Cloning and expression of CTP:phosphoethanolamine cytidylyltransferase cDNA from rat liver.
CTP:phosphoethanolamine cytidylyltransferase (ET) is a key regulatory enzyme in the CDP-ethanolamine pathway for phosphatidylethanolamine synthesis. As a first step in the elucidation of the structure-function relationship and the regulation of ET, an ET cDNA was cloned from rat liver. The cloned cDNA encodes a protein of 404 amino acid residues with a calculated molecular mass of 45.2 kDa. The deduced amino acid sequence is very similar to that of human ET (89% identity). Furthermore, it shows less, but significant, similarity to yeast ET as well as to other cytidylyltransferases, including rat CTP:phosphocholine cytidylyltransferase and Bacillus subtilis glycerol-3-phosphate cytidylyltransferase. Like human and yeast ET, rat ET has a large repetitive internal sequence in the N- and C-terminal halves of the protein. Both parts of the repeat contain the HXGH motif, the most conserved region in the N-terminal active domain of other cytidylyltransferases, indicating the existence of two catalytic domains in ET. The hydropathy profile revealed that rat ET is largely hydrophilic and lacks a hydrophobic stretch long enough to span a bilayer membrane. There was no prediction for an amphipathic alpha-helix. Transfection of COS cells with the cDNA clone resulted in an 11-fold increase in ET activity, corresponding to an increase in the amount of ET protein as detected on a Western blot. Determination of the ET activity during liver development showed a 2. 5-fold increase between day 17 of gestation and birth (day 22) and the amount of ET protein changed accordingly. Northern blot analysis showed that this was accompanied by an increase in the amount of ET mRNA. Between day 17 of gestation and birth, the amount of mRNA in fetal rat liver increased approx. 6-fold, suggesting the regulation of ET at both pretranslational and post-translational levels during rat liver development.
Bladergroen
B A
BA
Department of Biochemistry and Cell Biology, Faculty of Veterinary Medicine, University of Utrecht, Yalelaan 2, 3508 TD Utrecht, The Netherlands.
Houweling
M
M
Geelen
M J
MJ
van Golde
L M
LM
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D019556:COS Cells; D003001:Cloning, Molecular; D018076:DNA, Complementary; D005260:Female; D006801:Humans; D008099:Liver / Q000201:enzymology*; D008969:Molecular Sequence Data; D009713:Nucleotidyltransferases / Q000235:genetics* / Q000378:metabolism; D012316:RNA Nucleotidyltransferases; D012333:RNA, Messenger / Q000235:genetics; D051381:Rats; D017208:Rats, Wistar; D017386:Sequence Homology, Amino Acid
D018076:DNA, Complementary; D012333:RNA, Messenger; D009713:Nucleotidyltransferases; D012316:RNA Nucleotidyltransferases; C020126:Ethanolamine-phosphate cytidylyltransferase