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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Abbreviations
- CHAPS:
-
3-[(3-cholamidopropyl)dimethylamino]-1-propanesulfonate
- CPT:
-
cholinephosphotransferase
- DOC:
-
sodium deoxycholate
- EGTA:
-
ethyleneglycol-bis(β-aminoethylether)N,N,N′,N′-tetra-acetic acid
- EPT:
-
ethanolaminephosphotransferase
- n-OG:
-
n-octyl-β-D-glucopyranoside
- PMSF:
-
phenylmethylsulfonyl fluoride
- SDS-PAGE:
-
sodium dodecylsulfate-polyacrylamide gel electrophoresis
- TLC:
-
thin-layer chromatography
- TX-100:
-
Triton X-100
References
Yeagle, P.L. (1989)FASEB J. 3, 1833–1842.
Exton, J. (1990)J. Biol. Chem. 265, 1–4.
Billah, M.M., and Anthes, J.C. (1990)Biochem. J. 269, 281–291.
Pelech, S.L., and Vance, D.E. (1989)Trends Biochem. Sci. 14, 28–30.
Loffelholz, K. (1989)Biochem. Pharmacol. 38, 1543–1549.
Kiss, Z., and Anderson, W.B. (1990)J. Biol. Chem. 265, 7345–7350.
Ishidate, K., Nakagomi, K., and Nakazawa, Y. (1984)J. Biol. Chem. 259, 14706–14710.
Porter, T.J., and Kent, C. (1990)J. Biol. Chem. 265, 414–422.
Uchida, T., and Yamashita, S. (1990)Biochim. Biophys. Acta 1043, 281–288.
Ishidate, K., Furusawa, K., and Nakazawa, Y. (1985)Biochim. Biophys. Acta 836, 119–124.
Weinhold, P.A., Rounsifer, M.E., and Feldman, D.A. (1986)J. Biol. Chem. 261, 5104–5110.
Feldman, D.A., and Weinhold, P.A. (1987)J. Biol. Chem. 262, 9075–9081.
Sundler, R. (1975)J. Biol. Chem. 250, 8585–8590.
Kanoh, H., and Ohno, K. (1976)Eur. J. Biochem. 66, 201–210.
Cornell, R., and MacLennan, D.H. (1985)Biochim. Biophys. Acta 821, 97–105.
O, K.-M., and Choy, P.C. (1990)Lipids 25, 122–124.
Roberti, R., Vecchini, A., Freysz, L., Masoom, M., and Binaglia, L. (1989)Biochim. Biophys. Acta 1004, 80–88.
Ishidate, K., Tsuruoka, M., and Nakazawa, Y. (1980)Biochim. Biophys. Acta 620, 49–58.
Helenius, A., and Simons, K. (1975)Biochim. Biophys. Acta 415, 29–79.
Lowry, O.H., Rosebrough, N.J., Farr, A.L., and Randall, R.J. (1951)J. Biol. Chem. 193, 265–275.
Rouser, G., Saikotos, A.N., and Fleischer, S. (1966)Lipids 1, 85–86.
Kanoh, H., and Ohno, K. (1981)Methods Enzymol. 71, 536–546.
Folch, J., Lees, M., and Sloane-Stanley, G.H. (1957)J. Biol. Chem. 226, 497–509.
Vial, H.J., Thuet, M.J., and Philippot, J.R. (1984)Biochim. Biophys. Acta 795, 372–383.
Polokoff, M.A., Wing, D.C., and Raetz, R.H. (1981)J. Biol. Chem. 256, 7687–7690.
Hjelmstad, R.H., and Bell, R.M. (1987)J. Biol. Chem. 262, 3909–3917.
Hosaka, K., and Yamashita, S. (1987)Eur. J. Biochem. 162, 7–13.
Hjelmstad, R.H., and Bell, R.M. (1988)J. Biol. Chem. 263, 19748–19757.
Radominska-Pyrek, A., Pilarska, M., and Zimniak, P. (1978)Biochem. Biophys. Res. Commun. 85, 1074–1081.
Vecchini, A., Roberti, R., Freysz, L., and Binaglia, L. (1987)Biochim. Biophys. Acta 918, 40–47.
O, K.-M., Siow, Y.L., and Choy, P.C. (1989)Biochem. Cell Biol. 67, 680–686.
Kameyama, Y., Yoshioka, S., Hasegawa, I., and Nozawa, Y. (1981)Biochim. Biophys. Acta 665, 195–204.
Percy, A.K., Carson, M.A., Moore, J.F., and Waechter, C.J. (1984)Arch. Biochem. Biophys. 230, 69–81.
Coleman, B., and Bell, R.M. (1977)J. Biol. Chem. 252, 3050–3056.
Ansell, G.B., and Metcalfe, R.F. (1971)J. Neurochem. 18, 647–665.
Liteplo, R.G., and Sribney, M. (1977)Can. J. Biochem. 55, 1049–1056.
Cornell, R. (1989) inPhosphatidylcholine Metabolism (Vance, D.E., ed.) Chapter 4, pp. 47–64, CRC Press, Boca Raton.
Khan, Z.U., and Helmkamp, G.M., Jr. (1990)J. Biol. Chem. 265, 700–705.
Tijburg, L.B.M., Geelen, M.J.H., and van Golde, L.M.G. (1989)Biochim. Biophys. Acta 1004, 1–19.
Laemmli, U.K. (1970)Nature (Lond.) 227, 680–685.
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Ishidate, K., Matsuo, R. & Nakazawa, Y. 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. Lipids 28, 89–96 (1993). https://doi.org/10.1007/BF02535770
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DOI: https://doi.org/10.1007/BF02535770