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Inhibition of yeast inositol phosphorylceramide synthase by aureobasidin A measured by a fluorometric assay
Author(s) -
Zhong Wenyan,
Murphy Dennis J,
Georgopapadakou Nafsika H
Publication year - 1999
Publication title -
febs letters
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.593
H-Index - 257
eISSN - 1873-3468
pISSN - 0014-5793
DOI - 10.1016/s0014-5793(99)01633-6
Subject(s) - enzyme , atp synthase , ceramide , biochemistry , saccharomyces cerevisiae , yeast , non competitive inhibition , chemistry , sphingolipid , substrate (aquarium) , inositol , biosynthesis , fluorescence , stereochemistry , biology , receptor , apoptosis , ecology , physics , quantum mechanics
Inositol phosphorylceramide synthase (IPC synthase) is an essential and unique enzyme in fungal sphingolipid biosynthesis and is the target of the cyclic nonadepsipeptide antibiotic aureobasidin A. As a first step towards understanding the mechanism of aureobasidin A inhibition, we developed a fluorometric HPLC assay for IPC synthase using the Saccharomyces cerevisiae enzyme and the fluorescent substrate analog 6‐[ N ‐(7‐nitro‐2,1,3‐benzoxadiazol‐4‐yl)amino]‐hexanoyl ceramide (C 6 ‐NBD‐cer). The kinetic parameters for C 6 ‐NBD‐cer were comparable to those for the synthetic substrate N ‐acetylsphinganine used previously. Aureobasidin A acted as a tight‐binding, non‐competitive inhibitor with respect to C 6 ‐NBD‐cer and had a K i of 0.55 nM.