Evidence that mammalian phosphatidylinositol transfer protein regulates phosphatidylcholine metabolism.
Evidence that mammalian phosphatidylinositol transfer protein regulates phosphatidylcholine metabolism.
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有证据表明哺乳动物磷脂酰肌醇转移蛋白调节磷脂酰胆碱代谢。
DOI：
10.1042/bj3350175
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发表时间：
1998
期刊：
影响因子：
--
通讯作者：
P. Walden
中科院分区：
文献类型：
--
作者：
M. Monaco;R. J. Alexander;G. Snoek;N. Moldover;K. Wirtz;P. Walden
磷脂酰肌醇转移蛋白（PITP）及其酵母对应物（SEC 14 p）具有结合磷脂酰肌醇（PtdIns）并在体外在膜之间转移的能力。然而，这些蛋白在体内的生物化学功能尚不清楚。在本研究中，通过测定降低这种蛋白质的细胞含量的生化后果来研究PITP的生理作用。用含有以反义方向插入的全长大鼠PITPalpha cDNA的质粒转染WRK-1大鼠乳腺肿瘤细胞，并分析所得细胞克隆。将表达PITPalpha反义mRNA的三个克隆与用缺乏插入片段的表达载体转染的三个克隆进行比较。三个反义克隆具有比对照克隆平均少25%的PITPalpha蛋白。三个反义克隆中的两个也表现出生长速率降低。所有三个反义克隆表现出显着减少标记的前体掺入PtdCho在90分钟的孵育期。然而，在相同的条件下，前体掺入PtdIn中没有变化。进一步的实验表明，在反义克隆中观察到的前体掺入的减少不是由于周转率的增加。当使用平衡标记条件在一个对照（2-5）和一个反义（4-B）克隆中更广泛地分析胆碱代谢时（孵育48小时），观察到以下结果：（1）在短期实验中观察到的PtdCho放射性标记的减少也在长期实验中观察到，这表明在反义转染的克隆中PtdCho的总量较低（2）在细胞鞘磷脂、lysoPtdCho和甘油磷酸胆碱中观察到类似的减少;（3）在反义转染的克隆中观察到细胞磷酸胆碱平均增加两倍;（4）细胞胆碱平均减少;（5）细胞CDP胆碱没有显著改变。
Phosphatidylinositol transfer proteins (PITPs) and their yeast counterpart (SEC14p) possess the ability to bind phosphatidylinositol (PtdIns) and transfer it between membranes in vitro. However, the biochemical function of these proteins in vivo is unclear. In the present study, the physiological role of PITP was investigated by determining the biochemical consequences of lowering the cellular content of this protein. WRK-1 rat mammary tumour cells were transfected with a plasmid containing a full-length rat PITPalpha cDNA inserted in the antisense orientation and the resultant cell clones were analysed. Three clones expressing antisense mRNA for PITPalpha were compared with three clones transfected with the expression vector lacking the insert. The three antisense clones had an average of 25% less PITPalpha protein than control clones. Two of the three antisense clones also exhibited a decreased rate of growth. All three antisense clones exhibited a significant decrease in the incorporation of labelled precursors into PtdCho during a 90-min incubation period. Under the same conditions, however, there was no change in precursor incorporation into PtdIns. Further experimentation indicated that the decrease in precursor incorporation seen in antisense clones was not due to an increased rate of turnover. When choline metabolism was analysed more extensively in one control (2-5) and one antisense (4-B) clone using equilibrium-labelling conditions (48 h of incubation), the following were observed: (1) the decrease in radioactive labelling of PtdCho seen in short-term experiments was also observed in long-term experiments, suggesting that the total amount of PtdCho was lower in antisense-transfected clones (this was confirmed by mass measurements); (2) a similar decrease was seen in cellular sphingomyelin, lysoPtdCho and glycerophosphorylcholine; (3) an average two-fold increase in cellular phosphorylcholine was observed in the antisense-transfected clone; (4) cellular choline was, on average, decreased; and (5) cellular CDPcholine was not significantly altered.