Arachidonic acid is released from membrane phospholipids upon cell stimulation (for example, by immune complexes and calcium ionophores) and converted to leukotrienes by a 5-lipoxygenase that also has leukotriene A4 synthetase activity. Leukotriene A4, an unstable epoxide, is hydrolyzed to leukotriene B4 or conjugated with glutathione to yield leukotriene C4 and its metabolites, leukotriene D4 and leukotriene E4. The leukotrienes participate in host defense reactions and pathophysiological conditions such as immediate hypersensitivity and inflammation. Recent studies also suggest a neuroendocrine role for leukotriene C4 in luteinizing hormone secretion. Lipoxins are formed by the action of 5- and 15-lipoxygenases on arachidonic acid. Lipoxin A causes contraction of guinea pig lung strips and dilation of the microvasculature. Both lipoxin A and B inhibit natural killer cell cytotoxicity. Thus, the multiple interaction of lipoxygenases generates compounds that can regulate specific cellular responses of importance in inflammation and immunity.

Cyclooxygenase (COX; prostaglandin G/H synthase, EC 1.14.99.1) catalyzes the first two steps in the biosynthesis of prostaglandins (PGs). The two COX isoforms COX-1 and COX-2 are the targets of the widely used nonsteroidal anti-inflammatory drugs, indicating a role for these enzymes in pain, fever, inflammation, and tumorigenesis. The ubiquitous constitutive expression of COX-1 and inducible expression of COX-2 have led to the widely held belief that COX-1 produces homeostatic PGs, while PGs produced by COX-2 are primarily pathophysiological. However, recent discoveries call this paradigm into question and reveal as yet underappreciated functions for both enzymes. This review focuses on some of these new insights.-Rouzer, C. A., and L. J. Marnett. Cyclooxygenases: structural and functional insights. J. Lipid Res. 2009. 50: S29-S34. Supplementary key words prostaglandinThe cyclooxygenase isoforms (COX-1 and COX-2) are among the most thoroughly studied and best understood mammalian oxygenases. Possessing two separate but linked active sites, the COXs catalyze the bis-dioxygenation and subsequent reduction of arachidonic acid (AA) to prostaglandin (PG)G 2 and PGH 2 (Fig. 1A). The mechanism of oxygenation has been well characterized through kinetics, mutagenesis, and X-ray crystallography (1-3). PGH 2 is subject to metabolism by downstream enzymes yielding the family of PGs, each member of which exerts a range of physiologic effects through specific G-protein-coupled receptors (Fig. 1B) (4, 5). The discovery that the COXs are the target of the nonsteroidal anti-inflammatory drugs (NSAIDs), which play a primary therapeutic role in the treatment of pain, fever, and inflammation (6), promulgated the first wave of experimentation on the constitutively expressed COX-1 during the 1970s and 1980s. Then, just as interest began to wane, the discovery of the inducible isoform, COX-2, rekindled a massive new effort that ultimately led to new insights about both isoforms. A search of PubMed over the past 2 years indicates that there have been over 70 review articles containing "cyclooxygenase" in their title, leading one to question the need for yet another. However, despite the overwhelming mass of data available on these enzymes, recent discoveries suggest that some original assumptions concerning their roles in physiology and pathophysiology require reexamination. This review will emphasize these issues. STRUCTURE OF THE COX ENZYMESHuman COX-1 and COX-2 are homodimers of 576 and 581 amino acids, respectively. Both enzymes contain three high mannose oligosaccharides, one of which facilitates protein folding. A fourth oligosaccharide, present only in COX-2, regulates its degradation. Considering the 60% identity in sequence between COX-1 and COX-2, it is not surprising that their three-dimensional structures are nearly superimposable. Each subunit of the dimer consists of three domains, the epidermal growth factor domain (residues 34-72), the membrane binding domain (residues 73-116), and the catalytic domain comprisi...

