INCORPORATION OF SERINE-<sup>14</sup>C AND ETHANOLAMINE-<sup>14</sup>C INTO NITROGEN-CONTAINING PHOSPHATIDES AND EFFECTS OF MEDIUM CONTAINING ETHANOLAMINE ON PHOSPHATIDE BIOSYNTHESIS IN EXCISED TOMATO ROOTS

(7) (3) However, reaction 7 is clearly an exchange reaction and can only lead to net synthesis of PE if the organism can make PS (e.g. by reaction 2) and ethanolamine.The cells of higher plants have both procaryotic and eucaryotic characteristics exemplified by the nucleic acids of the nuclei and other subcellular organelles. It is, therefore, of some interest to examine the biosynthesis of PE in higher plants to see whether the mechanism of biosynthesis is by the prevalent method in animals, the method in bacteria, or both. The reaction studied in this paper is the formation of PE from CDPethanolamine (reaction 6). PS -> PE + CO2Our understanding of the biosynthesis of phospholipids in animal tissues stems from the basic findings of 9,12,19,20 MATERIALS AND METHODSMaterials. Plant materials were purchased from local grocery stores. CDP-ethanolamine was prepared by the method of Kennedy (19) or purchased from Sigma Chemical Company, St. Louis, Missouri, CDP-choline was purchased from Sigma Chemical Company. "4C-CDP-choline was purchased from International Chemical and Nuclear Corporation, Irvine, California. "4C-Phosphoryl ethanolamine was obtained from New England Nuclear."4C-CDP-ethanolamine was prepared from "4C-phosphoryl ethanolamine using enzyme preparations from chicken liver as described by Chojnacki and Metcalf (10). The product was isolated by ion exchange chromatography on Dowex-1-formate according to the method of Kennedy (19). Purity of the product was evaluated by paper chromatography in solvent systems recommended by Scheider (31) and scanning of the paper strip with a radioactivity scanner.Methods. Plant extracts were prepared by homogenizing a given weight of material with twice as much homogenizing medium. The homogenizing medium consisted of 0.25 M sucrose, 10 mM tris-HCl pH 7.5, 1 mm EDTA. The plant material and cold medium were homogenized in a Waring Blendor (three 5-sec bursts), and the homogenate was pressed through two layers of cheesecloth. In the preparation of subcellular fractions, the filtrate was first centrifuged for 5 min at 1,000g. The supernatant was centrifuged for 30 min at 20,000g and centrifuged for 60 min at 100,000g. The first two pellets (1,OOOg and 20,000g) were suspended in 20 ml each 10 mm tris-HCI pH 7.5, 1 mM EDTA, and the l00,OOOg pellet in 10 ml of the same medium. Reaction mixtures contained 2 mm DTT, 20 mM

Section: Discussionmentioning

confidence: 99%

“…If PE is normally made in vivo by decarboxylation of PS, we might expect that free ethanolamine would be a poor precursor of PE. In fact the opposite was true (37). It is possible that ethanolamine was introduced by an exchange reaction (35) rather than de novo synthesis.…”

Section: Discussionmentioning

confidence: 99%

Biosynthesis of Phosphatidylethanolamine by Enzyme Preparations from Plant Tissues

Macher

Mudd²

1974

“…However, the cytidylyltransferase catalyzing reaction 8 has been mentioned only very briefly as present in enzyme preparations from carrot root (20), and subsequent attempts in at least two laboratories to demonstrate this activity in extracts of higher plants were unsuccessful (25)(26)(27). Radiolabeled EA is incorporated into PtdEA (1 1,26,27,33,38,39). In certain systems, the characteristics of this incorporation are such that it was concluded that some or all of it occurred by base exchange (26,27,38), for example, reaction 2 (proceeding from right to left as written above).…”

mentioning

confidence: 99%

Synthesis of Ethanolamine and Its Regulation in Lemna paucicostata

Mudd¹,

Datko²

1989

The metabolism of ethanolamine and its derivatives in Lemna paucicostata has been investigated, with emphasis on the pathway for synthesis of phosphoethanolamine, a precursor of phosphatidylcholine in higher plants. In experiments involving labeling of intact plants with radioactive serine, ambiguities of interpretation due to entry of radioactivity into methyl groups of methylated ethanolamine derivatives were mitigated by pregrowth of plants with methionine. Difficulties due to labeling of diacylglyceryl moieties of phospholipids were avoided by acid hydrolysis of crucial samples and determination of radioactivity in isolated serine or ethanolamine moieties. The results obtained from such experiments are most readily reconciled with the biosynthetic sequence: seine --ethanolamine --phosphoethanolamine phosphatidylethanolamine. A possible altemative is: serine phosphatidylserine -. phosphatidylethanolamine -i ethanolamine --phosphoethanolamine. Cell-free extracts of L. paucicostata were shown to produce CO2 from the carbon originating as C-1 of serine at a rate sufficient to satisfy the demand for ethanolamine moieties. A number of experiments produced no support for a hypothetical role for phosphoserine in phosphoethanolamine formation. Uptake of exogenous ethanolamine commensurately down-regulates the synthesis of ethanolamine moieties (considered as a whole, and regardless of their state of derivatization at the time of their formation). In agreement with previous observations, uptake of exogenous choline down-regulates the methylation of phosphoethanolamine, without being accompanied by secondary accumulation of a marked excess of ethanolamine derivatives.findings bring to the fore the question of the biosynthetic origin in higher plants of P-EA, a compound crucial to initiation of each of these metabolic pathways. One route to P-EA, each of the component steps of which has been demonstrated in higher plants, involves the decarboxylation of PtdSerine:PtdSerine --PtdEA + CO2 (reaction 1)PtdSerine decarboxylase activity has been demonstrated in extracts of spinach leaves, chiefly in the 100,000g supernatant fraction (26,27 Plant Physiol. Vol. 91, 1989 10, 18), contribution to EA formation. EA is a widespread constituent of higher plants (23), but a decarboxylase unequivocally acting on free serine (5, 10) has not, to our knowledge, been reported.Once EA is formed, the pathway to P-EA may be completed by EA kinase:This kinase is well known in higher plants (29,30), including Lemna (32,33).An alternative route to P-EA, entirely speculative at the moment (32), would involve the decarboxylation of P-serine:Evidence (reviewed and summarized by Keys [21 ]) is available suggesting that P-serine may be formed in at least some higher plants from the photosynthetic and glycolytic intermediate, P-glycerate, by successive oxidation and transamination.An additional uncertainty related to P-EA metabolism in higher plants is whether this compound can be converted to PtdEA by a sequence analogous to that for the c...

