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https://doi.org/10.1016/0005-2760(92)90123-d
Copy DOIPublication Date: Feb 1, 1992 | |
Citations: 7 |
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′)-0-(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, Mg 2+ or Mn 2+-requiring and inhibited strongly by the presence of Ca 2+. 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).
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