Subcellular study of sphingoid base phosphorylation in rat tissues: evidence for multiple sphingosine kinases
Subcellular study of sphingoid base phosphorylation in rat tissues: evidence for multiple sphingosine kinases
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DOI：
10.1016/s1388-1981(01)00111-1
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发表时间：
2001-05-31
影响因子：
4.8
通讯作者：
Van Veldhoven, PP
中科院分区：
文献类型：
--
作者：
Gijsbers, S;Van der Hoeven, G;Van Veldhoven, PP
以D-β-[4，5-H-3]二氢鞘氨醇为底物，分析了大鼠组织中鞘氨醇碱的酶促磷酸化。优化后的测定，注意阻断鞘氨醇磷酸裂解酶和鞘氨醇磷酸酶，最高的ATP依赖性激酶活性存在于睾丸，其次是肾脏和肠粘膜。大约三分之二的肾脏活动是膜结合的，其余的是胞质的。肾脏和肝脏的经典细胞分级分离研究不能明确识别膜结合激酶的亚细胞位点。通过Percoll梯度和蔗糖密度梯度离心从肾匀浆中分离颗粒部分，发现激酶活性与来自内质网和质膜的囊泡相关。基于间接数据，如去污剂和二价离子的影响，细胞溶质和膜结合活性似乎存在于不同的蛋白质中。N，N-二甲基鞘氨醇对所有三种不同的激酶都有抑制作用，这些激酶主要对鞘氨醇和鞘氨醇的D-邻位异构体有活性。(C)2001爱思唯尔科技有限公司。保留所有权利。
The enzymatic phosphorylation of sphingoid bases was analysed in rat tissues, using D-erythro-[4,5-H-3]sphinganine as substrate. After optimisation of the assay, taking care to block sphingosine-phosphate lyase and sphingosine phosphatase, highest ATP-dependent kinase activities were present in testis, followed by kidney, and intestinal mucosa. Approximately two thirds of the kidney activity were membrane bound, the remaining being cytosolic. Classical cell fractionation studies of kidney and liver did not allow to identify unequivocally the subcellular site of the membrane bound kinase. Separation of a particulate fraction from kidney homogenates by Percoll gradient and sucrose density gradient centrifugation revealed that kinase activities are associated with vesicles derived from the endoplasmic reticulum and the plasma membrane. Based on indirect data, such as the effect of detergents and divalent ions, the cytosolic and both membrane bound activities appear to reside in different proteins. N,N-Dimethylsphingenine was inhibitory to all three different kinases, which were mainly active towards the D-erythro isomers of sphingenine and sphinganine. (C) 2001 Elsevier Science B.V. All rights reserved.