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Metabolism and Enzymology

Purification and Partial Characterization of a Membrane-Associated Lipoxygenase in Tomato Fruit ¹

Department of Molecular Biology and Genetics, University of Guelph, Guelph, Ontario, Canada N1G 2W1, Department of Horticultural Science, University of Guelph, Guelph, Ontario, Canada N1G 2W1, Department of Biology University of Waterloo, Waterloo, Ontario, Canada N1G 2G1

Membrane-associated lipoxygenase from green tomato (Lycopersicon esculentum L. cv Caruso) fruit has been purified 49-fold to a specific activity of 8.3 µmol·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 µM, and V_max of 103. µmol·min^–1·mg^–1 of protein with linoleic acid as substrate. Corresponding values for the partially purified soluble lipoxygenase from tomato are 3.8 µM and 1.3 µmol·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.

³ Present address: Department of Biology, Queens University, Kingston, Ontario, Canada K7L 3N6.

¹ This work was supported by grants to S.J.R. and J.E.T. from the Natural Sciences and Engineering Research Council of Canada.

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