Rapid replenishment of sphingomyelin in the plasma membrane upon degradation by sphingomyelinase in NIH3T3 cells overexpressing the phosphatidylinositol transfer protein beta.
Rapid replenishment of sphingomyelin in the plasma membrane upon degradation by sphingomyelinase in NIH3T3 cells overexpressing the phosphatidylinositol transfer protein beta.
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在过度表达磷脂酰肌醇转移蛋白β的 NIH3T3 细胞中，鞘磷脂酶降解后质膜中的鞘磷脂快速补充。
DOI：
10.1042/0264-6021:3460537
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发表时间：
2000
期刊：
影响因子：
--
通讯作者：
Wirtz,KW
中科院分区：
文献类型：
--
作者：
VanTiel,CM;Luberto,C;Snoek,GT;Hannun,YA;Wirtz,KW
为研究磷脂酰肌醇转移蛋白β（PI-TP β）在体内的功能，将编码PI-TP β的cDNA转染小鼠NIH 3T3成纤维细胞。分离到两个稳定的细胞系（SPI β 2和SPI β 8），其中PI-TP β水平分别增加了16倍和11倍。SPI β细胞的倍增时间约为野生型（wt）细胞的1.7倍。由于PI-TP β在体外表达对鞘磷脂（SM）的转移活性，因此研究了过表达的SM代谢。通过测定SM和磷脂酰胆碱（PtdCho）中氯化[甲基-3H]胆碱的掺入，表明转染细胞和野生型细胞中SM和PtdCho的合成速率相似。我们还确定了细胞的能力，以resynthesize SM从神经酰胺产生的质膜的作用，细菌鞘磷脂酶（bSMase）。在这些实验中，用[3H]胆碱将细胞标记至平衡（60 h）。在相对较低的bSMase浓度（50 munits/ml）下，wt NIH 3T3细胞中50%的[3H] SM被降解，而SPI β细胞中的[3H] SM水平似乎不受影响。由于wt NIH 3T3和SPI β细胞向培养基中释放的[3H]磷酸胆碱相当，因此这些结果强烈表明SPI β细胞中SM的分解被来自所形成的神经酰胺的SM的快速再合成所掩盖。当bSMase浓度增加到200 munits/ml时，SPI β细胞中[3H] SM的水平降低了50%。在6小时的恢复期（不存在bSMase）期间，发现这些SPI β细胞中SM的再合成比50%[3H] SM耗尽的wt NIH 3T3细胞中更明显。回收6 h后，约50%的SPI β细胞中再合成的SM可用于bSMase的第二次水解。当在恢复期存在莫能菌素时，bSMase处理的SPI β细胞中SM的再合成不受影响。然而，在这些条件下，100%的再合成SM可用于水解。基于这些结果，我们提出，在神经酰胺在质膜中形成的条件下，PI-TP β在恢复SM的稳态水平中起重要作用。
In order to study thein vivofunction of the phosphatidylinositol transfer protein β (PI-TPβ), mouse NIH3T3 fibroblasts were transfected with cDNA encoding mouse PI-TPβ. Two stable cell lines were isolated (SPIβ2 and SPIβ8) in which the levels of PI-TPβ were increased 16- and 11-fold respectively. The doubling time of the SPIβ cells was about 1.7 times that of the wild-type (wt) cells. Because PI-TPβ expresses transfer activity towards sphingomyelin (SM)in vitro, the SM metabolism of the overexpressors was investigated. By measuring the incorporation of [methyl-3H]choline chloride in SM and phosphatidylcholine (PtdCho), it was shown that the rate ofde novoSM and PtdCho synthesis was similar in transfected and wt cells. We also determined the ability of the cells to resynthesize SM from ceramide produced in the plasma membrane by the action of bacterial sphingomyelinase (bSMase). In these experiments the cells were labelled to equilibrium (60 h) with [3H]choline. At relatively low bSMase concentrations (50 munits/ml), 50% of [3H]SM in wt NIH3T3 cells was degraded, whereas the levels of [3H]SM in SPIβ cells appeared to be unaffected. Since the release of [3H]choline phosphate into the medium was comparable for both wt NIH3T3 and SPIβ cells, these results strongly suggest that breakdown of SM in SPIβ cells was masked by rapid resynthesis of SM from the ceramide formed. By increasing the bSMase concentrations to 200 munits/ml, a 50% decrease in the level of [3H]SM in SPIβ cells was attained. During a recovery period of 6 h (in the absence of bSMase) the resynthesis of SM was found to be much more pronounced in these SPIβ cells than in 50% [3H]SM-depleted wt NIH3T3 cells. After 6 h of recovery about 50% of the resynthesized SM in the SPIβ cells was available for a second hydrolysis by bSMase. When monensin was present during the recovery period, the resynthesis of SM in bSMase-treated SPIβ cells was not affected. However, under these conditions 100% of the resynthesized SM was available for hydrolysis. On the basis of these results we propose that, under conditions where ceramide is formed in the plasma membrane, PI-TPβ plays an important role in restoring the steady-state levels of SM.
影响因子：
4.1
作者：
DEVRIES, KJ;HEINRICHS, AAJ;SNOEK, GT
通讯作者：
SNOEK, GT
有证据表明哺乳动物磷脂酰肌醇转移蛋白调节磷脂酰胆碱代谢。
DOI：
10.1042/bj3350175
发表时间：
1998
期刊：
The Biochemical journal
影响因子：
--
作者：
M. Monaco;R. J. Alexander;G. Snoek;N. Moldover;K. Wirtz;P. Walden
通讯作者：
P. Walden