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A number of plasmids were constructed containing chimeric genes consisting of fragments of antisense-oriented lentil lipoxygenase cDNA. The different constructs were tested for their ability to lower lipoxygenase activity in lentil protoplasts. Plasmids containing a full length lentil lipoxygenase cDNA proved to be the most effective, reducing the activity of the target enzyme to 70% of the control value. On the other hand, the full length lentil lipoxygenase cDNA in the sense orientation yielded a 20% increase of lipoxygenase activity.

Protoplasts were isolated from lentil (Lens culinaris) roots and their suitability as a transient expression system was investigated. After transfecting the protoplasts with the beta-glucuronidase (GUS) gene by either electroporation or polyethylene glycol (PEG), the specific activity of the reporter enzyme and the cell viability were determined. Electroporation was more effective than PEG treatment as transfection procedure and its efficiency was affected by the plasmid length. The feasibility of electro-transferring at the same time (coelectroporation) inhibitory anti-lipoxygenase monoclonal antibodies and the GUS-carrying plasmid pBI 221 was investigated as well. The amount of transferred immunoglobulins was quantitated by ELISA and the inhibitory ability of monoclonal antibodies on the intracellular target enzyme was determined. Evidence is presented for the successful coelectroporation of immunoglobulins and plasmid DNA into lentil protoplasts, the two types of macromolecules acting independently of each other in the recipient cells.

Screening of a cDNA library constructed from the shoots of etiolated lentil seedlings resulted in finding a 2778 bp cDNA sequence, containing an open reading frame coding for a lipoxygenase of 866 amino acid residues. This lipoxygenase appears to be a novel type of vegetative lipoxygenase, different from the seed lipoxygenases of other leguminosae (complete homology < or = 72%).

The ability of soybean lipoxygenases-1 and -2 to oxygenate biomembranes isolated from soybean seedlings has been investigated. Constituents of the lipid bilayer were analyzed by means of reversed phase and chiral phase high performance liquid chromatography, gas chromatography/mass spectrometry, high performance thin layer chromatography and uv spectroscopy. Evidence is presented that soybean lipoxygenase-2, at variance with the type-1 enzyme, oxygenates the esterified unsaturated fatty acid moieties in biomembranes, whereas membrane-embedded free unsaturated fatty acid moieties were not a suitable substrate for either isoenzyme. The oxygenation products derived from the biomembranes were the 9- and 13-hydroperoxides of linoleic acid residues, in a molar ratio of 1.0 to 1.7, and the 9- and 13-hydroperoxides of alpha-linolenic acid residues, in a molar ratio of 1.0 to 0.1. The R/S ratios of 13-hydroperoxy-9Z,11E-octadecadienoic acid and 9-hydroperoxy-10E,12Z,15Z-octadecatrienoic acid were found to be 0.5 and 25.0, respectively. These stereospecificity values were much higher than those of hydroperoxides isolated after incubation of lipoxygenase-2 with non-membraneous fatty acids or their methyl esters. The hydroperoxy fatty acids produced were distributed in neutral lipids and phospholipids isolated from soybean membranes, the former being oxidized to a larger extent. Furthermore, both intracellular and plasma membranes were substrates for the enzymic oxygenation, with a preference for those of chloroplasts followed by those of Golgi apparatus, endoplasmic reticulum, plasma membrane and mitochondria. These data point towards a different action of the two lipoxygenases in soybean cells. We suggest that the type-2 enzyme plays a role in the in vivo remodelling of biomembranes. The physiological relevance of these findings is discussed.

