Nishijima M, Kuge O, Maeda M, Nakano A, Akamatsu Y
J Biol Chem. 1984 Jun 10;259(11):7101-8.
By means of an in situ autoradiographic assay for the base-exchange reaction of phospholipids with L-serine in Chinese hamster ovary cell colonies immobilized on filter paper ( Esko , J.D. and Raetz , C.R.H. (1978) Proc. Natl. Acad. Sci. U.S.A. 75, 1190-1193), a mutant (designated 89.1) was isolated in which the specific activity of the serine-exchange enzyme was about 2-fold less than in the parent. Unexpectedly, it was demonstrated that in extracts of the mutant the specific activities of both ATP:choline phosphotransferase (choline kinase) (EC 2.7.1.32) and the enzyme that catalyzes the base-exchange of phospholipids with choline (choline-exchange enzyme) were strikingly reduced (3- to 4-fold and 10- to 15-fold, respectively), while the specific activities of other enzymes of phosphatidylcholine synthesis were normal. Several lines of evidence presented here suggested that the partial defect of serine-exchange activity in this mutant was due to a decrease of acceptor phospholipid(s) for the reaction. The growth rates and phospholipid compositions of the mutant and parent were quite similar. However, mutant 89.1 exhibited a significant defect in its ability in vivo to synthesize phosphatidylcholine. The fact that the mutant was also defective in phosphorylcholine biosynthesis in vivo, together with the finding of an enzymatic lesion of the mutant in choline kinse in vitro as described above, clearly demonstrated that with respect to the reduced phosphatidylcholine biosynthesis the primary defect was at the level of choline kinase. In addition to the decreased synthetic rate of phosphatidylcholine, the turnover rate of phosphatidylcholine was also reduced approximately 2-fold in this mutant. These decreased rates of both synthesis and degradation of phosphatidylcholine probably account for the identical phosphatidylcholine contents between the mutant and parent. As a conclusion, it may be given that strain 89.1 is a pleiotropic mutant which possesses several alterations in phosphatidylcholine metabolism, and such mammalian mutants have not been isolated previously.
通过一种原位放射自显影分析方法，用于检测固定在滤纸上的中国仓鼠卵巢细胞集落中磷脂与L-丝氨酸的碱基交换反应（埃斯科，J.D.和雷茨，C.R.H.（1978年）《美国国家科学院院刊》75，1190 - 1193），分离出了一个突变体（命名为89.1），其中丝氨酸交换酶的比活性比亲本大约低2倍。出乎意料的是，研究表明在该突变体的提取物中，ATP：胆碱磷酸转移酶（胆碱激酶）（EC 2.7.1.32）以及催化磷脂与胆碱进行碱基交换的酶（胆碱交换酶）的比活性都显著降低（分别降低了3至4倍和10至15倍），而磷脂酰胆碱合成的其他酶的比活性正常。此处给出的几条证据表明，该突变体中丝氨酸交换活性的部分缺陷是由于反应的受体磷脂减少所致。突变体和亲本的生长速率和磷脂组成相当相似。然而，突变体89.1在体内合成磷脂酰胆碱的能力上表现出显著缺陷。该突变体在体内磷酸胆碱生物合成方面也存在缺陷，以及上述体外胆碱激酶中突变体的酶学损伤这一发现，清楚地表明就磷脂酰胆碱生物合成减少而言，主要缺陷在于胆碱激酶水平。除了磷脂酰胆碱合成速率降低外，该突变体中磷脂酰胆碱的周转率也大约降低了2倍。磷脂酰胆碱合成和降解速率的这些降低可能解释了突变体和亲本之间磷脂酰胆碱含量相同的原因。总之，可以得出结论，89.1菌株是一个多效性突变体，在磷脂酰胆碱代谢方面存在多种改变，并且此前尚未分离出这样的哺乳动物突变体。
