Crystalline Lipoxidase.

Theorell, Hugo; Holman, Ralph T.; Åkeson, Åke; Linnasalmi, Annikki; Laukkanen, Pentti

doi:10.3891/acta.chem.scand.01-0571

Cited by 165 publications

(58 citation statements)

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“…The biological purpose for lipoxygenase catalysis in plants is not so clear, but evidence for biosynthetic roles in the generation of growth-regulatory substances and pest-resistance compounds has been presented [3, 41. While the lipoxygenase found in soybeans has been known for some time [5], the details of the structure and mechanism of action of the enzyme are still in the process of being elucidated. The primary structures for the lipoxygenase 1 from soybeans [6] and the 5-lipoxygenase from leukocytes [7,81 have been published recently.…”

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confidence: 99%

The initial characterization of the iron environment in lipoxygenase by Mössbauer spectroscopy

Dunham

Carroll

Thompson

et al. 1990

European Journal of Biochemistry

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The incorporation of 7Fe into two lipoxygenase isoenzymes from soybeans has been achieved making possible the first observations of the iron environment in these proteins using Mossbauer spectroscopy. Immature soybean seeds were grown in tissue culture medium supplied with 57Fe. The iron in the active lipoxygenases that were isolated from the cultured seeds was readily detected in Mossbauer measurements. It was unequivocally demonstrated that the native enzyme contains high-spin Fe(I1). Based on the sign of the electric field gradient, the most likely ligand sphere for the iron in native lipoxygenase consists of oxygen and nitrogen ligands in a roughly octahedral field of symmetry. It was possible to detect Mossbauer signals in highly concentrated samples of native lipoxygenases containing 57Fe at natural abundance. The spectra obtained for enriched and natural abundance native enzyme had the same high-spin Fe(l1) Mossbauer parameters. This confirmed that the environment of the iron in enzymes isolated from cultured seeds and dry soybeans were the same. The Mossbauer spectra (4.2 -250 K) for samples of both isoenzymes after oxidation of the iron in native enzyme by the product of lipoxygenase catalysis were extremely broad (20 mmjs) with no obvious narrow resonance lines. This was the result of the existence of paramagnetically broadened spectra for such samples even at relatively high temperature as evidenced by the appropriate EPR signal. A small molecule containing an iron site sharing many of these Mossbauer and electron paramagnetic resonance properties with lipoxygenase was identified: Fe(II)/(III) . diethylenetriaminepentaacetic acid.Lipoxygenases are responsible for an important fraction of polyunsaturated fatty acid metabolism in both plants and animals. The inaugural step in the conversion of arachidonic acid into 5-hydroxyicosatetraenoic acid and the leukotrienes in the mammalian circulatory system is catalyzed by 5-lipoxygenase [I]. Lipoxygenase-derived arachidonic acid metabolites have potent, medically relevant physiological activities in the immune response [2]. The biological purpose for lipoxygenase catalysis in plants is not so clear, but evidence for biosynthetic roles in the generation of growth-regulatory substances and pest-resistance compounds has been presented [3, 41. While the lipoxygenase found in soybeans has been known for some time [5], the details of the structure and mechanism of action of the enzyme are still in the process of being elucidated. The primary structures for the lipoxygenase 1 from soybeans [6] and the 5-lipoxygenase from leukocytes [7, 81 have been published recently. The degree of similarity among the sequences indicates that there may be common aspects of the structure which are presumably related to the catalytic mechanism.All of the lipoxygenases available in sufficient quantity to be characterized have been found to contain 1 mol Fe/mol enzyme [9, 101. In lipoxygenase 1 the iron is necessary for catalysis and is thought to play an integral part in the mechanism...

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confidence: 99%

The initial characterization of the iron environment in lipoxygenase by Mössbauer spectroscopy

Dunham

Carroll

Thompson

et al. 1990

European Journal of Biochemistry

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“…In this experiment, a linear NaCl gradient of 0-60 mM in SO mM Tris, pH 8.0 in an elution volume of 100 ml was employed, followed by elution at pH 6 in Fig. 3 -5, and the purification factors obtained are given in Table 1.…”

Section: Using Other Derivatives Of Aminohexyl Agarosementioning

confidence: 99%

“…Linoleic and linolenic acids are therefore its main natural substrates. Several conventional methods for the isolation of this enzyme from a variety of plants have been published [I -51, a number of which have indicated the presence of isoenzymes; these are now generally classified as lipoxygenase-1, which has an activity optimum at about pH 9 (and was the original soybean lipoxygenase of Theorell [6]), and lipoxygenase-2, with an optimum at around pH 6.8.…”

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confidence: 99%

Affinity Chromatography of Lipoxygenases

Allen

Eriksson

Galpin³

1977

Eur J Biochem

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A number of aminohexyl agarose derivatives of unsaturated fatty acids have been prepared and evaluated as materials for the affinity chromatography of soybean and pea lipoxygenases. A practical method for a one-stage purification of soybean lipoxygenase-1, with a purification factor of 16, is described, using either linolenate or docosa-4,7,10,13,16,19-hexaenoate as ligands.Results show that alleged competitive inhibitors do not cause sharp elution from the affinity column, and that there is an increasing specificity of binding and sharpness of elution as the proportion of unsaturation in the ligand is increased. These results are discussed in terms of the relative importance of the types of bonding involved in enzyme-substrate binding.

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“…This heterogeneity has been studied by a variety oftechniques (6). Reports typically describe the type-1 isoenzyme, which was first isolated by Theorell et al ( 18) and is maximally active at pH 9, and one or more additional isoenzymes maximally active near neutral pH. While their pH profiles for catalytic activity and isoelectric points differed substantially, the isoenzymes have been found to share the same apparent mol wt and iron content (2).…”

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confidence: 99%

The Lipoxygenases in Developing Soybean Seeds, Their Characterization and Synthesis in Vitro

Funk

Carroll²,

Thompson³

et al. 1986

Plant Physiol.

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A number of lipoxygenase isoenzymes were identified in developing soybean (Glycine max L. Merrill cv Provar) seeds and two have been partially characterized. In a study of lipoxygenase level in developing soybean seeds, the enzyme content increased markedly during development. Comparisons of the lipoxygenases from mature soybean seeds and immature seeds by isoelectric focusing, chromatofocusing, sodium dodecyl sulfate polyacrylamide gel electrophoresis and peptide mapping identified two categories of isoenzyme. The isoenzymes from immature seeds were found by electron paramagnetic resonance spectroscopy to be isolated at least in part as the high spin iron(III) or active form of the enzyme in contrast to lipoxygenases from mature seeds which were isolated as electron paramagnetic resonance silent, high spin iron(II) species. The discovery of increased levels of lipoxygenases during seed development and their isolation in an active form suggests that the enzyme may play a physiological role during the maturation process. The incorporation of iron-59 from the nutrient medium into lipoxygenase during culture of immature seeds was indicative of de novo synthesis of the enzyme. The efficiency of the iron uptake was high, as indicated by the level of radioactivity found in the enzyme (one gram atom of iron per mole of lipoxygenase).

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Crystalline Lipoxidase.

Cited by 165 publications

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The initial characterization of the iron environment in lipoxygenase by Mössbauer spectroscopy

The initial characterization of the iron environment in lipoxygenase by Mössbauer spectroscopy

Affinity Chromatography of Lipoxygenases

The Lipoxygenases in Developing Soybean Seeds, Their Characterization and Synthesis in Vitro

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