Histochemical analyses using antibody or cDNA probes reveal

Histochemical analyses using antibody or cDNA probes reveal the precise cellular location of the 12S-lipoxygenases in tissues of interest. An immunohistochemical study in various rat tissues showed specific expression of the leukocyte-type 12S-lipoxygenase in macrophages in lung and spleen [27]. In situ hybridization showed high level expression of murine leukocyte-type 12S-lipoxygenase in 30–40% of resident peritoneal macrophages [22]; however, no specific expression was detected in other populations of mouse macrophages (alveolar and bone marrow-derived). We have demonstrated that rat leukocyte-type 12S-lipoxygenase is localized to a cells of pancreatic islet, zona glomerulosa cells of adrenal cortex, and neuronal cells of spinal cord and superior cervical ganglion [27]. The platelet-type 12S-lipoxygenase is present in open-type enteroendocrine cells of the epithelium of stomach and intestine as demonstrated by immunohistochemical staining [31]. Interestingly, the leukocyte-type 12S-lipoxygenase is found in defined brain regions (pituitary and pineal glands) of several animal species [12], [32]. When porcine anterior pituitary cells are examined by immunohistochemical double-staining using palovarotene against the 12S-lipoxygenase and pituitary hormones, approximately 7% of the cells producing luteinizing hormone and follicle-stimulating hormone were also positive for the leukocyte-type enzyme, while other cells contained scarcely any enzyme [33]. We demonstrated that the rat pineal 12S-lipoxygenase showed diurnal fluctuation being high in the dark and the lowest around 16.00h [34]. With regard to the intracellular localization of 12S-lipoxygenase, immunoelectron microscopy revealed a selective localization of 12S-lipoxygenase in the cytoplasm of porcine polymorphonuclear leukocytes [35]. Although the 12-lipoxygenase activity was detected in the cytosol of human and rat platelets, the platelet-type 12S-lipoxygenase was found in both cytosol and microsomal fractions of epidermal cells of human skin [36]. We have shown that the 12S-lipoxygenase of human platelets is activated by translocation from cytosol to membranes upon thrombin stimulation. Furthermore, an anti-platelet agent, OPC-29030, suppresses 12S-HETE production by inhibiting a step in the enzyme translocation [37]. The microsomal enzyme has been shown to be selectively overexpressed in germinal layer keratinocytes during psoriatic inflammation [36]. It is of interest that a microsomal 12S-lipoxygenase is expressed in cultured ovine tracheal epithelial cells, and is regulated by changes in cellular oxidation–reduction conditions [38].
Protein structure and enzymological properties Both mammalian and plant lipoxygenases are non-heme iron-containing dioxygenases [3], [6]. In fact, the purified 12S-lipoxygenase of porcine leukocytes contains approximately 1g atom of iron per mol of enzyme [39]. Alignment of amino acid sequences of all the lipoxygenases shows that five invariant histidines are conserved in the central region of the enzymes [6]. Three-dimensional X-ray structures are, as yet, only available for the soybean lipoxygenase and rabbit reticulocyte 15-lipoxygenase [40], [41]. Reviews on the molecular modeling indicate that the active site structure is well-conserved in plant and animal lipoxygenases [40], [41]. By analogy with the crystal structure of soybean and reticulocyte 15-lipoxygenases, the 12S-lipoxygenase is presumably composed of an N-terminal beta-barrel domain and a C-terminal domain containing a hydrophobic substrate-binding site. The non-heme iron atom is coordinated by three histidine residues and the carboxy-terminal isoleucine [40], [41]. Mutations of histidines at 361, 366 and 541 of porcine leukocyte 12S-lipoxygenase cause a complete loss of enzyme activity and iron binding, indicating that these histidines are essential for catalysis [42]. Mutagenesis and deletion of the highly conserved C-terminal isoleucine of murine 12S-lipoxygenase abolishes enzyme activity, suggesting that there is a stringent requirement for the proper spatial alignment and folding of the C-terminal chain [10]. The general structure and active site models of lipoxygenases are discussed in detail elsewhere.