Development of a quantitative biochemical and cellular sphingomyelin synthase assay using mass spectrometry

被引：8

作者：

Chen, YanQun ^{[1
]}

Yurek, David A. ^{[1
]}

Yu, Lan ^{[1
]}

Wang, He ^{[1
]}

Ehsani, Mariam E. ^{[1
]}

Qian, Yue-Wei ^{[1
]}

Konrad, Robert J. ^{[1
]}

Jiang, Xian-Cheng ^{[2
]}

Kuo, Ming-Shang ^{[1
]}

Cao, Guoqing ^{[1
]}

Wang, Jian ^{[1
]}

机构：

[1] Eli Lilly & Co, Lilly Res Labs, Indianapolis, IN 46285 USA

[2] Suny Downstate Med Ctr, Dept Anat & Cell Biol, Brooklyn, NY 11203 USA

来源：

ANALYTICAL BIOCHEMISTRY | 2013年 / 438卷 / 01期

关键词：

Sphingomyelin; Mass spectrometry; Assay; INCREASES; PLASMA; LIVER;

D O I：

10.1016/j.ab.2013.03.016

中图分类号：

Q5 [生物化学];

学科分类号：

071010 ; 081704 ;

摘要：

The last step in sphingolipid biosynthesis is the conversion of ceramide (Cer) to sphingomyelin (SM), which is catalyzed by sphingomyelin synthase (SMS). Two isoforms of SMS have been identified with differential subcellular localizations. It is not clear whether the two isoforms have any differences in biochemical or cellular SMS activities. This report describes a mass spectrometry (MS)-based method that was used to characterize biochemical and cellular SMS activities of the two isoforms of SMS, namely SMS1 and SMS2. Cellular extracts of SMSI or SMS2 expressed in SF9 cells displayed significant SMS activity. When these activities were measured by MS, both SMS1 and SMS2 demonstrated similar time- and substrate-dependent SMS activity. A previously reported SMS inhibitor, D609, inhibited both SMSI and SMS2 activity. In HEK293 cells, overexpression of either SMS1 or SMS2 significantly increased SMS activity. These studies using MS methods to measure SMS activity of SMSI and SMS2 represent the first quantitative measurement of SMS activities. The establishment of quantitative biochemical and cellular SMS assays may help to facilitate the discovery of novel SMS1- or SMS2-specific inhibitors. (C) 2013 Elsevier Inc. All rights reserved.

引用

页码：61 / 66

页数：6

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