Pallu Reddanna

Inhibition of 5-lipoxygenase by vitamin E

Purified 5‐lipoxygenase from potato tubers was inhibited strongly by vitamin E and its analogs. The inhibition by d‐α‐tocopherol was found to be irreversible and non‐competitive with respect to arachidonic acid. An IC50 of 5μM was calculated for d‐α‐tocopherol. The inhibition appears to be unrelated to its antioxidant function. Binding studies with 14C‐labelled d‐α‐tocopherol revealed that there is a strong interaction between vitamin E and 5‐lipoxygenase. Tryptic digestion and peptide mapping of 5‐lipoxygenase‐vitamin E complex indicate that vitamin E binds strongly to a single peptide. These studies suggest that cellular vitamin E levels may have profound influence on the formation of leukotrienes.

Purification of arachidonate 5-lipoxygenase from potato tubers

Publisher Summary The chapter briefly describes the assay, the purification, and the properties of arachidonate 5-1ipoxygenase (EC 1.13.11.34) from potato tubers. Lipoxygenases (EC 1.13. 11. 12) are a group of closely related enzymes that appear to be widely distributed in plants and mammalian tissues. One of the most physiologically important lipoxygenases and the subject of greater interest in recent years is 5-lipoxygenase, which catalyzes the first committed step in leukotriene (LT) biosynthesis. Information on the mechanistic details of this enzyme-catalyzed reaction is scant because of limitations in obtaining sufficient quantity of the purified enzyme in stable form from mammalian sources. Therefore, potato tubers are frequently employed as the source of large amounts of 5-1ipoxygenase. The mechanism of dioxygenation of polyunsaturated fatty acids (PUFA) catalyzed by lipoxygenases involves the following three main steps: (1) Abstraction of a hydrogen atom from the double allylic methylene carbon atom (the first and rate-limiting step), (2) conjugation of double bonds followed by rearrangement of the radical electron, and (3) addition of molecular oxygen (this occurs at a diffusion-controlled rate under ambient O 2 concentrations). Based on this mechanism, it is possible to determine the lipoxygenase activity by measuring the following: (1) absorbance of conjugated diene at 235 nm, (2) O 2 uptake by means of a Clark oxygen electrode, and (3) product formation by means of high-performance liquid chromatography (HPLC) or thin-layer chromatography (TLC). Although each one of these methods can be used for monitoring enzyme activity during the course of purification of 5 -lipoxygenase from potato tubers, the polarographic method is found to be the most convenient and reproducible.

Stereochemical nature of the products of linoleic acid oxidation catalyzed by lipoxygenases from potato and soybean

When linoleic acid was incubated with the purified potato lipoxygenase under O2 atmosphere, a mixture of 9 and 13-hydroperoxyoctadecadienoic acids was formed. Stereochemical analysis of the respective methyl-hydroxyoctadecadienoic acids revealed that the 9-isomer was in S-configuration whereas 13-hydroxyoctadecadienoic acid was a mixture of S (39%) and R (61%). Exactly the opposite was the case with the soybean lipoxygenase products, where the 13-isomer was found to be in S-configuration and 9-hydroxyoctadecadienoic acid - a mixture of S (73%) and R (27%). A general scheme is proposed for the stereochemical nature of oxidation products of enzymes which are predominantly either [+2] or [-2] lipoxygenases.

Isolation and characterization of 5-lipoxygenase from tulip bulbs

An unique membrane bound lipoxygenase was isolated and purified from purple star tulip bulbs with a specific activity of 5.2 mu moles O2 consumed.min-1.mg-1 protein. The purified tulip enzyme exhibits regiospecificity for O2 insertion at C-5 of the arachidonic acid molecule. Identification of the reaction product was confirmed as 5-hydroperoxyeicosatetraenoic acid by analytical criteria which included: cochromatography with the authentic compound, as well as mass spectral and 1H-NMR analysis. Thus, the enzyme from tulip bulbs appears to be different from the cytosolic lipoxygenase from potato tubers, which exhibits non-regiospecificity in terms of O2 incorporation. However, the purified tulip lipoxygenase showed a strong immunological crossreactivity with antiserum raised against the purified potato lipoxygenase, indicating close immunological relationship with the other plant lipoxygenases.

11-Hydroperoxyeicosatetraenoic acid is the major dioxygenation product of lipoxygenase isolated from hairy root cultures of Solanum tuberosum

The profile of primary dioxygenation products of arachidonic acid catalyzed by lipoxygenase isolated from hairy root cultures of Solanum tuberosum treated with a fungal elicitor was compared to that obtained for the enzyme from potato tubers. 11-Hydroperoxyeicosatetraenoic acid (11-HPETE) was the most abundant dioxygenation product formed followed by 8- and 5-HPETEs in the decreasing order of abundance. In contrast, 5-HPETE is the predominant oxidation product of lipoxygenase from potato tubers. Differences in the defense requirements of storage tuber as compared to roots may be the basis of the differences in regio-specificity demonstrated in this work.

The effect of selected arachidonic acid metabolites on natural killer cell activity

The effect of arachidonic acid (AA) metabolites of lipoxygenase(s) was evaluated on natural killer (NK) cell activity in Fischer F344 rat splenic lymphocytes and compared with prostaglandin E2 (PGE2), a known inhibitor of NK cell lytic activity. It was observed that 5(S),12(S)-dihydroxy-6,10-trans-8,14-cis-eicosatetraenoic acid (5(S),12(S)-diHETE, EZEZ) inhibited NK cell activity to a degree comparable to the inhibitory effects of PGE2. This compound maximally inhibited NK cell activity at concentrations of 10(-6) and 10(-8) M. PGE2 and 5(S),12(S)-diHETE (EZEZ) inhibited NK activity to an identical degree at all concentrations and effector:target (E:T) cell ratios tested. Of the other lipoxygenase pathway metabolites screened, 8(S),15(S)-all trans-diHETE and 8(S),15(S)-diHETE (EZEZ) also inhibited NK activity, but only at 10(-6) M and a 50:1 E:T cell ratio. These findings provide further evidence that the lipoxygenase and cyclooxygenase pathways produce metabolites which can modulate NK cell function, and that 5(S),12(S)-diHETE (EZEZ), which has not been previously tested for effects on NK cells, may have a significant immunoregulatory role.