STUDIES ON THE REGULATION OF CTP-PHOSPHOCHOLINE CYTIDYLYLTRANSFERASE USING PERMEABILIZED HEP G2 CELLS - EVIDENCE THAT BOTH ACTIVE AND INACTIVE ENZYME ARE MEMBRANE-BOUND

To obtain more insight into the mechanisms regulating CTP:phosphocholi ne cytidylyltransferase (CT), we determined the effect of oleate treat ment on the rate of CT release from permeabilized Hep G2 cells and the distribution of the CT remaining in the permeabilized cells. When we permeabilized untreated cells in pH 7.5 buffer containing 0.15 M KCl, the rate of CT release was much slower than the release of lactate deh ydrogenase. Oleate treatment caused a further decrease in CT release f rom cells. In untreated cells, 70-80% of the CT remaining in cells 10 min after permeabilization was recovered as soluble CT. Oleate treatme nt increased the amount of bound CT but over 50% of the CT in cells 10 min after permeabilization was recovered as soluble CT. In both contr ol and oleate-treated cells, the increase in CT release with time corr elated with a decrease in the amount of CT recovered from permeabilize d cells as soluble CT. These results suggested that CT existed in a fo rm that was not immediately available for release from permeabilized c ells, but was recovered in the soluble fraction after cell disruption. When cells were permeabilized in 10 mM imidazole-20% glycerol-5 mM Mg 2+ pH 6.5, over 80% of CT in control and over 90% of CT in oleate-trea ted cells was recovered bound to the particulate fraction. Essentially no CT was released from the cells. The recovery of CT in the particul ate fraction required Mg2+ to be present when permeabilization was ini tiated, The addition of Mg2+, after cells were disrupted, did not incr ease CT in the particulate fraction. In untreated cells, 50% of bound CT was active. Oleate treatment increased the amount of active CT in t he particulate fraction to over 70% of total. About 50% of particulate CT in untreated cells but only 15% in oleate-treated cells was extrac ted with 0.15 M KCl. Inactive CT was preferentially extracted by KCI. The bound CT was recovered in isolated nuclei. Overall, the results su ggested that both inactive and active CT are bound to nuclear membrane s? and that the activation of CT involves conversion of CT loosely bou nd to membrane to a form more tightly bound to membranes perhaps by hy drophobic interaction with phospholipids. This model does not involve translocation from a soluble pool. (C) 1998 Elsevier Science B.V.