BINDING OF CTP-PHOSPHOCHOLINE CYTIDYLYLTRANSFERASE TO LIPID VESICLES - DIACYLGLYCEROL AND ENZYME DEPHOSPHORYLATION INCREASE THE AFFINITY FOR NEGATIVELY CHARGED MEMBRANES

The regulation of membrane binding and activity of purified CDP:phosph ocholine cytidylyltransferase (CT) by lipid activators and enzyme deph osphorylation was examined, The binding of CT to membranes was analyze d using sucrose-loaded vesicles (SLVs). Binding to phosphatidylcholine vesicles was not detected even at a lipid:protein ratio of similar to 2000 (1 mM PC), CT bound to vesicles containing anionic lipids with a pparent molar partition coefficients between 2 x 10(5) and 2 x 10(6), depending on the vesicle charge. The vesicle binding and activation of CT showed very similar sigmoidal dependencies on the lipid negative c harge. In addition, diacylglycerol interacted synergistically with ani onic phospholipids to stimulate both binding and activation at lower m ole percent anionic lipid. These results demonstrate parallel requirem ents for binding and activity. Dephosphorylation of CT without destabi lization was accomplished using the catalytic subunit of protein phosp hatase 1. Dephosphorylated CT required a lower mole percent anionic ph ospholipid than phosphorylated CT for binding to and activation by SLV s. The combination of 10 mol % diacylglycerol and enzyme dephosphoryla tion shifted the mole percent phosphatidic acid required for half-maxi mal activation from 25% to 12%, These results suggest a mechanism wher eby large changes in CT activity can result from changes in the phosph orylation state combined with small alterations in the membrane conten t of diacylglycerol. We propose a mechanism whereby dephosphorylation on the domain adjacent to the membrane binding domain increases the af finity of the latter for a negatively charged membrane surface.