Rat basophilic leukemia cells exhibit 12-lipoxygenase activity only upon cell disruption. 12-Lipoxygenase may also possess 15-lipoxygenase activity, as is indicated by the formation of low amounts of 15(S)-HETE, in addition to the predominant product 12(S)-HETE, upon incubation of partially purified 12-lipoxygenase with arachidonic acid. With 5(S)-HPETE as substrate not only 5(S), 12(S)-diHETE and 5(S), 15(S)-diHETE are formed, but also LTA4, as was indicated by the presence of LTA4-derived LTB4-isomers. 12-Lipoxygenase from rat basophilic leukemia cells has many features in common with 12-lipoxygenase from bovine leukocytes. As was suggested for the latter enzyme, 12-lipoxygenase from rat basophilic leukemia cells may represent the remaining LTA4-synthase activity of 5-lipoxygenase, of which the 5-dioxygenase activity has disappeared upon cell disruption. Such a possible shift from 5-lipoxygenase activity to 12-lipoxygenase activity could not simply be induced by interaction of cytosolic 5-lipoxygenase with a membrane fraction after cell disruption, but may involve release of membrane-associated 5-lipoxygenase upon disruption of activated rat basophilic leukemia cells.

12-Lipoxygenase from rat basophilic leukemia cells was purified about 300-fold by protein-HPLC in a single run. Maximal 12-lipoxygenase activity was observed at pH 7.5, while the enzyme became almost inactive at pH 6 and 9. Although Ca2+ was not essential for 12-lipoxygenase activity, the partially purified enzyme was stimulated approx. 2-fold in the presence of 0.1-5.0 mM Ca2+. Contrary to 5-lipoxygenase from RBL-1 cells, 12-lipoxygenase was not inactivated by preincubation with Ca2+ for 1-10 min, nor was it stimulated by 0.1-10 mM ATP.

12-Lipoxygenase and 5-lipoxygenase from rat basophilic leukemia cells were separated by protein-HPLC in a single step. Upon incubation in the presence of Ca2+, 12-lipoxygenase converted arachidonic acid into 12(S)-hydroxyeicosatetraenoic acid and linoleic acid into 13(S)-hydro(pero)xyoctadecadienoic acid. The reaction products were analyzed by reversed-phase and chiral straight-phase HPLC with ultraviolet-detection. Using the cytosolic fraction of rat basophilic leukemia cells, optimal 12-lipoxygenase activity was observed at 10 degrees C. At 37 degrees C 12-lipoxygenase was very rapidly inactivated by its own product, hydroperoxy fatty acid, at low concentrations (10-100 nM).

In this study we present evidence for the existence of an intrinsic 12-lipoxygenase in the bovine polymorphonuclear leukocyte which differs from the well-known platelet 12-lipoxygenase. Intact bovine polymorphonuclear leukocytes synthesize predominantly 5-lipoxygenase products. However, this 5-lipoxygenase activity disappears completely upon sonication of the cells, whereas a 12-lipoxygenase activity then becomes apparent. This 12-lipoxygenase resembles the platelet 12-lipoxygenase in metabolizing arachidonic acid into 12(S)-hydroxyeicosatetraenoic acid and in being independent of Ca2+ as well as of ATP. The most striking difference between the two 12-lipoxygenases is their behaviour towards linoleic acid. While the platelet 12-lipoxygenase does not convert linoleic acid, the 12-lipoxygenase from bovine polymorphonuclear leukocytes, apparent only in the cell-free system, converts linoleic acid into 13-hydroxyoctadecadienoic acid as efficiently as it converts arachidonic acid into 12-hydroxyeicosatetraenoic acid. This provides a convenient method to distinguish both 12-lipoxygenase activities. The fact that this new 12-lipoxygenase is able to metabolize linoleic acid into 13-hydroxyoctadecadienoic acid suggests that this enzyme, in contrast to platelet 12-lipoxygenase, resembles 5-lipoxygenases in showing a preference for hydrogen abstraction at a position which is determined by the distance to the carboxylic end of the fatty acid.

Lipoxygenase-1 from soybeans is incubated with an isomer of linoleic acid, 13-cis, 16-cis-octadecadienoic acid. Analysis of the oxygenation products indicates that molecular oxygen is stereospecifically introduced mainly at C-17 (n-2) of the fatty acid (in the LS-configuration), and only to a minor extent at C-13 (n-6). These findings contradict previous suggestions about the postional specificity of lipoxygenase-1.

