Purification, tandem mass characterization, and inhibition studies of oxidosqualene-lanosterol cyclase enzyme from bovine liver

Tung-Kung Wu*, Chiao Yin Huang, Cheng Yu Ko, Cheng Hsiang Chang, Yu Ju Chen, Hsin Kai Liao

*Corresponding author for this work

Research output: Contribution to journalArticlepeer-review

9 Scopus citations

Abstract

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 ∼70 and ∼140kDa, 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 37°C, respectively. The apparent K M and kcat/KM values were estimated to be 11μM and 1.45mM-1min-1, respectively. Inhibition studies using both Ro48-8071 and N-(4-methylenebenzophenonyl)pyridinium bromide showed potent inhibition of OSC with an IC50 of 11nM and 0.79μM, 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.

Original languageEnglish
Pages (from-to)42-53
Number of pages12
JournalArchives of Biochemistry and Biophysics
Volume421
Issue number1
DOIs
StatePublished - 1 Jan 2004

Keywords

  • 5,5′-Dithio-bis-(2- nitrobenzoic) acid
  • Diethyl pyrocarbonate
  • Ergosterol biosynthesis
  • Oxidosqualene cyclase

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