Reticulocyte 15-lipoxygenase

It has been reported that a single methionine residue of rabbit reticulocyte 15-lipoxygenase can be oxidized to its sulphoxide by treatment of the enzyme with 13-hydroperoxy-octadecadienoic acid (a 15-lipoxygenase product from linoleic acid) under anaerobic conditions [59]. Since under this condition the enzyme functioned as lipohydroperoxidase , splitting the hydroperoxide, and resulted in self-inactivation , a central role of the methionine residue was presumed for the catalysis of the enzyme. Recently, this particular methionine has been identified as Met-590 in human 15-lipoxygenase and as Met-591 in rabbit 15-lipoxygenase. When Met-590 in the human enzyme is replaced by leucine by the site-directed mutagenesis, the mutant enzyme is still inactivated by 13-hydroperoxyoctadeca-dienoic acid, 15-HpETE or AA. The result shows that the enzyme inactivation is not attributable to methionine oxidation [60]. 

Lipoxygenase in homogenates from rat platelets [121] and human erythroleukaemia (HEL) cells [148] is translocated from the cytosol to membrane in a calcium-dependent manner. The calcium-dependent translocation has also been observed with rabbit reticulocyte 15-lipoxygenase [149]. 

Different isoforms of 15-lipoxygenase have been purified to homogeneity from mammalian sources, including reticulocytes and leukocytes, and complementary DNAs encoding them have been cloned. The reticulocyte 15-lipoxygenase is a cytosolic protein, whose amino acid sequence is 65% similar to human 5-lipoxygenase and 45% similar to type I soybean lipoxygenase (whose tridimensional structure has been resolved by... 

Kuhn, H., Belkner, J., Wiesner, R. and Brash, A.R (1990). Oxygenation of biological membranes by the pure reticulocyte lipoxygenase. J. Biol. Chem. 265, 18351-18361. 

Schewe, T., Rapoport, S.M., and Kuhn, H. (1986). Enzymolc and physiology of reticulocyte lipoxygenase comparison with other lipoxygenases. 191-272. 

LOX catalyzed the oxidation of arachidonoylphosphatidylcholine at both carbon-12 and carbon-15. Later on, it has been found [21] that reticulocyte lipoxygenase oxidized rat liver mitochondrial membranes, beef heart submitochondrial particles, rat liver endoplasmic membranes, and erythrocyte plasma membranes without preliminary release of unsaturated acids by phospholipases. 

Figure 6. (A) - The oxygenation of dilinoleoylphosphatidilcholine (DLPC) liposomes by C-15 plant (from soybeans) or animal (from rabbit reticulocytes) lipoxygenase ...

Increased content of conjugated dienes in linoleate acyls in the mixed liposomes composed 95% of DPPC and 5% of DLPC caused the increase in their microviscosity (Figure 11, curve 1). The microviscosity of liposome membranes containing 100% DLPC was considerably decreased upon the enzymatic oxidation by C-15 reticulocyte lipoxygenase (Figure 11, curve 2). 

V.Z. Lankin, N.T. Gordeeva, A.K. Tikhaze and A.M. Vikhert, Animal lipoxygenases. The nature of substrate and changes in conformation of reticulocyte lipoxygenase in its interaction with membranes, Biokhimiia (Moscow) 50 (1985) 1894-1900 (English translation in Biochemistry). 

Figure 5. The oxygenation of cholesteryl arachidonate by animal or plant C-15 lipoxygenases (1), oxidation by rabbit reticulocyte lipoxygenase (2), oxidation by soybean lipoxygenase.

Figure 6. Stimulation by human LDL of the arachidonic acid oxidation catalyzed by C-15 animal lipoxygenase (reticulocyte lipoxygenase) (1), in the absence of LDL (2), in the presence of LDL.

J.Belkner, R.Wiesner R., H.Kuhn H. and V.Z.Lankin, The oxygenation of cholesterol esters by the reticulocyte lipoxygenase, FEBS Lett. 279 (1991) 110-114. 

Murray JJ, Brash AR. Rabbit reticulocyte lipoxygenase catalyzes specific 12(S) and 15(S) oxygenation of arachidonate-containing phospholipids. Arch Biochem Biophys 1988 265 514-531. 

A series of indolizines 281 and azaindolizines 282 were screened as possible inhibitors of 15-lipoxygenase (15-LO) from soybeans and rabbit reticulocytes. Most compounds studied were significantly more active than quercitin (IC50 51 pi) <2003BML5409>. The indolizine and azaindolizine sulfonates were particularly studied and showed high activity <2005BMC5409>. 

