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Enzymes of the Primary Phosphatidylethanolamine Biosynthetic Pathway in Postgermination Castor Bean Endosperm (Developmental Profiles and Partial Purification of the Mitochondrial CTP:Ethanolaminephosphate Cytidylyltransferase)

Authors :
Thomas S. Moore
F. Tang
Source :
Plant Physiology. 115:1589-1597
Publication Year :
1997
Publisher :
Oxford University Press (OUP), 1997.

Abstract

Ethanolamine kinase, CTP:ethanolaminephosphate cytidylyltransferase (ECT), and ethanolaminephosphotransferase, which sequentially catalyze the primary pathway for phosphatidylethanolamine synthesis, were measured in castor bean (Ricinus communis L. var Hale) endosperm for 6 d after the onset of imbibition. Ethanolamine kinase (EC 2.7.1.82) activity was cytosolic, increasing slowly during the first 5 d and then declining. Total ECT (EC 2.7.7.14) activity increased until the 4th d, but the endoplasmic reticulum fraction of the activity peaked at d 3, and the mitochondrial activity peaked at d 4. Diacylglycerol:CDPethanolamine ethanolaminephosphotransferase (EC 2.7.8.1) increased during the first 2 d after imbibition began, after which it declined. The lowest activity of ethanolamine kinase during postgermination was more than 5-fold higher than the maximum activity of ECT, and the total activity of diacylglycerol:CDPethanolamine ethanolaminephosphotransferase at d 2 was at least triple that of ECT of the endoplasmic reticulum. We have partially purified ECT from mitochondrial fractions of postgermination castor bean endosperm starting with mitochondria purified by sucrose (Suc) density gradient centrifugation and broken by osmotic shock and homogenization. The membrane-bound ECT was solubilized with 1.5% 3-[(3-cholamidopropyl)dimethylammonio]-2-hydroxy-1-propanesulfonate and purified approximately 118-fold by polyethylene glycol precipitation, chromatography on Sephacryl S-200, and then Suc gradient centrifugation. The continuous presence of both salt (0.5 M NaCl) and detergent (1% [w/v] 3-[(3-cholamidopropyl)dimethylammonio]-2-hydroxy-1-propanesulfonate) was necessary to prevent aggregation. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the final activity peak resulted in a prominent protein band at 35 kD, which correlated with bands from peak ECT activity fractions after both Suc gradient centrifugation and gel filtration on Sephacryl S-200. The activity of this enzyme was enhanced by the addition of several phospholipids.

Details

ISSN :
15322548 and 00320889
Volume :
115
Database :
OpenAIRE
Journal :
Plant Physiology
Accession number :
edsair.doi.dedup.....cb5adc0e33fe6302847b0e7972fc5fbc
Full Text :
https://doi.org/10.1104/pp.115.4.1589