title: Purification and some properties of the squalene-tetrahymanol cyclase from Tetrahymena thermophila.
abstract: The membrane-bound enzyme from Tetrahymena thermophila responsible for the conversion of squalene into the quasi-hopanoid tetrahymanol was purified 297-fold to near homogeneity. Purification involved solubilization by octylthioglucoside, chromatography on DEAE-trisacryl, hydroxyapatite and FPLC ion-exchange on Mono Q. The apparent KM was found to be 18 microM. 2,3-Iminosqualene and N,N-dimethyldodecylamine-N-oxide are effective inhibitors of the cyclase with I50 values of 50 and 30 nM, respectively. The cyclase has a molecular mass of 72 kDa as judged by electrophoresis in polyacrylamide gels under denaturating conditions. The optimal enzymatic activity was obtained at pH 7.0 and 30 degrees C. The solubilized enzyme needs the presence of detergent for maintaining activity. The influence of different detergents on cyclase activity was studied. Triton X-100 proved to be a strong inactivator of the enzyme. Solubilization of the cyclase in Tween 80 and digitonin inactivates the enzyme. However, its activity can be recovered by complementation of the assay buffer with octylthioglucoside above its critical micellar concentration. We suggest that this approach might be applicable to other membrane-bound proteins.
authors.affiliation: Botanisches Institut, Universität Tübingen, F.R.G.
authors.forename: J
authors.initials: J
authors.lastname: Saar
authors.forename: J C
authors.initials: JC
authors.lastname: Kader
authors.forename: K
authors.initials: K
authors.lastname: Poralla
authors.forename: G
authors.initials: G
authors.lastname: Ourisson
mesh_terms: D000818:Animals; D002462:Cell Membrane / Q000201:enzymology; D002853:Chromatography, Liquid; D003902:Detergents; D004591:Electrophoresis, Polyacrylamide Gel; D004791:Enzyme Inhibitors / Q000494:pharmacology; D008190:Lyases / Q000037:antagonists & inhibitors / Q000302:isolation & purification* / Q000378:metabolism; D013768:Tetrahymena / Q000201:enzymology*; D014315:Triterpenes / Q000737:chemistry
chemical_list: D003902:Detergents; D004791:Enzyme Inhibitors; D014315:Triterpenes; C029766:diplopterol; C100046:tetrahymanol; D008190:Lyases; C022651:squalene cyclase