Add time:08/23/2019         Source:sciencedirect.com

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 (C6-NBD-cer). The kinetic parameters for C6-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 C6-NBD-cer and had a Ki of 0.55 nM.

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