Antiserum directed against lipoxygenase-1 from soybean (Glycine Max (L). Merr. var. Williams) was developed in rabbits with electrophoretically pure lipoxygenase-1 as an antigen. To remove traces of lipoxygenase-1 from a lipoxygenase-2 preparation the latter was chromatographed over a column of Sepharose 4B to which antibodies directed against lipoxygenase-1 were coupled. During affinity chromatography of lipoxygenase-2 no protein and only a small amount of activity were lost. Affinity-purified enzyme was used for immunization of rabbits to produce lipoxygenase-2 antibodies. Results with double gel immunodiffusion tests with the two soybean lipoxygenases and antisera directed against them demonstrated that there is no immunological relationship between the isoenzymes. Lipoxygenases from different species of leguminosae crossreacted only with antiserum directed against soybean lipoxygenase-2, and not with anti soybean lipoxygenase-1 serum. No crossreaction could be detected between soybean lipoxygenase antisera and lipoxygenases from species of Gramineae, Linaceae and Solanaceae.

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.

The leukotriene production by bovine polymorphonuclear leukocytes isolated from peripheral blood has been studied. Cells were incubated in the presence of arachidonic acid, glutathione, calcium ionophore A23187 and Ca2+. The leukotrienes then formed are leukotriene C4, leukotriene B4, two all-trans isomers of leukotriene B4 and the double dioxygenation product 12-epi-6-trans-8-cis-leukotriene B4. Leukotriene C4 is formed in such a large quantity by the bovine polymorphonuclear leukocyte that it might constitute an excellent and inexpensive source for the biosynthetic preparation of this spasmogenic leukotriene.

The zero-field splitting constants (D) of the different components building up the high-spin Fe(III) EPR spectrum of lipoxygenase from soybeans were determined by two methods: (1) temperature dependence studies using the low-spin Fe(III) signal of cytochrome c at g 3 for accurate measuring of the temperature in the sample; (2) by establishing g-shift upon increasing the microwave frequency. The ranges of D for the axial and rhombic species contributing to the complex signal at g 6 are found to be 1.5-3.0 K and 1.8-4.4 K, respectively. The occurrence of such large ranges is attributed to variations in amount and number of species in the different samples. The combination of the applied methods offers a more generally applicable approach to the determination of zero-field splitting constants.

1. The EPR spectrum at 15 degrees K of soybean lipoxygenase-1 in borate buffer pH 9.0 has been studied in relation to the presence of substrate (linoleic acid), product (13-L-hydroperoxylinoleic acid) and oxygen. 2. The addition of 13-L-hydroperoxylinoleic acid to lipoxygenase-1 at pH 9.0 gives rise to the appearance of EPR lines at g equals 7.5, 6.2, 5.9 and 2.0, and an increased signal at g equals 4.3. 3. In view of the effect of the end product on both the kinetic lag period of the aerobic reaction and the fluorescence of the enzyme, it is concluded that 13-L-hydroperoxylinoleic acid is required for the activation of soybean lipoxygenase-1. Thus it is proposed that the enzyme with iron in the ferric state is the active species. 4. A reaction scheme is presented in which the enzyme alternatingly exists in the ferric and ferrous states for both the aerobic and anaerobic reaction.

The iron coordination in native, Fe(II), lipoxygenase has been studied by Extended X-Ray Absorption Fine Structure (EXAFS). The ligands are 6 +/- 1 nitrogen and/or oxygen ligands at 2.05-2.09 A, with a maximum variance of 0.09 A. The number of imidazole ligands is estimated at 4 +/- 1 using multiple scattering simulations. The remaining ligands are proposed to be carboxylate oxygens.

1. The addition of 13-Ls-hydroperoxylinoleic acid to lipoxygenase-1 (linoleate: oxygen oxidoreductase EC 1.13.11.12) from soybeans at pH 9 and 25 degrees C causes a quenching of the fluorescence of the enzyme at 328 nm when exciited at 280 nm and gives rise to an increase of the absorbance of the enzyme in the 300 nm to 450 nm region. 2. In the absence of 02, addition of linoleic acid to enzyme treated with 13-Ls-hydroperoxylinoleic acid, causes an increase of the fluorescence at 328 nm and a decrease of the absorbance in the 300 nm to 450 nm region. 3. The fluorescence changes are suggested to be directly coupled to the absorbance changes via a non-radioactive energy transfer process. 4. It is proposed that the observed fluorescence and absorbance changes are related to changes in the formal change of iron in the protein.