Additional lipoxygenases are known which oxygenate different positions on the arachidonic acid chain. 12-LO, resulting in the formation of 12-HETE (7), is best known in platelets, while the 15-LO from soybean has been studied in detail for many years [8]. 15-HETE (8) is also produced by mammalian cells the enzymes from neutrophils and particularly rabbit reticulocytes are the best characterized. 

In particular the C-15 animal lipoxygenase may oxidize unsaturated acyls of membrane phospholipids [6,7] (Figure 3) and this process plays the leading role in the internal cell membranes decomposition during maturation of reticulocyte to erythrocyte [6]. 

Figure 3. The oxidation of various native membrane preparations by animal (rabbit reticulocyte) C-15 lipoxygenase (1), erythrocyte ghosts (2), liver microsomes (3), liver mitochondria.

Figure 4, The cooxidation of P-carotene (X.=450 nm) by secondary lipid free radicals which formed during arachidonic acid peroxidation (A,=233 nm) by animal (rabbit reticulocyte) C-15 lipoxygenase in the water...

V.Z. Lankin, H. Kuhn, C. Hiebsch, T. Schewe, S.M. Rapoport, A.K. Tikhaze and N.T. Gordeeva, On the nature of the stimulation of the lipoxygenase from rabbit reticulocytes by biological membranes, Biomed.Biochim.Acta 44 (1985) 655-664. 

S. Stvolinsky et al., Effect of carnosine on lipoxygenase activity of rabbit reticulocytes. Bull. Exp. Biol Med. (Russian), 119(1996)40-43. 

Rapoport SM, Schewe T, Wiesner R, Halangk W, Ludwig P, Janicke-Hohne M, Tannert C, Hiebsch C, Klatt D. The lipoxygenase of reticulocytes. Purification, characterization and biological dynamics of the lipoxygenase its identity with the respiratory inhibitors of the reticulocyte. Eur J Biochem. 96 (1979) 545-561. 

Schewe T, Halangk W, Hiebsch C, Rapoport SM. A lipoxygenase in rabbit reticulocytes which attacks phospholipids and intact mitochondria. FEBS Lett. 60 (1975) 149-152. 

The acetylenic analog of Mead Acid, 5,8,11-eicosatriynoic acid, was reported to be a selective inhibitor of platelet 12-lipoxygenase. Acetylenic fatty acids also inhibited 15-lipoxygenases of plant (soybean) as well as of animal (rabbit reticulocyte) origin. The nature of the acetylenic compound significantly affected its activity on the soybean enzyme, but not the rabbit enzyme. For the former, 7,10,13-eicosatriynoic acid was the most powerful inactivator. Addition of a fourth triple bond at position 4 or 5 strongly reduced the rate of inactivation. On the other hand, the rabbit reticulocyte enzyme was inactivated almost equally well by the various acetylenic fatty acids that were tried. The mechanism of inactivation of lipoxygenases was also suicide inhibition . ... 

HETE. The 15-lipoxygenase (15-LOX) has been the most extensively characterized pathway in the reticulocytes, leukocytes, and airway epidermal cells (24-27). An outline in Figure 5 shows that 15-LOX can, on the one hand, catalyze the abstraction of a proton from C-13 of 20-carbon AA to produce 155-hydroperoxyeicosatetraenoic acid (155-HPETE), whereas on the other hand, the 18-carbon linoleic acid is converted mainly to 13-hydroperoxyoctadecadienoic acid (13-HPODE) and 9-hydroperoxy-10,12- , Z-octadecadienoic acid (9-HPODE) in the ratio of 10 1 (28). Both the 155-HPETE (intermediate) from AA and 13-HPODE (intermediate) from linoleic acid can be further metabolized by glutathione peroxidase to mainly monohydroxylated 15S-HETE and 13-hydroxy-octadecadienoic acid (HODE), respectively. 

Rapoport, S M., Schewe, T., Wiesner, R., Halangk, W., Ludwig, P, Janicke-Hohne, M., Tannert, L., Hiebsch, C., and Klatt, D. (1979) The Lipoxygenase of Reticulocytes. Purification, Characterization and Biological Dynamics of the Lipoxygenase Its Identity with Respiratory Inhibitors of the Reticulocyte, Eur. J. Biochem. 96,545-561. 

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