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Several inflammatory diseases, including asthma, arthritis and psoriasis are associated with the production of leukotrienes by neutrophils, mast cells and macrophages. The initial enzymatic step in the formation of leukotrienes is the oxidation of arachidonic acid by 5-lipoxygenase (5-LO) to leukotriene A4. Osteosarcoma cells transfected with 5-LO express active enzyme in broken cell preparations, but no leukotriene metabolites are produced by these cells when stimulated with the calcium ionophore A23187, indicating that an additional component is necessary for cellular 5-LO activity. A new class of indole leukotriene inhibitor has been described that inhibits the formation of cellular leukotrienes but has no direct inhibitory effect on soluble 5-LO activity. We have now used these potent agents to identify and isolate a novel membrane protein of relative molecular mass 18,000 which is necessary for cellular leukotriene synthesis.

Oxidation of endogenous macromolecules can generate electrophiles capable of forming mutagenic adducts in DNA. The lipid peroxidation product malondialdehyde, for example, reacts with DNA to form M 1 G, the mutagenic pyrimidopurinone adduct of deoxyguanosine. In addition to free radical attack of lipids, DNA is also continuously subjected to oxidative damage. Among the products of oxidative DNA damage are base propenals. We hypothesized that these structural analogs of malondialdehyde would react with DNA to form M 1 G. Consistent with this hypothesis, we detected a dose-dependent increase in M 1 G in DNA treated with calicheamicin and bleomycin, oxidizing agents known to produce base propenal. The hypothesis was proven when we determined that 9-(3-oxoprop-1-enyl)adenine gives rise to the M 1 G adduct with greater efficiency than malondialdehyde itself. The reactivity of base propenals to form M 1 G and their presence in the target DNA suggest that base propenals derived from oxidative DNA damage may contribute to the mutagenic burden of a cell.

A high glucose concentration in vivo or an increased glucose or glucose 6-phosphate concentration in vitro has been found to lead to the glycosylation of Eamino groups of lysine residues in bovine and rat lens crystallins. In vitro, this glycosylation imparts an increased susceptibility of the crystallins to sulfhydryl oxidation. Disulfide crosslinks result in the formation of high molecular weight aggregates and an opalescence in the crystallin solutions. The addition of reducing agents prevents as well as reverses the formation of high molecular weight aggregates and the opalescence of the crystallins. These phenomena suggest a new interpretation of previous results on cataract formation and a new approach for development of drugs to prevent cataracts. The secondary complications of diabetes mellitus now account for most of the morbidity and mortality associated with this disease (1). These sequelae which affect most major organs (e.g., kidney, peripheral nerve, and eye) are of unknown etiology, although many workers believe that they may be due to an increased amount of glycoproteins in capillary basement membranes (2, 3). Several years ago it was proposed (4) that the glycosylation of hemoglobin to form hemoglobin Aic may serve as a biochemical model for the glycosylation purported to occur in diabetes (5). It was found that the glycosylation of hemoglobin AIC occurred as a postsynthetic modification throughout the life of the erythrocyte and that the rate of glycosylation was 2.7 times faster when the erythrocytes circulated in a diabetic mouse than in a normal mouse. Subsequent work has shown this modification to be I-amino-l-deoxyfructose which is specifically attached to the amino-terminal valine in the (3-chain (6, 7 ). This addition occurs nonenzymatically as either the reaction of glucose directly (8) or of glucose 6-phosphate (9, 10) which is then dephosphorylated. It is not clear which is predominant in vivo. The mechanism proposed for both is formation of a Schiff base with the amino group of valine and a subsequent Amadori rearrangement to form a fairly stable product that is reducible with sodium borohydride.Reasoning that a similar type of glycosylation might be occurring in other tissues, we initiated a study of the ocular lens. The lens, like the erythrocyte, is not dependent on insulin for glucose uptake and hence the intracellular glucose concentration reflects the extracellular milieu (11). In the present communication we report that the crystallin proteins of the lens may be glycosylated both in mvo and in vitro at the C-amino groups of lysine residues.MATERIALS AND METHODS Lens Cultures. Lenses from rats were cultured on wire grids in medium 199 containing 5% fetal calf serum and 30 mM glucose according to the method of Obazawa et al. (13). The medium was replaced daily. After 7 and 11 days in culture, the crystallim were extracted from the whole lens in 1 ml of buffer as described below. Freshly isolated normal rat lenses served as uncultured controls.Preparation of Crystallins. ...