Characterization of murine sphingosine-1-phosphate phosphohydrolase
Characterization of murine sphingosine-1-phosphate phosphohydrolase
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DOI：
10.1074/jbc.m109968200
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发表时间：
2002-03-15
影响因子：
4.8
通讯作者：
Spiegel, S
中科院分区：
文献类型：
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作者：
Le Stunff, H;Peterson, C;Spiegel, S
在本研究中，我们的特点是哺乳动物鞘氨醇-1-磷酸水解酶（SPP 1），一种酶，特异性去磷酸化鞘氨醇1-磷酸（SIP）和不同于以前描述的脂质磷酸磷酸水解酶。基于与鼠SPP 1的序列同源性，我们克隆了人同源物。用鼠或人SPP 1转染人胚肾293和中国仓鼠卵巢细胞导致用于检查其酶学性质的膜组分中SPP 1活性显著增加。与其他已知的2型脂质磷酸磷酸水解酶（LPPs）不同，但与酵母直系同源物相似，哺乳动物SPP 1对长链鞘氨醇碱磷酸具有高度特异性，并降解SIP、二氢SIP和植物S1 P。SPP 1表现出明显的Michaelis-Menten动力学与SIP作为基板的表观K-m为38.5妈妈和最佳活性在pH 7.5。与其他LPPs类似，SPP 1活性也不依赖于任何阳离子需求，包括Mg 2+，并且不受EDTA抑制，但受NaF和Zn 2+显著抑制。然而，SPP 1与LPPs相比具有一些显著不同的酶学性质：脂肪族阳离子心得安是SPP 1的有效抑制剂，它是1型磷脂酸磷酸水解酶活性的有效抑制剂，并且作为LPPs的抑制剂仅是适度有效的，是SPP 1的有效抑制剂;重要的是，低浓度的Triton X-100和其它非离子去污剂具有强烈的抑制作用。因此，与聚类分析一致，聚类分析显示，在共有基序之外，SPP 1与其他2型脂质磷酸水解酶之间的同源性非常小，SPP 1与哺乳动物LPP显著不同且不同。
In the present study we have characterized mammalian sphingosine-1-phosphate phosphohydrolase (SPP1), an enzyme that specifically dephosphorylates sphingosine 1-phosphate (SIP) and which differs from previously described lipid phosphate phosphohydrolases. Based on sequence homology to murine SPP1, we cloned the human homolog. Transfection of human embryonic kidney 293 and Chinese hamster ovary cells with murine or human SPP1 resulted in marked increases in SPP1 activity in membrane fractions that were used to examine its enzymological properties. Unlike other known type 2 lipid phosphate phosphohydrolases (LPPs), but similar to the yeast orthologs, mammalian SPP1s are highly specific toward long chain sphingoid base phosphates and degrade SIP, dihydro-SIP, and phyto-S1P. SPP1 exhibited apparent Michaelis-Menten kinetics with SIP as substrate with an apparent K-m of 38.5 mum and optimum activity at pH 7.5. Similar to other LPPs, SPP1 activity was also independent of any cation requirements, including Mg2+, and was not inhibited by EDTA but was markedly inhibited by NaF and Zn2+. However, SPP1 has some significantly different enzymological properties than the LPPs: the aliphatic cation propanolol, which is an effective inhibitor of type 1 phosphatidate phosphohydrolase activities and is only modestly effective as an inhibitor of LPPs, is a potent inhibitor of SPP1; the activity was partially sensitive to N-ethylmaleimide but not to the thioreactive compound iodoacetamide; and importantly, low concentrations of Triton X-100 and other non-ionic detergents were strongly inhibitory. Thus, in agreement with Cluster analysis which shows that outside of the consensus motif there is very little homology between SPP1s and the other type 2 lipid phosphohydrolases, SPP1s are significantly different and divergent from the mammalian LPPs.