Monaco M E, Alexander R J, Snoek G T, Moldover N H, Wirtz K W, Walden P D
DVA Medical Center (151A), 423 East 23rd Street, New York, NY 10010, USA.
Biochem J. 1998 Oct 1;335 ( Pt 1)(Pt 1):175-9. doi: 10.1042/bj3350175.
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.
磷脂酰肌醇转移蛋白（PITPs）及其酵母对应物（SEC14p）具有在体外结合磷脂酰肌醇（PtdIns）并在膜之间转移它的能力。然而，这些蛋白在体内的生化功能尚不清楚。在本研究中，通过确定降低该蛋白细胞含量的生化后果来研究PITP的生理作用。用一个含有以反义方向插入的全长大鼠PITPα cDNA的质粒转染WRK-1大鼠乳腺肿瘤细胞，并对所得细胞克隆进行分析。将三个表达PITPα反义mRNA的克隆与三个用缺乏插入片段的表达载体转染的克隆进行比较。这三个反义克隆的PITPα蛋白平均比对照克隆少25%。三个反义克隆中的两个也表现出生长速率降低。在90分钟的孵育期内，所有三个反义克隆在将标记前体掺入PtdCho中的量均显著减少。然而，在相同条件下，前体掺入PtdIns中的量没有变化。进一步的实验表明，反义克隆中观察到的前体掺入减少不是由于周转率增加所致。当使用平衡标记条件（孵育48小时）在一个对照克隆（2-5）和一个反义克隆（4-B）中更广泛地分析胆碱代谢时，观察到以下情况：（1）在短期实验中看到的PtdCho放射性标记减少在长期实验中也观察到，这表明反义转染克隆中PtdCho的总量较低（这通过质量测量得到证实）；（2）在细胞鞘磷脂、溶血PtdCho和甘油磷酸胆碱中观察到类似的减少；（3）在反义转染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；以及（5）细胞CDP胆碱没有显著改变。
