Molecular cloning and primary structure of human 15-lipoxygenase

A full-length cDNA encoding 15-lipoxygenase has been isolated from a human reticulocyte cDNA library. The predicted primary structure of the enzyme exhibits a sequence similarity of 61% and 45% with human 5-lipoxygenase and the soybean lipoxygenase isoenzyme I, respectively. When all three lipoxygenases are aligned, there are two distinct regions of significant sequence identity including a cluster of five histidine residues conserved in all three lipoxygenases. Because histidines can serve as ligands for the enzymatically active iron, this region may be critical to enzymatic function. These results provide a basis for exploring functional domains of lipoxygenases.     

Immunohistochemical study of arachidonate 12-lipoxygenase in porcine tissues

12-Lipoxygenase oxygenates the 12 position of arachidonic acid and produces its 12-hydroperoxy derivative. The enzyme is found in greatest amounts in porcine leukocytes and is distributed widely in various other tissues. An anti-12-lipoxygenase antibody was raised in rabbits with the immunoaffinity-purified enzyme as an antigen and was used in immunohisto- and cytochemical studies on the enzyme, the physiological significance of which remains to be clarified. When peripheral blood cells were examined by immunoelectron microscopy, the enzyme was found in neutrophils and monocytes but was not detected in lymphocytes, platelets, and erythrocytes. In immunostained neutrophils and monocytes the enzyme was localized in the cytosol but was not clearly detected in the plasma membrane, nuclear membrane, endoplasmic reticulum, and other organelles. Several other organs known to contain considerable amounts of 12-lipoxygenase were also investigated immunohistochemically, i.e., alimentary tract (ileum and jejunum), lymphatic organs (spleen, lymph node, and thymus), ovary, lung, liver, and others. In these organs, resident mast cells and granulocytes infiltrating the interstitial tissues were positively immunostained. The enzyme was not detected in parenchymal cells of these organs under our experimental conditions.     

Lipoxin synthesis by arachidonate 12-lipoxygenase purified from porcine leukocytes

Arachidonate 12-lipoxygenase purified from porcine leukocytes shows 14R-oxygenase and 14,15-leukotriene A synthase activities with 15-hydroperoxy-arachidonic acid as substrate. The enzyme transformed 5,15-dihydroperoxy-arachidonic acid to several compounds with a conjugated tetraene. A major product was identified as 5S,14R,15S-trihydroperoxy-6,10,12-trans-8-cis-eicosatetraenoic acid, which was reduced to 5S,14R,15S-8-cis-lipoxin B. A requirement of molecular oxygen and the results of H₂¹⁸O experiments suggested that formation of the latter compound was attributed mostly to the 14R-oxygenase activity of the enzyme. There were several other minor products identified as lipoxin A and B isomers. They were produced presumably by hydrolysis of 14,15-epoxy compound formed by the leukotriene A synthase activity of 12-lipoxygenase.     

Analysis of non-heme iron in arachidonate 12-lipoxygenase of porcine leukocytes

Arachidonate 12-lipoxygenase of porcine leukocytes, which was purified to homogeneity by immunoaffinity chromatography, was analyzed for iron content by atomic absorption spectrophotometry. The enzyme contained 0.70 +/- 0.09 g atom of iron per mol of enzyme (mean +/- S.D., n = 4). Inorganic iron, which was added to the enzyme solution as an internal standard, was recovered in almost 100% yield. Among various iron chelators tested, only 2,2'-dipyridyl at 1 mM inactivated the enzyme by 87%, but the enzyme was not reactivated by the addition of excess ferrous or ferric iron.     

Localization of arachidonate 12-lipoxygenase in parenchymal cells of porcine anterior pituitary

12-Lipoxygenases oxygenate arachidonic acid producing its 12S-hydroperoxy derivative and are well known as platelet and leukocyte enzymes. When a peroxidase-linked immunoassay of the enzyme according to the avidin-biotin method was applied to the cytosol fractions from various parts of porcine brain, a considerable amount of the enzyme was found in the anterior pituitary. The enzyme level (about 200 ng/mg cytosol protein) corresponded to about 6% of the enzyme content in porcine peripheral leukocytes. Posterior and intermediate lobes showed about one-tenth of the enzyme level of anterior pituitary. Other parts of porcine brain contained the 12-lipoxygenase in amounts below 7 ng/mg cytosol protein. The cytosol fraction (0.7 mg of protein) of anterior pituitary produced 12S-hydroxy-5,8,10,14-eicosatetraenoic acid from 25 microM arachidonic acid in about 34% conversion at 24 degrees C for 5 min, giving a specific enzyme activity about 3 nmol/min/mg protein. Furthermore, various octadecapolyenoic acids were oxygenated almost as fast as the arachidonate 12-oxygenation. When anterior pituitary was investigated immunohistochemically with anti-12-lipoxygenase antibody, most of the immunostained cells were certain parenchymal cells with granules, which were not blood cells. These biochemical and immunohistochemical results provide a good reason for considering that 12-lipoxygenase does play an important role in pituitary function.     

