title: Affinity labeling of vertebrate oxidosqualene cyclases with a tritiated suicide substrate.
abstract: Pig and rat liver oxidosqualene cyclase (OSC) enzymes were purified to homogeneity and showed single bands on SDS-polyacrylamide gel electrophoresis with molecular masses of 75 kDa (pig) and 78 kDa (rat). Pig liver OSC was purified for the first time (441-fold with a yield of 39%). Chemical affinity labeling of pure or crude preparations of the liver cyclases using the mechanism-based irreversible inhibitor of OSC, [3H]29-methylidene-2,3-oxidosqualene ([3H]29-MOS), showed a single radioactive band at 75 kDa (pig) and 78 kDa (rat). Affinity labeling experiments were also performed with dog and human microsomal preparations and with yeast and plant cyclases. All of the vertebrate OSC enzymes were specifically labeled with [3H]29-MOS and gave a single band with molecular masses ranging from 70 to 80 kDa (rat, 78 kDa; dog, 73 kDa; pig, 75 kDa; and human, 73 kDa). In contrast, yeast lanosterol cyclase and plant cycloartenol cyclase were not labeled, demonstrating subtle differences in the active sites of animal, plant, and fungal enzymes.
authors.affiliation: Department of Chemistry, State University of New York, Stony Brook 11794-3400.
authors.forename: I
authors.initials: I
authors.lastname: Abe
authors.forename: M
authors.initials: M
authors.lastname: Bai
authors.forename: X Y
authors.initials: XY
authors.lastname: Xiao
authors.forename: G D
authors.initials: GD
authors.lastname: Prestwich
mesh_terms: D000345:Affinity Labels; D000818:Animals; D001665:Binding Sites; D003500:Cyclization; D004285:Dogs; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D019751:Intramolecular Transferases; D007535:Isomerases / Q000032:analysis / Q000737:chemistry* / Q000302:isolation & purification; D008099:Liver / Q000201:enzymology; D015394:Molecular Structure; D008970:Molecular Weight; D010944:Plants / Q000201:enzymology; D051381:Rats; D012441:Saccharomyces cerevisiae / Q000201:enzymology; D013185:Squalene / Q000031:analogs & derivatives* / Q000737:chemistry / Q000494:pharmacology; D013552:Swine; D014316:Tritium
chemical_list: D000345:Affinity Labels; D014316:Tritium; C076537:29-methylidene-2,3-oxidosqualene; D013185:Squalene; D007535:Isomerases; D019751:Intramolecular Transferases; C021924:lanosterol synthase