title: Purification and partial characterization of a membrane-associated lipoxygenase in tomato fruit.
abstract: Membrane-associated lipoxygenase from green tomato (Lycopersicon esculentum L. cv Caruso) fruit has been purified 49-fold to a specific activity of 8.3 mumol.min(-1).mg(-1) of protein by solubilization of microsomal membranes with Triton X-100, followed by anion- exchange and size-exclusion chromatography. The apparent molecular mass of the enzyme was estimated to be 97 and 102 kD by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and size-exclusion chromatography, respectively. The purified membrane lipoxygenase preparation consisted of a single major band following sodium dodecyl sulfate-polyacrylamide gel electrophoresis, which cross-reacts with immunoserum raised against soluble soybean lipoxygenase 1. It has a pH optimum of 6.5, an apparent K(m) of 6.2 mum, and V(max) of 103. mumol.min(-1).mg(-1) of protein with linoleic acid as substrate. Corresponding values for the partially purified soluble lipoxygenase from tomato are 3.8 mum and 1.3 mumol.min(-1).mg(-1) of protein, respectively. Thus, the membrane-associated enzyme is kinetically distinguishable from its soluble counterpart. Sucrose density gradient fractionation of the isolated membranes indicated that the membrane-associated lipoxygenase sediments with thylakoids. A lipoxygenase band with a corresponding apparent mol wt of 97,000 was identified immunologically in sodium dodecyl sulfate-polyacrylamide gel electrophoresis-resolved proteins of purified thylakoids prepared from intact chloroplasts isolated from tomato leaves and fruit.
authors.affiliation: Department of Molecular Biology and Genetics, University of Guelph, Guelph, Ontario, Canada N1G 2W1.
authors.forename: C G
authors.initials: CG
authors.lastname: Bowsher
authors.forename: B J
authors.initials: BJ
authors.lastname: Ferrie
authors.forename: S
authors.initials: S
authors.lastname: Ghosh
authors.forename: J
authors.initials: J
authors.lastname: Todd
authors.forename: J E
authors.initials: JE
authors.lastname: Thompson
authors.forename: S J
authors.initials: SJ
authors.lastname: Rothstein