Molecular cloning and expression of human L-pipecolate oxidase

In higher eukaryotes L-lysine can be degraded via two distinct routes including the saccharopine pathway and the L-pipecolate pathway. The saccharopine pathway is the primary route of degradation of lysine in most tissues except the brain in which the L-pipecolate pathway is most active. L-pipecolate is formed from L-lysine via two enzymatic reactions and then undergoes dehydrogenation to Delta(1)-piperideine-6-carboxylate. At least in humans and monkeys, this is brought about by the enzyme L-pipecolate oxidase (PIPOX) localized in peroxisomes. In literature, several patients have been described with hyperpipecolic acidaemia. The underlying mechanism responsible for the impaired degradation of pipecolate has remained unclear through the years. In order to resolve this question, we have now cloned the human L-pipecolate oxidase cDNA which codes for a protein of 390 amino acids and contains an ADP-betaalphabeta-binding fold compatible with its identity as a flavoprotein. Furthermore, the deduced protein ends in -KAHL at its carboxy terminus which constitutes a typical Type I peroxisomal-targeting signal (PTS I).     

Pharmacological inhibition of arachidonate 12-lipoxygenase ameliorates myocardial ischemia-reperfusion injury in multiple species

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Hinokitiol, a selective inhibitor of the platelet-type isozyme of arachidonate 12-lipoxygenase

Hinokitiol (4-isopropyltropolone), a constituent of Japanese cypress, reversibly inhibited platelet-type 12-lipoxygenase with an IC(50) of 0.1 microM, and the enzyme activity was almost lost at 1 microM. The compound was much less active with other lipoxygenase enzymes with higher IC(50) values (leukocyte-type 12-lipoxygenase, 50 microM; soybean lipoxygenase, 17 microM; 15-lipoxygenase-1, >100 microM; 5-lipoxygenase, 17 microM). Hinokitiol up to 100 microM had almost no effect on cyclooxygenases-1 and -2. Their structure-activity relationship examined with various tropolone derivatives indicated the requirements of the 2-hydroxyl group and 4-alkyl group for the potent and selective inhibition of platelet-type 12-lipoxygenase.     

Molecular cloning and expression of human bile acid beta-glucosidase

A novel microsomal beta-glucosidase was recently purified and characterized from human liver that catalyzes the hydrolysis of bile acid 3-O-glucosides as endogenous compounds. The primary structure of this bile acid beta-glucosidase was deduced by cDNA cloning on the basis of the amino acid sequences of peptides obtained from the purified enzyme by proteinase digestion. The isolated cDNA comprises 3639 base pairs containing 524 nucleotides of 5'-untranslated and 334 nucleotides of 3'-untranslated sequences including the poly(A) tail. The open reading frame predicts a 927-amino acid protein with a calculated M(r) of 104,648 containing one putative transmembrane domain. Data base searches revealed no homology with any known glycosyl hydrolase or other functionally identified protein. The cDNA sequence was found with significant identity in the human chromosome 9 clone RP11-112J3 of the human genome project. The recombinant enzyme was expressed in a tagged form in COS-7 cells where it displayed bile acid beta-glucosidase activity. Northern blot analysis of various human tissues revealed high levels of expression of the bile acid beta-glucosidase mRNA (3.6-kilobase message) in brain, heart, skeletal muscle, kidney, and placenta and lower levels of expression in the liver and other organs.     

Molecular cloning and expression of a human chondroitin synthase

We have identified a human chondroitin synthase from the HUGE (human unidentified gene-encoded large proteins) protein data base by screening with two keywords: "one transmembrane domain" and "galactosyltransferase family." The identified protein consists of 802 amino acids with a type II transmembrane protein topology. The protein showed weak homology to the beta1,3-galactosyltransferase family on the amino-terminal side and to the beta1,4-galactosyltransferase family on the carboxyl-terminal side. The expression of a soluble recombinant form of the protein in COS-1 cells produced an active enzyme, which transferred not only the glucuronic acid (GlcUA) from UDP-[(14)C]GlcUA but also N-acetylgalactosamine (GalNAc) from UDP-[(3)H]GalNAc to the polymer chondroitin. Identification of the reaction products demonstrated that the enzyme was chondroitin synthase, with both beta1,3-GlcUA transferase and beta1,4-GalNAc transferase activities. The coding region of the chondroitin synthase was divided into three discrete exons and localized to chromosome 15. Northern blot analysis revealed that the chondroitin synthase gene exhibited ubiquitous but markedly differential expression in the human tissues examined. Thus, we demonstrated that analogous to human heparan sulfate polymerases, the single polypeptide chondroitin synthase possesses two glycosyltransferase activities required for chain polymerization.     

Molecular cloning and expression of the human interferon-gamma receptor

A cDNA encoding the human interferon-gamma receptor was isolated from a lambda gt11 expression library using a polyclonal antireceptor antiserum. The gene for this receptor was identified in a cosmid library and transfected into mouse cells. The human interferon-gamma receptor expressed in mouse cells displayed the same binding properties as in human cells. However, transfected cells were not sensitive to human IFN-gamma, suggesting the need for species-specific cofactors in receptor function. As inferred from the cDNA sequence, the human interferon-gamma receptor shows no similarities to known proteins and represents a novel transmembrane receptor. It is most likely the product of a single mRNA and a gene located on chromosome 6q.