A membrane-associated lipoxygenase from breaker-stage fruit of tomato (Lycopersicon esculentum Mill.) was purified and partially sequenced. Using degenerate oligonucleotides corresponding to portions of this sequence, a cDNA was amplified by PCR and used to screen a breaker fruit cDNA library. Two clones, tomloxA and tomloxl, were isolated and one of these (tomloxA) corresponded to the isolated protein. Genomic clones were isolated and sequence data from these were used to obtain the 5' ends of the cDNAs. The 2.8-kb cDNAs encode proteins that are similar in size and sequence to each other and to other plant lipoxygenases. DNA blot analysis indicated that tomato contains three or more genes that encode lipoxygenase. RNA blot analysis showed that tomloxA is expressed in germinating seeds as well as in ripening fruit, where it reached its peak during breaker stage. tomloxB appears to be fruit specific and is at its highest level in ripe fruit.
Membrane-associated lipoxygenase from green tomato (Lycopersicon esculentum L. cv Caruso) fruit has been purified 49-fold to a specific activity of 8.3 ,mol *min' . mg-' 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 Km of 6.2 Mm, and Vm,x of 10.3 jAmol-min-' mg-' of protein with linoleic acid as substrate. Corresponding values for the partially purified soluble lipoxygenase from tomato are 3.8 Mm and 1.3 Amol min-'.mg-' of protein, respectively. Thus, the membraneassociated 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.The predominant mechanism for fatty acid catabolism is fl-oxidation, which occurs in mitochondria or glyoxysomes. Although this pathway metabolizes most of the fatty acids derived from stored fats and oils, several other routes are utilized for the oxidation of specialized fatty acids. One of these alternative pathways involves the enzyme lipoxygenase (EC 1.13.11.12), which catalyzes the oxidation of a class of unsaturated fatty acids having a cis,cis-1,4-pentadiene structure. These fatty acids are primarily present as constituents of membrane phospholipids. The existence of this enzyme 'This work was supported by grants to S
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