Phosphocholine synthesis in spinach: Characterization of phosphoethanolamine <i>N</i>‐methyltransferase
Bibliographic record
Abstract
Phosphocholine is a precursor for phosphatidylcholine or it may be hydrolysed to choline. Choline can be oxidized to form the compatible osmolyte glycine betaine which is accumulated by many plants under conditions of osmotic stress. In Spinacia oleracea phosphocholine is synthesized by 3 sequential N‐methylations of phosphoethanolamine with the first step catalysed by the enzyme phosphoethanolamine N‐methyltransferase (EC 2.1.1.103). This enzyme has been partially purified 5400‐fold from spinach leaves using a combination of ammonium sulphate fractionation, followed by chromatographic separations on DEAE‐Sepharose, phenyl‐Sepharose, Ω‐aminohexyl‐agarose, Mono Q and adenosine‐agarose. Sodium dodecyl sulphate‐polyacrylamide gel electrophoresis (SDS‐PAGE) separation and silver‐staining of the final preparation revealed several polypeptides present, only one of which with an estimated molecular mass of 54 kDa could be photoaffinity cross‐linked to the substrate [3H] S‐adenosyl‐l‐methionine. HPLC gel permeation chromatography was used to obtain an estimate for the native molecular mass of 77 kDa. Enzyme activity was optimal at pH 7.8 in HEPES‐KOH buffer, it was inhibited by S‐adenosyl‐l‐homocysteine, phosphocholine, phosphate, Mn2+ and Co2+ but not by ethanolamine, methylethanolamine, dimethylethanolamine, choline, glycine betaine or Mg2+. Using phosphoethanolamine as substrate, the final preparation had a specific activity of 189 nmol mg−1 protein min−1. The reaction products were identified and their relative abundance estimated following separation by TLC as phosphomethylethanolamine (87%), phosphodimethylethanolamine (10%) and phosphocholine (2%). Thus, a highly purified preparation of phosphoethanolamine N‐methyltransferase was shown to catalyse 3 successive N‐methylations of phosphoethanolamine. Photoaffinity cross‐linking of proteins extracted from leaves of spinach followed by SDS‐PAGE and autoradiography shows that a 54‐kDa radiolabelled polypeptide was more prominent in extracts from salinized plants and barely visible in extracts from plants exposed to prolonged dark periods, a pattern which corresponds to the salt and light‐responsive changes in phosphoethanolamine N‐methylating activity. Thus, the production of phosphocholine for glycine betaine accumulation in spinach can be mediated by a single phosphobase N‐methyltransferase which is more abundant in salt‐stressed plants.
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How this classification was reachedexpand
Full frame machine prediction
Teacher imitationNot calibrated prevalence, not ground truth. Human validation pending. The Gemma side is a direct model label for every work in the frame, read from the title-only record. The Codex side is a classifier learned from the 10,348 direct Codex labels and calibrated to design-weighted sample rates; fields without enough sample support carry no Codex call. Candidate is the union of the two sides; consensus is their intersection. These outputs are machine_predicted_unvalidated and are not human labels.
Distilled classifier scores by category (both heads)
| Category | Codex | Gemma |
|---|---|---|
| Metaresearch | 0.000 | 0.000 |
| Meta-epidemiology (narrow) | 0.001 | 0.000 |
| Meta-epidemiology (broad) | 0.000 | 0.000 |
| Bibliometrics | 0.000 | 0.000 |
| Science and technology studies | 0.000 | 0.000 |
| Scholarly communication | 0.000 | 0.000 |
| Open science | 0.000 | 0.000 |
| Research integrity | 0.000 | 0.000 |
| Insufficient payload (model declined to judge) | 0.001 | 0.001 |
Machine scores (provisional)
The two teacher heads of the student model, read on this work. A score orders the frame for review; it never asserts a category, and the validation status ships verbatim with every row.
Baseline scores from an immature model (maturity gate not passed, 7 training rounds). Scores rank; they never assert a category.
score_only:v0-immature-baseline · verbatim from the scoring run: score_only means the number may rank works, and no category label ships from itClassification
machine, unvalidatedMachine predicted; a candidate call from one source (direct Gemma or distilled Codex), not a consensus.
How this classification was reached, model by model and score by score, is at the end of the page under "How this classification was reached".