Cookies

We use cookies to ensure that we give you the best experience on our website. By continuing to browse this repository, you give consent for essential cookies to be used. You can read more about our Privacy and Cookie Policy.


Durham Research Online
You are in:

A plate-based assay system for analyses and screening of the Leishmania major inositol phosphorylceramide synthase.

Mina, J.G. and Mosely, J.A. and Ali, H.Z. and Shams-Eldin, H. and Schwarz, R.T. and Steel, P.G. and Denny, P.W. (2010) 'A plate-based assay system for analyses and screening of the Leishmania major inositol phosphorylceramide synthase.', International journal of biochemistry and cell biology., 42 (9). pp. 1553-1561.

Abstract

Sphingolipids are key components of eukaryotic membranes, particularly the plasma membrane. The biosynthetic pathway for the formation of these lipid species is largely conserved. However, in contrast to mammals, which produce sphingomyelin, organisms such as the pathogenic fungi and protozoa synthesize inositol phosphorylceramide (IPC) as the primary phosphosphingolipid. The key step involves the reaction of ceramide and phosphatidylinositol catalysed by IPC synthase, an essential enzyme with no mammalian equivalent encoded by the AUR1 gene in yeast and recently identified functional orthologues in the pathogenic kinetoplastid protozoa. As such this enzyme represents a promising target for novel anti-fungal and anti-protozoal drugs. Given the paucity of effective treatments for kinetoplastid diseases such as leishmaniasis, there is a need to characterize the protozoan enzyme. To this end a fluorescent-based cell-free assay protocol in a 96-well plate format has been established for the Leishmania major IPC synthase. Using this system the kinetic parameters of the enzyme have been determined as obeying the double displacement model with apparent Vmax = 2.31 pmol min−1 U−1. Furthermore, inhibitory substrate analogues have been identified. Importantly this assay is amenable to development for use in high-throughput screening applications for lead inhibitors and as such may prove to be a pivotal tool in drug discovery.

Item Type:Article
Full text:(AM) Accepted Manuscript
Download PDF
(7708Kb)
Status:Peer-reviewed
Publisher Web site:https://doi.org/10.1016/j.biocel.2010.06.008
Publisher statement:NOTICE: this is the author's version of a work that was accepted for publication in International journal of biochemistry and cell biology.
Record Created:28 Jun 2010 15:05
Last Modified:15 Aug 2017 16:54

Social bookmarking: del.icio.usConnoteaBibSonomyCiteULikeFacebookTwitterExport: EndNote, Zotero | BibTex
Look up in GoogleScholar | Find in a UK Library