Nishijima M, Kuge O, Akamatsu Y
J Biol Chem. 1986 May 5;261(13):5784-9.
The effect of phosphatidylserine exogenously added to the medium on de novo biosynthesis of phosphatidylserine was investigated in cultured Chinese hamster ovary cells. When cells were cultured for several generations in medium supplemented with phosphatidylserine and 32Pi, the incorporation of 32Pi into cellular phosphatidylserine was remarkably inhibited, the degree of inhibition being dependent upon the concentration of added phosphatidylserine. 32Pi uptake into cellular phosphatidylethanolamine was also partly reduced by the addition of exogenous phosphatidylserine, consistent with the idea that phosphatidylethanolamine is biosynthesized via decarboxylation of phosphatidylserine. However, incorporation of 32Pi into phosphatidylcholine, sphingomyelin, and phosphatidylinositol was not significantly affected. In contrast, the addition of either phosphatidylcholine, sphingomyelin, phosphatidylethanolamine, or phosphatidylinositol to the medium did not inhibit endogenous biosynthesis of the corresponding phospholipid. Radiochemical and chemical analyses of the cellular phospholipid composition revealed that phosphatidylserine in cells grown with 80 microM phosphatidylserine was almost entirely derived from the added phospholipid. Phosphatidylserine uptake was also directly determined by using [3H]serine-labeled phospholipid. Pulse and pulse-chase experiments with L-[U-14C] serine showed that when cells were cultured with 80 microM phosphatidylserine, the rate of synthesis of phosphatidylserine was reduced 3-5-fold whereas the turnover of newly synthesized phosphatidylserine was normal. Enzyme assaying of extracts prepared from cells grown with and without phosphatidylserine indicated that the inhibition of de novo phosphatidylserine biosynthesis by the added phosphatidylserine appeared not to be caused by a reduction in the level of the enzyme involved in the base-exchange reaction between phospholipids and serine. These results demonstrate that exogenous phosphatidylserine can be efficiently incorporated into Chinese hamster ovary cells and utilized for membrane biogenesis, endogenous phosphatidylserine biosynthesis thereby being suppressed.
研究了向培养基中外源添加磷脂酰丝氨酸对培养的中国仓鼠卵巢细胞中磷脂酰丝氨酸从头生物合成的影响。当细胞在补充有磷脂酰丝氨酸和32Pi的培养基中培养几代时，32Pi掺入细胞磷脂酰丝氨酸的过程受到显著抑制，抑制程度取决于添加的磷脂酰丝氨酸的浓度。添加外源磷脂酰丝氨酸也会部分降低细胞磷脂酰乙醇胺对32Pi的摄取，这与磷脂酰乙醇胺通过磷脂酰丝氨酸脱羧生物合成的观点一致。然而，32Pi掺入磷脂酰胆碱、鞘磷脂和磷脂酰肌醇的过程没有受到显著影响。相反，向培养基中添加磷脂酰胆碱、鞘磷脂、磷脂酰乙醇胺或磷脂酰肌醇不会抑制相应磷脂的内源性生物合成。对细胞磷脂组成的放射化学和化学分析表明，在含有80微摩尔磷脂酰丝氨酸的培养基中生长的细胞中的磷脂酰丝氨酸几乎完全来自添加的磷脂。还使用[3H]丝氨酸标记的磷脂直接测定了磷脂酰丝氨酸的摄取。用L-[U-14C]丝氨酸进行的脉冲和脉冲追踪实验表明，当细胞在含有80微摩尔磷脂酰丝氨酸的培养基中培养时，磷脂酰丝氨酸的合成速率降低了3至5倍，而新合成的磷脂酰丝氨酸的周转正常。对在有和没有磷脂酰丝氨酸的培养基中生长的细胞制备的提取物进行酶活性测定表明，添加的磷脂酰丝氨酸对磷脂酰丝氨酸从头生物合成的抑制似乎不是由参与磷脂与丝氨酸之间碱基交换反应的酶水平降低引起的。这些结果表明，外源磷脂酰丝氨酸可以有效地掺入中国仓鼠卵巢细胞并用于膜生物合成，从而抑制内源性磷脂酰丝氨酸的生物合成。
