PLANT PHYSIOLOGY , Vol 115, Issue 4 1589-1597, Copyright © 1997 by American Society of Plant Biologists
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BIOCHEMISTRY AND ENZYMOLOGY |
Enzymes of the Primary Phosphatidylethanolamine Biosynthetic Pathway in Postgermination Castor Bean Endosperm (Developmental Profiles and Partial Purification of the Mitochondrial CTP:Ethanolaminephosphate Cytidylyltransferase)
F. Tang and T. S. Moore Jr
Department of Plant Biology, Louisiana State University, Baton Rouge, Louisiana 70803-1705
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.
