Chieko Yokoyama

Partial purification and characterization of arachidonate 12-lipoxygenase from rat lung

Incubation of rat lung cytosol with arachidonic acid produced 12-hydroxy-5,8,10,14-eicosatetraenoic acid as a major product, which was identified by gas chromatography-mass spectrometry. By ammonium sulfate fractionation and DEAE-cellulose chromatography the arachidonate 12-lipoxygenase was purified about 30-fold from the rat lung cytosol. The partially purified enzyme was mostly free of the glutathione peroxidase activity and transformed arachidonic acid to its 12-hydroperoxide. 5,8,11,14,17-Eicosapentaenoic acid was also an active substrate, and the oxygenation at C-12 was confirmed by mass spectrometry. A significant amount of 12-lipoxygenase activity was also found in the microsomes and other particulate fractions.

Regulation of two isozymes of prostaglandin endoperoxide synthase and thromboxane synthase in human monoblastoid cell line U937

The mechanism responsible for the rapid increase of thromboxane A2 synthesis by cells of the human monoblastoid cell line U937, which were differentiated with 12-O-tetradecanoyl-phorbol-13-acetate, induced by lipopolysaccharide (LPS) was studied. Both RNA blot and immunoblot analyses showed that LPS increased the levels of prostaglandin endoperoxide synthase-1 (PES-1) and -2 (PES-2) in a time-dependent manner, and the modes of induction of the two isozymes differed. The maximum PES-1 mRNA level was 1.6 times higher 36 h after than before stimulation by LPS, and that of PES-2 mRNA was elevated about 20-fold at its peak at 12 h after stimulation. Consequently, the immunoreactive PES-1 and PES-2 protein levels also increased time-dependently after LPS stimulation. However, the effects of LPS on the thromboxane synthase mRNA and protein levels were much less marked. These results indicate that LPS-induced thromboxane synthesis by the differentiated cells was regulated at the levels of the two PES isozymes, predominantly at the PES-2 level.

Molecular evolution of cyclooxygenase and lipoxygenase

Four oxygenases of the arachidonic acid cascade (cyclooxygenase, 5-lipoxygenase, 12-lipoxygenase and 15-lipoxygenase) were investigated by the method of computer-assisted sequence comparison. From the calculations, some aspects of evolution and function of these enzymes were revealed. (1) The evolutionary origin of cyclooxygenase was different from that of lipoxygenases. (2) Cyclooxygenase was a distantly related member of a peroxidase family. (3) Enzymes with 12-lipoxygenase activity were created independently twice by gene duplication.

Biochemical Studies on Mammalian Lipoxygenasesa,b

The arachidonate cascade provides biochemists with a number of materials of enzymological interest. Mammalian tissues have several lipoxygenases, each with a positional and stereochemical specificity of arachidonate oxygenation. Some lipoxygenases are involved in the synthesis of bioactive oxyeicosanoids, while others are still of unknown physiological significance. Fatty acid cyclooxygenase is an atypical lipoxygenase incorporating two molecules of oxygen into arachidonic acid and producing a prostaglandin peroxide.

Mechanistic studies of the dioxygenase and leukotriene synthase activities of the porcine leukocyte 12S-lipoxygenase

Lipoxygenases react with hydroperoxy fatty acids and catalyze dioxygenase or dehydrase (leukotriene A4 (LTA4) synthase) types of reactions. In the present investigation we studied the mechanism of reaction of the purified porcine leukocyte 12S-lipoxygenase with 15S-hydroperoxyeicosatetraenoic acid (15S-HPETE). Oxygen-18 labeling experiments with GC-MS analysis were used to distinguish dioxygenase and leukotriene synthase activities of the enzyme; 8S,15S-DiHPETE and 14R,15S-DiHPETE were formed by oxygenation, and a series of 8,15- and 14,15-diols were formed via enzymatic synthesis of 14,15-LTA4 and nonenzymatic hydrolysis of the epoxide. 10D-3H- and 10L-3H-labeled substrates were used to study the stereospecificity of the C-10 hydrogen abstraction in the synthesis of these products. Formation of 14,15-DiHPETE and 14,15-LTA4 was associated with stereoselective abstraction of hydrogen from the 10L position of 15S-HPETE. The same type of measurements on the 8S,15S-DiHPETE product indicated a variable (50-250%) retention of the 10L-3H label, and a consistent 90% retention of the 10D-3H. In contrast, the synthesis of 8S,15S-DiHPETE by the soybean lipoxygenase was associated with the expected stereoselective abstraction of the 10D hydrogen. It appears that the porcine leukocyte 12S-lipoxygenase synthesizes 8S,15S-DiHPETE by a different mechanism.

Lipoxin Syntheses by Arachidonate 12- and 5-Lipoxygenases Purified from Porcine Leukocytes

Recently, we were successful in the purification of 12-lipoxygenase1 and 5-lipoxygenase2 from porcine leukocytes by immunoaffinity chromatography using their monoclonal antibodies which were raised with the crude enzyme preparations as antigen3, 4. In this chapter the lipoxin biosynthesis will be discussed as catalytic properties of these purified lipoxygenases.

ARACHIDONATE 12-LIPOXYGENASE OF HUMAN PLATELETS

Thromboxane A2 is well known as a proaggregatory and vasoconstrictive compound [1]. It is produced from arachidonic acid in platelets, and fatty acid cyclooxygenase responsible for the thromboxane synthesis is localized in the microsomal fraction of human platelets [2]. There is another metabolic pathway of arachidonic acid in human platelets. The pathway is initiated by the reaction of arachidonate 12-lipoxygenase, which is localized predominantly in the cytosol of human platelets [3].

Two Types of Arachidonate 12-Lipoxygenase Demonstrated by Enzymological Immunological and Molecular Biological Studies

Each branch of the arachidonate cascade is initiated by a lipoxygenase reaction, by which a certain carbon atom of arachidonic acid is oxygenated. Several lipoxygenases have been found in mammalian tissues, and their physiological and pathological roles have been studied (1,2). Unlike cyclooxygenase and 5-lipoxygenase, which initiate the synthesis of prostaglandins and leukotrienes, general physiological functions of other mammalian lipoxygenases remain still unclarified.

Immunobiochemical and Molecular Biological Studies on Arachidonate 12-Lipoxygenase of Porcine Leukocytes

Arachidonate 12-lipoxygenase is a dioxygenase which incorporates one molecule of oxygen into arachidonic acid regiospecifically and stereospecifically, and produces 12S-hydroperoxy-5,8,10,14-eicosatetraenoic acid (Figure 1). Since the enzyme was found in platelets as the first mammalian lipoxygenase (1,2), the 12-lipoxygenase has been found in a variety of tissues of a number of animal species (3,4). In addition to 12-lipoxygenase, several more lipoxygenases have been found in mammalian tissues. It is well known that the leukotriene synthesis is initiated by 5-lipoxygenase and the biosynthesis of prostaglandin and thromboxane by cyclooxygenase. These compounds have specific biological activities which regulate the functions of various animal tissues, and their biological functions have been generalized for a variety of animal species and tissues. However, no bioactive compound with such a general function has so far been found in the 12-lipoxygenase pathway of arachidonate metabolism (5).