CDP-choline: 1,2-diacylglycerol cholinephosphotransferase from rat liver microsomes. II. Photoaffinity labeling by radioactive CDP-choline analogs.
Photoaffinity labeling of cholinephosphotransferase from rat liver microsomes directly by its substrate, [32P]CDP-choline or by a synthetic photoreactive CDP-choline analog, 3'(2')-O-(4-benzoyl)benzoyl [32P]CDP-choline (BB-[32P]CDP-choline), was examined for the possible identification of its molecular form on subsequent SDS-PAGE followed by 32P-autoradiography. When the partially purified cholinephosphotransferase was photoirradiated in the presence of [32P]CDP-choline, a considerable amount of 32P-radioactivity was incorporated into the TCA-insoluble component. This incorporation was dependent on irradiation time, Mg2+ or Mn(2+)-requiring and inhibited strongly by the presence of Ca2+. Either CDP-choline or CDP-ethanolamine inhibited the ultraviolet irradiation-dependent incorporation of 32P-radioactivity into the TCA-insoluble component in a dose-dependent manner, whereas neither phosphocholine or 5'-CDP had any effect on this process. These results strongly suggested that the observed 32P-incorporation from [32P]CDP-choline into the protein component could be a consequence of the covalent interaction between cholinephosphotransferase and its substrate, [32P]CDP-choline. Two polypeptides, 25 kDa and 18 kDa, with high 32P-radioactivity were clearly identified on a SDS gel after the direct photoaffinity labeling with [32P]CDP-choline for more than 5 min of ultraviolet irradiation. On the other hand, when BB-[32P]CDP-choline was used as a photoaffinity ligand, a single polypeptide with apparent molecular size of 55 kDa could be rapidly photolabeled within 2.5 min, then this band gradually lost its 32P-radioactivity with increasing time of ultraviolet irradiation. Thus, the overall results strongly indicated that cholinephosphotransferase in rat liver microsomes exists most likely as a 55 kDa polypeptide (or subunit) and that 25 kDa and 18 kDa peptides identified after the direct photoaffinity labeling with [32P]CDP-choline were probably the photo-cleavage products of cholinephosphotransferase during the prolonged ultraviolet irradiation, both of which could contain the catalytic domain of the original enzyme protein(s).
Ishidate
K
K
Medical Research Institute, Tokyo Medical and Dental University, Japan.
Matsuo
R
R
Nakazawa
Y
Y
D000818:Animals; D001345:Autoradiography; D002851:Chromatography, High Pressure Liquid; D003566:Cytidine Diphosphate Choline / Q000031:analogs & derivatives / Q000378:metabolism*; D002798:Diacylglycerol Cholinephosphotransferase / Q000302:isolation & purification / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D008862:Microsomes, Liver / Q000201:enzymology* / Q000528:radiation effects; D051381:Rats; D014466:Ultraviolet Rays
D003566:Cytidine Diphosphate Choline; D002798:Diacylglycerol Cholinephosphotransferase