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  • Phosphatase Inhibitor Cocktail 2 (100X in ddH2O) br Developm

    2020-02-21


    Development of a liquid chromatography-mass spectrometry method to improve detection of PA species detection Liquid chromatography-mass spectrometry (LC-MS) is a powerful tool to detect different molecular species of phospholipids in Phosphatase Inhibitor Cocktail 2 (100X in ddH2O) (Houjou et al., 2005, Pulfer and Murphy, 2003). However, there were still problems detecting PA molecular species using LC-MS. Because PA is a minor component of phospholipids and contains a variety of fatty acids, extensively broad PA peaks inevitably overlap with other major phospholipid peaks, causing ion suppression, which leads to inferior detection, quantification and reproducibility. Therefore, we optimized LC conditions using a silica column LC and mobile phases containing high concentrations of ammonia (Mizuno et al., 2012). To test the developed LC-MS method, we examined phospholipid mixtures from various mammalian cells and confirmed that PA species from m/z 591.41 (14:0/14:0-PA) to m/z 759.59 (18:0/22:0-PA) were quantitatively and reproducibly detected.
    DG species utilized by DGK isozymes
    Targeting of individual DG and PA molecular species
    1-Monoacylglycerol (MG) kinase (MGK) and 2-MGK activities of DGK isozymes We have recently revealed that type I DGKs (α, β and γ), type II DGKs (δ, η and κ) and type III DGK (ε) have 8–19% 2-MGK activity, which produces 2-lysoPA (LPA), compared to their DGK activities in vitro, whereas their 1-MGK activities were less than 3% (Sato et al., 2016). Both the 2-MGK and 1-MGK activities of the type IV DGKs (ζ and ι) were less than 1% of the corresponding DGK activity. Interestingly, the type V DGKθ has approximately 6% 1-MGK activity, which generates 1-LPA, and less than 2% 2-MGK activity, compared to DGK activity. These results indicate that the ten DGK isozymes are more enzymatically diverse than expected, and demonstrate a new aspect of DGK. The simultaneous production of PA molecular species with/without 1-LPA or 2-LPA adds complexity to products and may be important to maximize a variety of physiological functions of DGKs.
    Conclusion Unlike the dogma described above, our recent studies strongly suggest that DGK isozymes utilize not only PI turnover-derived DG species but also various DG species derived from pathways independent of PI turnover. Different DG species supplied form distinct pathways may be utilized by different DGK isozymes based on different stimuli present in different types of cells, and individual PA/DG molecular species would have specific targets and exert their own physiological functions. The DG supply pathways probably determine apparent DG species selectivity of DGK isozymes (DGKα, β, γ, δ, η, κ, ζ, ι and θ) that show no selectivity against substrate (DG species) in vitro. There may be various new and unknown DG supply pathways that are independent of the PI turnover. It is possible that exploring new and unknown DG supply pathways and PA species-selective binding proteins bring us to a new lipid world.
    Competing interests
    Acknowledgments We thank all members of the Sakane Lab for discussion and suggestions. This work was supported in part by Grants-in-Aid from The Ministry of Education, Culture, Sports, Science and Technology of Japan (FS). KAKENHI Grant Numbers 22370047 (Grant-in-Aid for Scientific Research (B)), 23116505 (Grant-in-Aid for Scientific Research on Innovative Areas), 25116704 (Grant-in-Aid for Scientific Research on Innovative Areas), 26291017 (Grant-in-Aid for Scientific Research (B)), and 15K14470 (Grant-in-Aid for Challenging Exploratory Research), 17H03650 (Grant-in-Aid for Scientific Research (B)).
    Introduction Diacylglycerol kinase (DGK) phosphorylates diacylglycerol (DG) to yield phosphatidic acid (PA) [1], [2], [3], [4], [5]. DG and PA have been well recognized as lipid second messengers, and DGK appears to participate in various physiological events by modulating the amounts of these two bioactive lipids. Ten mammalian DGK isozymes (α, β, γ, δ, ε, ζ, η, θ, ι and κ), containing two or three characteristic zinc finger-like C1 domains and the catalytic region in common, are divided into five groups according to their structural features [1], [2], [3], [4], [5]. Moreover, the occurrence of alternative splicing has been reported for at least six mammalian DGK genes (the β [6], γ [7], δ [8], ζ [9], η [10] and ι [11] isoforms).