Purification and characterization of leukotriene A4 epoxide hydrolase from dog lung.

Leukotriene A4 epoxide hydrolase from dog lung, a soluble enzyme catalyzing the hydrolysis of leukotriene A4 (LTA4) to leukotriene B4 (LTB4) was partially purified by anion exchange HPLC. The enzymatic reaction obeys Michaelis- Menten kinetics. The apparent Km ranged between 15 and 25 microM and the enzyme exhibited an optimum activity at pH 7.8. An improved assay for the epoxide hydrolase has been developed using bovine serum albumin and EDTA to increase the conversion of LTA4 to LTB4. This method was used to produce 700 mg of LTB4 from LTA4 methyl ester. The partial by purified enzyme was found to be uncompetitively inhibited by divalent cations. Ca+2, Mn+2, Fe+2, Zn+2 and Cu+2 were found to have inhibitor constants (Ki) of 89 mM, 3.4 mM, 1.1 mM, 0.57 mM, and 28 microM respectively Eicosapentaenoic acid was shown to be a competitive inhibitor of this enzyme with a Ki of 200 microM. From these inhibition studies, it can be theorized that the epoxide hydrolase has at least one hydrophobic and one hydrophilic binding site.

Organization and expression of leghaemoglobin genes.

Leghaemoglobin genes in soybean (Glycine max) are present as a moderately reiterated family of sequences. Since there are identical restriction site patterns of these sequences in DNA isolated from leaf, root, or nodule tissue, the data suggest that no major changes in the organization or methylation of leghaemoglobin genes occur during their induction. Cloned soybean leghaemoglobin-cDNA cross hybridized with RNA from root nodules of kidney bean (Phaseolus vulgaris), and to a lesser extent, of pea (Pisum sativum) indicating sequence homology in the leghaemoglobin genes of these species. Hybridization to the genomic DNA restriction fragments of two other species, Glycine soja and Vicia faba, also indicated interspecies sequence homologies. Several restriction fragments appear to be common to all species examined. The induction of these genes occurs following infection of the plant by Rhizobium and is independent of the appearance of nitrogenase activity in the nodules. The level of expression is, however, influenced by various mutations in Rhizobium that result in the development of ineffective (nonnitrogen fixing) nodules.