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:phosphocholine cytidylyltransferase (CT), we determined the effect of oleate treatment 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 dehydrogenase. Oleate treatment caused a further decrease in CT release from cells. In untreated cells, 70-80% of the CT remaining in cells 10 min after permeabilization was recovered as soluble CT. Oleate treatment 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 control and oleate-treated cells, the increase in CT release with time correlated with a decrease in the amount of CT recovered from permeabilized cells as soluble CT. These results suggested that CT existed in a form that was not immediately available for release from permeabilized cells, but was recovered in the soluble fraction after cell disruption. When cells were permeabilized in 10 mM imidazole-20% glycerol-5 mM Mg2+ pH 6.5, over 80% of CT in control and over 90% of CT in oleate-treated cells was recovered bound to the particulate fraction. Essentially no CT was released from the cells. The recovery of CT in the particulate fraction required Mg2+ to be present when permeabilization was initiated. The addition of Mg2+, after cells were disrupted, did not increase CT in the particulate fraction. In untreated cells, 50% of bound CT was active. Oleate treatment increased the amount of active CT in the particulate fraction to over 70% of total. About 50% of particulate CT in untreated cells but only 15% in oleate-treated cells was extracted with 0.15 M KCl. Inactive CT was preferentially extracted by KCl. The bound CT was recovered in isolated nuclei. Overall, the results suggested that both inactive and active CT are bound to nuclear membranes, and that the activation of CT involves conversion of CT loosely bound to membrane to a form more tightly bound to membranes perhaps by hydrophobic interaction with phospholipids. This model does not involve translocation from a soluble pool.
Weinhold
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PA
Veterans Affairs Medical Center and Department of Biological Chemistry, University of Michigan Medical School, Ann Arbor, MI, USA. pwein@umich.edu
Barrett
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D
D001692:Biological Transport / Q000187:drug effects; D006528:Carcinoma, Hepatocellular; D002463:Cell Membrane Permeability / Q000187:drug effects; D019916:Choline-Phosphate Cytidylyltransferase / Q000493:pharmacokinetics*; D004789:Enzyme Activation / Q000187:drug effects; D006801:Humans; D007424:Intracellular Fluid / Q000201:enzymology; D008565:Membrane Proteins / Q000378:metabolism*; D008954:Models, Biological; D009685:Nuclear Envelope / Q000201:enzymology; D019301:Oleic Acid / Q000494:pharmacology; D012995:Solubility; D014407:Tumor Cells, Cultured
D008565:Membrane Proteins; D019301:Oleic Acid; C013173:osteum; D019916:Choline-Phosphate Cytidylyltransferase