see Fig.…”

Synthesis of Ethanolamine and Its Regulation in Lemna paucicostata

“…Other Methods Chromatographic systems and solvents previously designated as A through E were used as described (32) of radioactivity in particular compounds which had been subjected to several sequential purification steps have been described (7,8,31,32 ,32P]GP-EA was assumed to have come into isotopic equilibrium) were used to calculate the "'C specific radioactivity (dpm/nmol) of the ['C,`2P]GP-EA.…”