Immunological characterization, lipid dependence, and subcellular localization of CTP:phosphoethanolamine cytidylyltransferase purified from rat liver. Comparison with CTP:phosphocholine cytidylyltransferase.
CTP:phosphoethanolamine cytidylyltransferase (ET) (ethanolamine-phosphate cytidylyltransferase, EC 2.7.7.14), which is generally considered as the rate-regulatory enzyme of phosphatidylethanolamine synthesis via the CDP-ethanolamine pathway, was purified to homogeneity from a rat liver postmicrosomal supernatant. A polyclonal antibody was raised against the enzyme in rabbits and subsequently purified by affinity chromatography. The affinity-purified antibody recognized one single immunoreactive 49.6-kDa protein band on SDS-polyacrylamide gel. The enzyme showed an isoelectric point at a pH of 6.5 and was sensitive to various sulfhydryl reagents. Cross-reactivity experiments of ET and CTP:phosphocholine cytidylyltransferase (CT) (choline-phosphate cytidylyltransferase, EC 2.7.7.15) with their corresponding antibodies showed that these enzymes were immunologically distinct. In contrast with the well known lipid dependence of CT, the activities of both purified and cytosolic ETs were not affected by the presence of various phospholipid preparations. Differential centrifugation studies as well as release experiments with digitonin-permeabilized hepatocytes demonstrated that ET, unlike CT, is not associated with cellular organelles. However, amino acid analysis of ET revealed a high content of hydrophobic amino acids, suggesting a possible association of this enzyme with some kind of cellular structure in the hepatocyte.
Laboratory of Veterinary Biochemistry, Utrecht University, The Netherlands.
P S
PS
Vermeulen
L B
LB
Tijburg
M J
MJ
Geelen
L M
LM
van Golde
D000596:Amino Acids / Q000032:analysis; D000818:Animals; D019916:Choline-Phosphate Cytidylyltransferase; D003429:Cross Reactions; D015151:Immunoblotting; D050356:Lipid Metabolism; D008099:Liver / Q000201:enzymology*; D008297:Male; D009713:Nucleotidyltransferases / Q000737:chemistry / Q000276:immunology / Q000378:metabolism*; D012316:RNA Nucleotidyltransferases; D051381:Rats; D017208:Rats, Wistar
D000596:Amino Acids; D009713:Nucleotidyltransferases; D012316:RNA Nucleotidyltransferases; C020126:Ethanolamine-phosphate cytidylyltransferase; D019916:Choline-Phosphate Cytidylyltransferase