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Binding of CTP:Phosphocholine Cytidylyltransferase to Lipid Vesicles:  Diacylglycerol and Enzyme Dephosphorylation Increase the Affinity for Negatively Charged Membranes

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Institute of Molecular Biology and Biochemistry and Department of Chemistry, Simon Fraser University, Burnaby, British Columbia, Canada, V5A 1S6, and Department of Biochemistry and Molecular Biology, Indiana University School of Medicine, Indianapolis, Indiana 46202
Cite this: Biochemistry 1997, 36, 20, 6149–6156
Publication Date (Web):May 20, 1997
https://doi.org/10.1021/bi970023z
Copyright © 1997 American Chemical Society

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    Abstract

    The regulation of membrane binding and activity of purified CDP:phosphocholine cytidylyltransferase (CT) by lipid activators and enzyme dephosphorylation was examined. The binding of CT to membranes was analyzed using sucrose-loaded vesicles (SLVs). Binding to phosphatidylcholine vesicles was not detected even at a lipid:protein ratio of ∼2000 (1 mM PC). CT bound to vesicles containing anionic lipids with apparent molar partition coefficients between 2 × 105 and 2 × 106, depending on the vesicle charge. The vesicle binding and activation of CT showed very similar sigmoidal dependencies on the lipid negative charge. In addition, diacylglycerol interacted synergistically with anionic phospholipids to stimulate both binding and activation at lower mole percent anionic lipid. These results demonstrate parallel requirements for binding and activity. Dephosphorylation of CT without destabilization was accomplished using the catalytic subunit of protein phosphatase 1. Dephosphorylated CT required a lower mole percent anionic phospholipid than phosphorylated CT for binding to and activation by SLVs. The combination of 10 mol % diacylglycerol and enzyme dephosphorylation shifted the mole percent phosphatidic acid required for half-maximal activation from 25% to 12%. These results suggest a mechanism whereby large changes in CT activity can result from changes in the phosphorylation state combined with small alterations in the membrane content of diacylglycerol. We propose a mechanism whereby dephosphorylation on the domain adjacent to the membrane binding domain increases the affinity of the latter for a negatively charged membrane surface.

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     This research was supported by a grant (12130) to R.B.C. from the Medical Research Council of Canada and by an NIH grant (DK36569) to A.A.D.-R.

     Simon Fraser University.

    §

     Present address:  Department of Biochemistry, Emory University School of Medicine, Atlanta, GA 30322.

     Indiana University School of Medicine.

    *

     To whom correspondence should be addressed. FAX:  (604) 291-5583. Email:  [email protected].

     Abstract published in Advance ACS Abstracts, May 1, 1997.

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