Bleasdale J E, Johnston J M
Biochim Biophys Acta. 1982 Mar 12;710(3):377-90. doi: 10.1016/0005-2760(82)90121-7.
Rabbit lung microsomes were found to catalyze CMP-dependent incorporation of [14C]glycerol 3-phosphate into a total lipid extract. The radioactively labeled products in the lipid extract were identified as phosphatidylglycerol and phosphatidylglycerol phosphate. CMP-dependent incorporation of [14C]glycerol 3-phosphate by lung microsomes proceeded optimally at pH 7.4 and required Mn2+. The apparent Km value for CMP in this reaction was calculated to be 0.19 mM. No other cytidine nucleotide could substitute completely for CMP in supporting [14C]glycerol 3-phosphate incorporation into lipid. Cytosine-beta-D-arabinofuranoside-5'-monophosphate-dependent incorporation of [14C]glycerol 3-phosphate was observed at pH 8.5 but not at pH 6.8 CMP-dependent incorporation of [14C]glycerol 3-phosphate by microsomes was inhibited by inositol. The optimal in vitro rates of CMP-dependent and CDP diacylglycerol-dependent incorporation of [14C]glycerol 3-phosphate into lipid were similar (approximately 1 nmol . mg-1 protein . h-1) and were not additive. Both CMP -dependent and CDP diacylglycerol-dependent incorporation of [14C]glycerol 3-phosphate by lung microsomes appeared to involve CDPdiacylglycerol:glycerol-3-phosphate phosphatidyltransferase. However, the specific activity of this enzyme in a particular subcellular fraction did not relate directly in the extent of CMP-dependent [14C]glycerol 3-phosphate incorporation in that fraction. Preincubation of lung microsomes with 5 mM CMP plus 3 mM phosphatidylinositol increased CMP-dependent incorporation of [14C]glycerol 3-phosphate. When lung microsomes were depleted specifically of phosphatidylinositol by incubating with a phosphatidylinositol-specific phospholipase C, CMP-dependent incorporation was diminished. The Mn2+ requirement for CMP-dependent incorporation of [14C] glycerol 3-phosphate, its phosphatidylinositol requirement and its inhibition by Triton X-100 (0.2%) were not features shared by CDPdiacylglycerol-dependent incorporation of [14C]glycerol 3-phosphate but were characteristics of the reverse reaction catalyzed by CDPdiacylglycerol: inositol phosphatidyltransferase. Together with the previous finding of a developmental increase in the CMP content of fetal rabbit lung, these observations are consistent with a role for CMP in the regulation of the phosphatidylinositol and phosphatidylglycerol content of lung surfactant during lung maturation.
研究发现兔肺微粒体可催化[14C]甘油3-磷酸在CMP依赖下掺入总脂质提取物中。脂质提取物中的放射性标记产物被鉴定为磷脂酰甘油和磷脂酰甘油磷酸。肺微粒体催化的[14C]甘油3-磷酸在CMP依赖下的掺入在pH 7.4时最佳，且需要Mn2+。该反应中CMP的表观Km值经计算为0.19 mM。在支持[14C]甘油3-磷酸掺入脂质方面，没有其他胞嘧啶核苷酸能完全替代CMP。在pH 8.5时观察到胞嘧啶-β-D-阿拉伯呋喃糖苷-5'-单磷酸依赖的[14C]甘油3-磷酸掺入，但在pH 6.8时未观察到。微粒体催化的[14C]甘油3-磷酸在CMP依赖下的掺入受到肌醇的抑制。[14C]甘油3-磷酸在CMP依赖和CDP二酰甘油依赖下掺入脂质的最佳体外速率相似（约1 nmol·mg-1蛋白质·h-1）且无累加性。肺微粒体催化的[14C]甘油3-磷酸在CMP依赖和CDP二酰甘油依赖下的掺入似乎都涉及CDP二酰甘油：甘油-3-磷酸磷脂酰转移酶。然而，该酶在特定亚细胞组分中的比活性与该组分中CMP依赖的[14C]甘油3-磷酸掺入程度并无直接关联。肺微粒体与5 mM CMP加3 mM磷脂酰肌醇预孵育可增加[14C]甘油3-磷酸在CMP依赖下的掺入。当肺微粒体与磷脂酰肌醇特异性磷脂酶C孵育以特异性去除磷脂酰肌醇时，CMP依赖的掺入减少。[14C]甘油3-磷酸在CMP依赖下掺入所需的Mn2+、其对磷脂酰肌醇的需求以及其被Triton X-100（0.2%）抑制等特性并非[14C]甘油3-磷酸在CDP二酰甘油依赖下掺入所具有，而是CDP二酰甘油：肌醇磷脂酰转移酶催化的逆反应的特征。连同先前关于胎兔肺中CMP含量发育性增加的发现，这些观察结果与CMP在肺成熟过程中调节肺表面活性物质的磷脂酰肌醇和磷脂酰甘油含量方面的作用一致。
