T. Shimizu, Z. Honda, I. Miki
1990
Citations
1
Influential Citations
34
Citations
Journal
Methods in enzymology
Abstract
Publisher Summary The chapter describes the purification and characterization of potato lipoxygenase, and details procedures for the syntheses of 5-hydroperoxyeicosatetraenoic acid (5-HPETE) and 5( S ), 12( S )-diHETE. Synthesis of biologically active leukotrienes (LTs) is initiated by 5-lipoxygenation of arachidonic acid to yield 5( S )-hydroperoxy-6- trans -8, 11, 14- cis -eicosatetraenoic acid (5-HPETE), which is further transformed by dehydration to LTA 4 , a key intermediate in the formation of LTs. It is mentioned that enzyme activity varies significantly from potato to potato. Some lots of potatoes have no detectable enzyme activity, presumably because of storage conditions (duration and temperature, radiation, and so on.) rather than to species differences. Before starting purification of the enzyme, or preparation of 5-HPETE, first the enzyme activity should be checked using the ammonium sulfate fraction. The use of fresh potatoes obtained directly from farms provides the most successful results. Like other lipoxygenases, the potato lipoxygenase activity can be determined by the following four assay methods: (1) Spectrophotometry, (2) oxygen monitoring, (3) thin-layer chromatography (TLC), and (4) high-performance liquid chromatography (HPLC). The chapter summarizes the characteristics of each method.