The circular dichroism spectra of the three forms of lipoxygenase-1 from soybeans show characteristic differences in the region between 300 and 600 nm. Native lipoxygenase-1 only shows a negative dichroic band around 330 nm. Yellow lipoxygenase-1, obtained by addition of an equimolar amount of 13-F-hydroperoxylinoleic acid to the native enzyme, shows a positive Cotton effect at 425 nm, while the negative band band at 330 nm has increased in intensity. The blue enzyme, representing a complex of yellow enzyme with 13-L-hydroperoxylinoleic acid exhibits a negative dichroic band at 580 nm and positive bands at 410 and 391 nm. The near-ultraviolet CD spectra of the three forms of lipoxygenase are very similar, showing several well resolved positive dichroic bands at 0 degrees C. Using the method of Chen et al. (Chen, Y.-H., Yang, J.T. and Martinez, H.M. (1972) Biochemistry 11, 4120--4131) the contents of alpha-helix, beta- and unordered form of native lipoxygenase-1 were estimated to be 34, 27 and 39% respectively.

The steady-state kinetics of the anaerobic reaction of soybean lipoxygenase-1 with linoleic acid and 13-L-hydroperoxylinoleic acid were studied. Initial rates of the formation of oxodienoic acids**, absorbing at 285 nm, were measured at pH 10. About 50% of the consumed 13-L-hydroperoxylinoleic acid was converted into oxodienoic acids regardless of the initial ratio of the two substrates. A linear inhibition by both linoleic acid and 13-L-hydroperoxylinoleic acid was observed in the concentration range studied, which is on the upper side limited by the concentrations at which micelle- or acid-soap formation starts. A kinetic scheme is proposed based on one active site in lipoxygenase-1 which alternately binds the two substrates. Values for the kinetic constants were calculated by fitting simultaneously the complete set of data to the appropriate rate equation.

The interaction of soybean lipoxygenase-1 with 13-Ls-hydroperoxy-9-cis,11-trans-octadecadienoic acid, the product of the enzymic dioxygenation of linoleic acid, results in the formation of either a yellow or a purple coloured enzyme form depending on the amount of product used. The composition of the high-spin Fe(III) signals in the EPR spectra of both enzyme forms has been studied and the amount of EPR-visible iron determined by integration and simulation. Sets of g values of the species building up the high-spin Fe(III) signal around g 6 are derived from both third-order perturbation calculation and exact numerical diagonalization of the spin Hamiltonian describing the system. The results of these calculations are generally applicable to systems having S = 5/2. The iron in the native, colourless enzyme is almost EPR-nondetectable. The yellow form of the enzyme shows a complex EPR signal around g 6 which consists of contributions of at least three species with different ligand symmetry. The signal corresponds to approx. 75% of the total iron content. The g 6 signal of the purple Fe(III) enzyme corresponds roughly to the same amount of iron but the ratio between the different species is not the same as in the yellow enzyme. This enzyme form also shows an additional g 4.3 signal with a large amplitude but a relatively low integrated intensity (approx. 10% of the total iron content). The results are consistent with the suggested mechanism of the catalytic function of iron in lipoxygenase which was based on qualitative EPR results (De Groot, J.J.M.C., Veldink, G.A., Vliegenthart, J.F.G., Boldingh, J., Wever, R. and van Gelder, B.F. (1975) Biochim. Biophys. Acta 377, 71--79).

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.

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%).

The interaction of nitric oxide with the non-heme iron dioxygenase lipoxygenase is reported. This apparently resulted in a novel type of complex where an electron is donated to the NO molecule. In addition a new position for an EPR transition from iron was discovered which, it is suggested results from high spin ferric iron in a field of axial symmetry characterised by a very low value for D.