Purification of a mammalian 5-lipoxygenase from rat basophilic leukemia cells |
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Authors: | Andrew M. Goetze Liz Fayer Jennifer Bouska Dirk Bornemeier George W. Carter |
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Affiliation: | Immunosciences Research, D-47K, Abbott Laboratories, North Chicago, IL 60064, U.S.A. |
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Abstract: | 5-Lipoxygenase (5-lipox) has been purified to homogeneity from the 20,000 xg supernatant of sonicated rat basophilic leukemia (RBL-1) cells using a 4-step procedure. Purification was achieved primarily through the use of anion-exchange HPLC on two different media. Using the supernatant from 1 × 10 9 cells, approximately 33 μg of the enzyme can be routinely isolated with an estimated net yield of 5–10%. Purified 5-lipox consists of a single Mr 73,000 band on SDS gels (reduced or unreduced). When the purified enzyme was incubated with radiolabeled arachidonic acid and products analyzed by both straight phase and reversed phase HPLC, 5-hydroperoxyeicosate-traenoic acid (5-HPETE) was the only enzymatic product detected. The purified enzyme exhibits the same characteristic lag phase and premature cessation of reaction as does the 5-lipox activity seen in crude cell homogenates. |
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