Hanada K, Nishijima M, Kiso M, Hasegawa A, Fujita S, Ogawa T, Akamatsu Y
Department of Chemistry, National Institute of Health, Tokyo, Japan.
J Biol Chem. 1992 Nov 25;267(33):23527-33.
We previously isolated a temperature-sensitive Chinese hamster ovary cell mutant (strain SPB-1) with thermolabile serine palmitoyltransferase, which is involved in the first step of sphingolipid synthesis (Hanada, K., Nishijima, M., and Akamatsu, Y. (1990) J. Biol. Chem. 265, 22137-22142). In this study, sphingolipid-deficient culture medium was used to examine the effect of exogenous sphingolipids on the cell growth of SPB-1. When cultivated in the sphingolipid-deficient medium, SPB-1 cells ceased growing at non-permissive temperatures. Under these conditions, de novo sphingolipid synthesis ceased in the SPB-1 cells, resulting in a decrease in levels of sphingomyelin and ganglioside sialyl lactosylceramide (GM3), whereas the parental CHO-K1 cells grew logarithmically with normal sphingolipid synthesis. Exogenous sphingosine restored the contents of both sphingomyelin and GM3 in the SPB-1 cells near to the parental levels through metabolic utilization and allowed the mutant cells to grow even at the non-permissive temperature. Similarly, exogenous sphingomyelin restored the sphingomyelin levels and only partly the GM3 levels and also suppressed the temperature-sensitivity of the SPB-1 cell growth. In contrast, exogenous glucosylceramide, which restored the GM3 levels but not the sphingomyelin levels, failed to suppress the temperature sensitivity of the SPB-1 cell growth. Combination of exogenous sphingomyelin with ceramide, glucosylceramide, GM3, or sphingoid bases did not show any synergistic or additive effect on the SPB-1 cell growth enhancement, compared with sphingomyelin alone. The results indicated that the temperature sensitivity of the SPB-1 cell growth was due to the lack of cellular sphingolipids, possibly that of sphingomyelin.
我们之前分离出了一种对温度敏感的中国仓鼠卵巢细胞突变体（SPB - 1株），其丝氨酸棕榈酰转移酶不耐热，该酶参与鞘脂合成的第一步（花田和夫、西岛正、赤松洋（1990年）《生物化学杂志》265卷，22137 - 22142页）。在本研究中，使用缺乏鞘脂的培养基来检测外源性鞘脂对SPB - 1细胞生长的影响。当在缺乏鞘脂的培养基中培养时，SPB - 1细胞在非允许温度下停止生长。在这些条件下，SPB - 1细胞中从头合成鞘脂的过程停止，导致鞘磷脂和神经节苷脂唾液酸乳糖神经酰胺（GM3）水平下降，而亲本CHO - K1细胞以正常的鞘脂合成对数生长。外源性鞘氨醇通过代谢利用使SPB - 1细胞中鞘磷脂和GM3的含量恢复到接近亲本水平，并使突变细胞即使在非允许温度下也能生长。同样，外源性鞘磷脂恢复了鞘磷脂水平，仅部分恢复了GM3水平，并且也抑制了SPB - 1细胞生长的温度敏感性。相比之下，外源性葡萄糖神经酰胺恢复了GM3水平但未恢复鞘磷脂水平，未能抑制SPB - 1细胞生长的温度敏感性。与单独使用鞘磷脂相比，外源性鞘磷脂与神经酰胺、葡萄糖神经酰胺、GM3或鞘氨醇碱基的组合对SPB - 1细胞生长增强没有显示出任何协同或相加作用。结果表明，SPB - 1细胞生长的温度敏感性是由于细胞内鞘脂缺乏，可能是鞘磷脂缺乏所致。
