Hogan M, Zimmermann L J, Wang J, Kuliszewski M, Liu J, Post M
Department of Paediatrics, Hospital for Sick Children Research Institute, University of Toronto, Ontario, Canada.
Am J Physiol. 1994 Jul;267(1 Pt 1):L25-32. doi: 10.1152/ajplung.1994.267.1.L25.
We previously reported that phosphatidylcholine synthesis increased in fetal rat lung type II cells with advancing gestation. This increase was accompanied by an increase in CTP:phosphocholine cytidylyltransferase activity, which catalyses a rate regulatory step in de novo phosphatidylcholine synthesis by fetal type II cells. To determine whether this increase in cytidylyltransferase activity is due to an increase in cytidylyltransferase protein levels, the gene and protein expression of cytidylyltransferase was investigated in maturing type II cells. The cytidylyltransferase cDNA was cloned from fetal rat type II cells and showed 99% sequence homology with rat liver cDNA. The cDNA detected two mRNA transcripts (1.8 and 7.5 kb) in fetal rat lung. By reverse-transcriptase polymerase chain reaction (RT-PCR) analysis, cytidyltransferase mRNA content increased three-fold in fetal type II cells with advancing gestation, whereas cytidylyltransferase mRNA levels in fibroblasts remained constant. An antibody against rat liver cytidylyltransferase was used to assess cytidylyltransferase protein. Western blotting revealed that cytidylyltransferase protein content increased threefold in the microsomal fraction of type II cells with advancing gestation. The enzyme protein levels in the cytosolic fraction did not significantly change with development. Enzyme activity studies confirmed these latter observations. We conclude that the increase in surfactant phosphatidylcholine synthesis by type II cells at late fetal gestation is due in part to an increase in the amount of cytidylyltransferase protein.
我们先前报道，随着胎龄增加，胎鼠肺II型细胞中磷脂酰胆碱合成增加。这种增加伴随着CTP：磷酸胆碱胞苷转移酶活性的增加，该酶催化胎鼠II型细胞从头合成磷脂酰胆碱过程中的一个速率调节步骤。为了确定胞苷转移酶活性的增加是否归因于胞苷转移酶蛋白水平的增加，我们研究了成熟II型细胞中胞苷转移酶的基因和蛋白表达。从胎鼠II型细胞中克隆出胞苷转移酶cDNA，其与大鼠肝脏cDNA的序列同源性为99%。该cDNA在胎鼠肺中检测到两种mRNA转录本（1.8和7.5 kb）。通过逆转录聚合酶链反应（RT-PCR）分析，随着胎龄增加，胎鼠II型细胞中胞苷转移酶mRNA含量增加了三倍，而成纤维细胞中胞苷转移酶mRNA水平保持不变。使用抗大鼠肝脏胞苷转移酶的抗体来评估胞苷转移酶蛋白。蛋白质印迹法显示，随着胎龄增加，II型细胞微粒体部分的胞苷转移酶蛋白含量增加了三倍。胞质部分的酶蛋白水平在发育过程中没有显著变化。酶活性研究证实了这些观察结果。我们得出结论，胎鼠妊娠后期II型细胞表面活性物质磷脂酰胆碱合成的增加部分归因于胞苷转移酶蛋白量的增加。
