Weinhold P A, Charles L, Feldman D A
VA Medical Center, Ann Arbor, MI.
Biochim Biophys Acta. 1994 Jan 20;1210(3):335-47. doi: 10.1016/0005-2760(94)90238-0.
We studied the effect of choline depletion on the biosynthesis of phosphatidylcholine (PC) and the distribution and phosphorylation of cytidylyltransferase (CT) in HepG2 cells. Phosphocholine concentrations decreased within 24 h of choline depletion to values less than 2% of controls. The incorporation of [3H]glycerol into PC was reduced in choline-depleted (CD) cells. The apparent turnover of PC was similar in CD and choline-supplemented (CS) cells (T1/2 = 20 h). The methylation pathway for PC synthesis increased nearly 10-fold in CD cells. Cell growth was similar in CD and CS cells. Over 95% of CT activity in CS cells was in the soluble pool. Choline depletion resulted in a progressive decrease in CT activity and immunodetected enzyme in the soluble pool and a corresponding increase in membrane CT over a 48-h period. Choline supplementation of CD cells caused a rapid release of membrane CT (complete release by 3 h). Two phosphorylated forms of CT were identified. One form contained a higher level of phosphorylation (HPCT) than the other form (LPCT). HPCT migrated slightly slower than LPCT on SDS gels. CD cells contained only LPCT in both soluble and membrane pools. CS cells contained only HPCT. During choline depletion PC content decreased nearly 20% but CT binding did not occur until LPCT was generated in cytosol. Conversely, choline supplementation released LPCT into cytosol and HPCT was formed only after the release. We conclude that both the induction of binding sites, perhaps by depletion of PC and dephosphorylation of HPCT to LPCT, are required for CT translocation to membranes. The release of CT from membranes is initiated by changes in membrane binding sites followed by trapping of the CT in the soluble pool by phosphorylation of LPCT to HPCT.
我们研究了胆碱缺乏对HepG2细胞中磷脂酰胆碱（PC）生物合成以及胞苷酰转移酶（CT）分布和磷酸化的影响。胆碱缺乏24小时内，磷酸胆碱浓度降至低于对照值2%的水平。在胆碱缺乏（CD）的细胞中，[3H]甘油掺入PC的量减少。CD细胞和胆碱补充（CS）细胞中PC的表观周转率相似（半衰期=20小时）。CD细胞中PC合成的甲基化途径增加了近10倍。CD细胞和CS细胞的生长相似。CS细胞中超过95%的CT活性存在于可溶性组分中。胆碱缺乏导致可溶性组分中CT活性和免疫检测到的酶逐渐降低，同时在48小时内膜CT相应增加。向CD细胞补充胆碱导致膜CT迅速释放（3小时内完全释放）。鉴定出CT的两种磷酸化形式。一种形式的磷酸化水平高于另一种形式（HPCT）。在SDS凝胶上，HPCT的迁移速度略慢于LPCT。CD细胞的可溶性和膜组分中仅含有LPCT。CS细胞仅含有HPCT。胆碱缺乏期间，PC含量下降近20%，但直到胞质溶胶中产生LPCT才发生CT结合。相反，补充胆碱使LPCT释放到胞质溶胶中，并且仅在释放后才形成HPCT。我们得出结论，CT向膜的转位需要结合位点的诱导，可能是通过PC的消耗以及HPCT去磷酸化为LPCT。CT从膜上的释放是由膜结合位点的变化引发的，随后LPCT磷酸化为HPCT将CT捕获在可溶性组分中。
