Affinity chromatography of antibodies directed against soybean lipoxygenase-1 and -2 and an enzyme-linked immunosorbent assay (ELISA) for antibodies and lipoxygenases

Crude immunoglobulin G (IgG) fractions of antisera directed against soybean lipoxygenase-1 and -2 were purified by being passed through an immunoadsorbent column containing lipoxygenase coupled to CNBr-activated Sepharose 4B. Bound immunoglobulin was desorbed with pulses of 2 M or 3 M ammonium thiocyanate or 0.1 M glycine-HCl buffer (pH 2.5). The total column recoveries of anti-lipoxygenase-1 IgG and anti-lipoxygenase-2 IgG were 45% and 58%, respectively. The affinity for lipoxygenase of immunospecific antibodies was determined in an enzyme-linked immunosorbent assay (ELISA). In a reaction with lipoxygenase-1, anti-lipoxygenase-1 IgG, which was eluted with glycine-HCl buffer (pH 2.5) with recovery of 24%, had a 6.5-times higher affinity than the whole IgG fraction of antiserum. The affinity of anti-lipoxygenase-2 IgG for lipoxygenase-2 increased 2.2-times after chromatography of IgG over an immunoadsorbent column using 2 M ammonium thiocyanate as eluent (recovery 21%).     

Evidence for lipoxin formation by bovine polymorphonuclear leukocytes via triple dioxygenation of arachidonic acid

Incubation of bovine polymorphonuclear leukocytes (PMNs) with arachidonic acid leads to the formation of four lipoxins. The same lipoxins are also formed upon incubation of bovine PMNs with 5(S)-hydroperoxy-6-trans-8,11,14-cis-eicosatetraenoic acid, 5-hydroxy-6-trans-8,11,14-cis-eicosatetraenoic acid, 5(S)-hydroperoxy, 15(S)-hydroxy-6,13-trans-8,11-cis-eicosatetraenoic acid or 5(S),15(S)-dihydroxy-6,13-trans-8,11-cis-eicosatetraenoic acid. A 5,6-epoxide as intermediate in lipoxin formation in the bovine PMN is highly improbable because the 5-hydroxy compounds are as good substrates as the 5-hydroperoxy compounds. Moreover, the two main lipoxins were found to coelute with the two lipoxins produced via a triple dioxygenation of arachidonic acid by soybean lipoxygenase-1. Hence the bovine PMN is the first cell for which evidence is presented that the formation of lipoxins proceeds mainly via triple dioxygenation and not via 15-hydroxy-leukotriene A4 as is proposed for human and porcine PMNs.     

Detecting non-neutral heterogeneity across a region of DNA sequence in the ratio of polymorphism to divergence

Natural selection, in the form of balancing selection or selective sweeps, can result in a decoupling of the amounts of molecular polymorphism and divergence. Thus natural selection can cause some areas of DNA sequence to have greater silent polymorphism, relative to divergence between species, than other areas. It would be useful to have a statistical test for heterogeneity in the polymorphism to divergence ratio across a region of DNA sequence, one that could identify heterogeneity greater than that expected from the neutral processes of mutation, drift, and recombination. The only currently available test requires that a region be arbitrarily divided into sections that are compared with each other, and the subjectivity of this division could be problematic. Here a test is proposed in which runs of polymorphic and fixed sites are counted, where a "run" is a set of one or more sites of one type preceded and followed by the other type. The number of runs is smaller than otherwise expected if polymorphisms are clumped together. By simulating neutral evolution and comparing the observed number of runs to the simulations, a statistical test is possible which does not require any a priori decisions about subdivision.      

The effect of modification of sulfhydryl groups in soybean lipoxygenase-1

Soybean lipoxygenase-1 was found to contain five free sulfhydryl groups and no disulfide bridges. Three sulfhydryl groups react readily with methylmercuric halides. This modification results in significant changes of the catalytic properties of the enzyme. Comparison of modified and native lipoxygenase-1 shows the following: 1. The catalytic constant of the oxygenation of linoleic acid is reduced by approximately 50%, whereas the affinity towards linoleic acid remains unaltered. 2. At high concentrations of substrate and low concentrations of enzyme the kinetic lag phase in the oxygenation is considerably longer. 3. The regio- and stereospecificities of the oxygenation are significantly lower. 4. Besides hydroperoxides, oxo-octadecadienoic acids (4%) are formed during the oxygenation. 5. The cooxidation capacity is considerably enhanced. Treatment of methylmercury-modified lipoxygenase-1 with NaHS results in the complete recovery of the sulfhydryl groups and of the catalytic properties.     

Purification of a lipoxygenase from ungerminated barley. Characterization and product formation

Lipoxygenase was purified from ungerminated barley (variety 'Triumph'), yielding an active enzyme with a pI of 5.2 and a molecular mass of approximately 90 kDa. In addition to the 90 kDa band SDS-PAGE showed the presence of two further proteins of 63 kDa. Western blot analysis showed cross-reactivity of each of these proteins with polyclonal antisera against lipoxygenases from pea as well as from soybean, suggesting a close immunological relationship. The 63 kDa proteins appear to be inactive degradation products of the active 90-kDa enzyme. This barley lipoxygenase converts linoleic acid mainly into (9S)-(10E,12Z)-9-hydroperoxy-10,12-octadecadienoic acid, and arachidonic acid into (5S)-(6E,8Z,11Z,14Z)-5-hydroperoxy-6,8,11,14-eic osatetraenoic acid.     

Properties of a complex of Fe(III)-soybean lipoxygenase-1 and 4-nitrocatechol

Fe(III)-soybean lipoxygenase-1 yields with 4-nitrocatechol a green coloured 1 : 1 complex, which shows at pH 7.0 absorption maxima at 385 nm and 650 nm. The formation of this complex is reversible. The circular dichroism spectrum of the complex of Fe(III)-lipoxygenase-1 and 4-nitrocatechol has a positive band at around 380 nm and a negative band at around 450 nm and is significantly different from that of the Fe(III)-enzyme as such. 4-Nitrocatechol can be displaced from the green complex by 13-L-hydroperoxy-cis-9, trans-11-octadecadienoic acid, resulting in the formation of the blue complex between the Fe(III)-enzyme and 13-L-hydroperoxy-cis-9,trans-11-octadecadienoic acid both under aerobic and anaerobic conditions. Also linoleic acid competes with 4-nitrocatechol for the binding site on the Fe(III)-enzyme, as can be demonstrated under anaerobic conditions, ultimately leading to reduction of the Fe(III)-enzyme. The oxygenation of linoleic acid by Fe(III)-lipoxygenase-1 is inhibited by 4-nitrocatechol. From steady-state kinetics a non-competitive inhibition pattern is obtained. Probably it has to be considered as pseudo non-competitive because of the slow establishment of the complex equilibrium. An inhibition constant (K4NC) of 16.3 microM is found. On prolonged incubation of Fe(III)-lipoxygenase-1 and 4-nitrocatechol the green complex converts into a brown species. This conversion is found to be coupled with a change in the nature of the inhibition from reversible to irreversible. A complex between native lipoxygenase-1 and 4-nitrocatechol is found to be unlikely.