CDPcholine:1,2-diacylglycerol cholinephosphotransferase from rat liver microsomes. I. Solubilization and characterization of the partially purified enzyme and the possible existence of an endogenous inhibitor |
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Authors: | Kozo Ishidate Ritsuko Matsuo Yasuo Nakazawa |
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Affiliation: | (1) The Medical Research Institute, Tokyo Medical and Dental University, 2-3-10 Kanda-surugadai, Chiyoda-ku, 101 Tokyo, Japan |
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Abstract: | The solubilization and partial purification of cholinephosphotransferase (CDPcholine:1,2-diacylglycerol cholinephosphotransferase,
EC 2.7.8.2) from rat liver microsomes were examined in the presence of ionic (sodium deoxycholate), nonionic (Triton X-100,n-octylglycoside), or zwitter ionic (CHAPS) detergents. Among the four detergents tested, only sodium deoxycholate was found
to be an efficient solubilizer of cholinephosphotransferase activity from microsomal membranes, whereas the other three detergents
caused irreversible inactivation of the enzyme at the solubilization step. Addition of phospholipids at the solubilization
step, or after solubilization of the membrane proteins, could not preserve or reconstitute activity to any extent. The sodium
deoxycholate-solubilized activity was partially purified by gel permeation chromatography (Superose 12HR). The partially purified
preparation appeared to consist of a large aggregate containing phospholipids; further dissociation of the protein-phospholipid
complex caused complete inactivation of the enzyme. The partially purified cholinephosphotransferase showed a specific activity
of 100–130 nmol/min/mg protein, which is the highest activity reported to date from any tissue source; this amounts to a 4-fold
enrichment of cholinephosphotransferase activity from the original KCl-washed rat liver microsomes. Ethanolaminephosphotransferase
(CDPethanolamine:1,2-diacylglycerol ethanolaminephosphotransferase, EC 2.7.8.1) activity was copurified and 6-fold enriched
with a total recovery of 60%. During the purification of cholinephosphotransferase activity, a putative endogenous inhibitor
of cholinephosphotransferase was also solubilized and was isolated from the microsomal membranes. This heat-labile, nondialyzable
inhibitor was shown to act specifically on cholinephosphotransferase and not on ethanolaminephosphotransferase. Further characterization
of the inhibitory activity revealed that it may act at the binding step of the cholinephosphotransferase to its lipid substrate,
diacylglycerol. |
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