標題: Purification, tandem mass characterization, and inhibition studies of oxidosqualene-lanosterol cyclase enzyme from bovine liver
作者: Wu, TK
Huang, CY
Ko, CY
Chang, CH
Chen, YJ
Liao, HK
生物科技學系
Department of Biological Science and Technology
關鍵字: oxidosqualene cyclase;ergosterol biosynthesis;5,5 '-dithio-bis-(2-nitrobenzoic) acid;diethyl pyrocarbonate
公開日期: 1-Jan-2004
摘要: The oxidosqualene-lanosterol cyclase (OSC) from bovine liver has been isolated from the microsomal membrane fraction and purified to homogeneity by ultracentrifugation, Q-Sepharose, hydroxyapatite, and HiTrap heparin chromatographies. The purified protein required Triton X-100 to retain its highest activity. The cyclase had a molecular mass of similar to70 and similar to140kDa, as evidenced by a single protein band on silver-stained SDS-PAGE and Coomassie-stained PAGE, respectively. Results from Edman degradation of OSC suggested that it might have a blocked N-terminus. Further peptide mapping coupled with tandem mass spectrometric determination identified three peptide fragments, ILGVGPDDPDLVR, LSAEEGPLVQSLR, and NPDGGFATYETK, which are highly homologous to human, rat, and mouse OSCs. The purified cyclase showed pH and temperature optima at pH 7.4 and 37degreesC, respectively. The apparent K-M and k(cat)/K-M values were estimated to be 11 muM and 1.45 mM(-1) min(-1), respectively. Inhibition studies using both Ro48-8071 and N-(4-methylenebenzophenonyl)pyridinium bromide showed potent inhibition of OSC with an IC50 of 11 nM and 0.79 muM, respectively. Results from DTNB modification and DTNB coupled with Ro48-8071 competition study suggest that two sulfhydryl groups are involved in the catalysis but not located in the substrate binding pocket or catalytic active site. The purified OSC was maximally inactivated by diethyl pyrocarbonate near neutral pH and re-activated by hydroxylamine, indicating the modification of histidine residues. The stoichiometry of histidine modification and the extent of inactivation showed that two essential histidine residues per active site are necessary for complete bovine liver OSC activity. (C) 2003 Elsevier Inc. All rights reserved.
URI: http://dx.doi.org/10.1016/j.abb.2003.09.036
http://hdl.handle.net/11536/27185
ISSN: 0003-9861
DOI: 10.1016/j.abb.2003.09.036
期刊: ARCHIVES OF BIOCHEMISTRY AND BIOPHYSICS
Volume: 421
Issue: 1
起始頁: 42
結束頁: 53
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