“…This regulation may be complex, however, since PC synthesis involves more than one pathway (10,12). For instance, in an early study of the incorporation of ['4C]serine into intact tomato roots, Willemot and Boll (18) concluded that PE was synthesized by the decarboxylation of PS, and PC by the methylation of PE. This pathway also has been found in castor bean endosperm (10), but the activity appeared to be only a fraction ofthe activity ofthe enzymes of the nucleotide pathway, in which PC is synthesized from free choline.…”

mentioning

confidence: 99%

“…Furthermore, there appears to be very little known about the serine decarboxylation reaction in any plant tissue. Although in some mammalian tissues it is clear that PS is a major precursor of PE (17), studies in plant tissues have been complicated due to apparently high turnover rates (18), low levels of PS (1 1), or an apparent lack of incorporation of radiolabeled precursors into the PS fraction (2). Marshall and Kates (9) reported the presence of a PS decarboxylase enzyme in spinach leaves, but its localization in a cytoplasmic fraction raises the possibility that the in vivo enzyme may also be involved in the decarboxylation of free serine.…”

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confidence: 99%

Phosphatidylcholine Synthesis in Castor Bean Endosperm

Kinney¹,

Moore²

1987

Endosperm halves from 3-day-old castor bean (Ricinus communis var Hale) were incubated for 30 minutes with L-14Cjserine, after which label was observed in ethanolamine, choline, phosphatidylserine, phosphatidylethanolamine, phosphatidylcholine, ethanolaminephosphate, and CDPethanolamine, but not in cholinephosphate or CDPcholine. Only later did significant amounts of isotope become incorporated into cholinephosphate and CDPcholine. The choline kinase inhibitor hemicholinium-3 prevented the incorporation of label from serine into cholinephosphate and CDPcholine, reduced the incorporation of I'4Cicholine into phosphatidylcholine by 65%, but inhibited the incorporation of label into phosphatidylcholine from serine by only 15%. The inhibitor did not prevent the incorporation of labeled methyl groups from S-adenosyl-Lmethionine into phosphatidyldimethylethanolamine plus phosphatidylcholine. The amount of incorporation of label from the methyl donor was only 8% of that from choline into phosphatidylcholine. The implications of these results for the pathway and regulation of phosphatidylcholine synthesis from the water-soluble precursors are discussed.Phosphatidylcholine is the major phospholipid in most eukaryotic cell membranes, with phosphatidylethanolamine generally being second most abundant (1). Some evidence exists for regulation of the levels of PC2 independent ofthe other phospholipids in plants. For example, PC increases as a proportion of the total phospholipid over the 8-d postgermination period in castor bean endosperm (13,16). This regulation may be complex, however, since PC synthesis involves more than one pathway (10, 12). For instance, in an early study of the incorporation of ['4C]serine into intact tomato roots, Willemot and Boll (18) concluded that PE was synthesized by the decarboxylation of PS, and PC by the methylation of PE. This pathway also has been found in castor bean endosperm (10), but the activity appeared to be only a fraction ofthe activity ofthe enzymes of the nucleotide pathway, in which PC is synthesized from free choline. Thus, at least two pathways must be considered as sources for PC in plants.The source of the water-soluble precursors also is unclear. It seems likely that in many plant tissues the biosynthesis ofcholine involves the methylation of ethanolamine, and that ethanolamine is formed by the decarboxylation of serine (6). It is not firmly established, however, whether these reactions occur predominantly at the level of free bases, phosphoryl bases, or phosphatides. In other words, it is not clear if the syntheses involve 'Supported by National Science Foundation grant PCM-8402001 to T. S. M.2Abbreviations: PC, phosphatidylcholine; PE, phosphatidylethanolamine; PS, phosphatidylserine; SAM, S-adenosyl-L-methionine.only the water soluble substrates or a complex interrelationship between cytosolic and membrane-bound intermediates. Studies in some plant tissues, utilizing both precursor incorporation (8,14) and enzyme studies (9, 10), appear to indicate that the m...