Effect of membrane perturbants on the activity and phase distribution of inositol phosphorylceramide synthase; development of a novel assay.

Paul A Aeed, Andrea E Sperry, Casey L Young, Marek M Nagiec, Ake P Elhammer

Index: Biochemistry 43(26) , 8483-93, (2004)

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Abstract

The effect of 26 different membrane-perturbing agents on the activity and phase distribution of inositol phosphorylceramide synthase (IPC synthase) activity in crude Candida albicans membranes was investigated. The nonionic detergents Triton X-100, Nonidet P-40, Brij, Tween, and octylglucoside all inactivated the enzyme. However, at moderate concentrations, the activity of the Triton X-100- and octylglucoside-solubilized material could be partially restored by inclusion of 5 mM phosphatidylinositol (PI) in the solubilization buffer. The apparent molecular mass of IPC synthase activity solubilized in 2% Triton X-100 was between 1.5 x 10(6) and 20 x 10(6) Da, while under identical conditions, octylglucoside-solubilized activity remained associated with large presumably membrane-like structures. Increased detergent concentrations produced more drastic losses of enzymatic activity. The zwitterionic detergents Empigen BB, N-dodecyl-N,N-(dimethylammonio)butyrate (DDMAB), Zwittergent 3-10, and amidosulfobetaine (ASB)-16 all appeared capable of solubilizing IPC synthase. However, these agents also inactivated the enzyme essentially irreversibly. Solubilization with lysophospholipids again resulted in drastic losses of enzymatic activity that were not restored by the inclusion of PI. Lysophosphatidylinositol also appeared to compete, to some extent, with the donor substrate phosphatidylinositol. The sterol-containing agent digitonin completely inactivated IPC synthase. By contrast, sterol-based detergents such as 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS), 3-[(3-cholamidopropyl)dimethylammonio]-2-hydroxy-1-propanesulfonate (CHAPSO), and taurodeoxycholate (tDOC) had little or no effect on the enzyme activity. The IPC synthase activity in C. albicans membranes remained largely intact and sedimentable at CHAPS concentrations (4%) where >90% of the phospholipids and 60% of the total proteins were extracted from the membranes. At 2.5% CHAPS, a concentration where approximately 50% of the protein and 80% of the phospholipids are solubilized, there was no detectable loss of enzyme activity, and it was found that the detergent-treated membranes had significantly improved properties compared to crude, untreated membranes as the source of IPC synthase activity. In contrast to assays utilizing intact membranes or Triton X-100 extracts, assays using CHAPS- or tDOC-washed membranes were found to be reproducible, completely dependent on added acceptor substrate (C(6)-7-nitro-2-1,3-benzoxadiazol-4-yl (NBD)-ceramide), and >95% dependent on added donor substrate (PI). Product formation was linear with respect to both enzyme concentration and time, and transfer efficiency was improved more than 20-fold as compared to assays using crude membranes. Determination of kinetic parameters for the two IPC synthase substrates using CHAPS-washed membranes resulted in K(m) values of 3.3 and 138.0 microM for C(6)-NBD-ceramide and PI, respectively. In addition, the donor substrate, PI, was found to be inhibitory at high concentrations with an apparent K(i) of 588.2 microM.