15-deoxy-Delta12,14-PGJ2 (15d-PGJ2) has been identified as an endogenous ligand for PPARgamma, inducing adipogenesis in vitro. Additional roles for this molecule in the propagation and resolution of inflammation, ligation of NF-kappaB, and mediation of apoptosis have been proposed. However, quantitative, physiochemical evidence for the formation of 15d-PGJ2 in vivo is lacking. We report that 15d-PGJ2 is detectable using liquid chromatography-mass spectrometry-mass spectrometry at low picomolar concentrations in the medium of 3T3-L1 preadipocytes. However, despite induction of COX-2, production of PGs, including 15d-PGJ2, does not increase during adipocyte differentiation, a process unaltered by COX inhibition. 15d-PGJ2 is detectable as a minor product of COX-2 in human urine. However, its biosynthesis is unaltered during or after COX activation in vivo by LPS. Furthermore, the biosynthesis of 15d-PGJ2 is not augmented in the joint fluid of patients with arthritis, nor is its urinary excretion increased in patients with diabetes or obesity. 15d-PGJ2 is not the endogenous mediator of PPARgamma-dependent adipocyte activation and is unaltered in clinical settings in which PPARgamma activation has been implicated.

The P450 2E1-catalyzed oxidation of ethanol to acetaldehyde is characterized by a kinetic deuterium isotope effect that increases K m with no effect on k cat , and rate-limiting product release has been proposed to account for the lack of an isotope effect on k cat (Bell, L. C., and Guengerich, F. P. (1997) J. Biol. Chem. 272, 29643-29651). Acetaldehyde is also a substrate for P450 2E1 oxidation to acetic acid, and k cat /K m for this reaction is at least 1 order of magnitude greater than that for ethanol oxidation to acetaldehyde. Acetic acid accounts for 90% of the products generated from ethanol in a 10-min reaction, and the contribution of this second oxidation has been overlooked in many previous studies. The noncompetitive intermolecular kinetic hydrogen isotope effects on acetaldehyde oxidation to acetic acid ( H (k cat / K m )/ D (k cat /K m ) ‫؍‬ 4.5, and D k cat ‫؍‬ 1.5) are comparable with the isotope effects typically observed for ethanol oxidation to acetaldehyde, and k cat is similar for both reactions, suggesting a possible common catalytic mechanism. Rapid quench kinetic experiments indicate that acetic acid is formed rapidly from added acetaldehyde (ϳ450 min ؊1 ) with burst kinetics. Pulse-chase experiments reveal that, at a subsaturating concentration of ethanol, ϳ90% of the acetaldehyde intermediate is directly converted to acetic acid without dissociation from the enzyme active site. Competition experiments suggest that P450 2E1 binds acetic acid and acetaldehyde with relatively high K d values, which preclude simple tight binding as an explanation for rate-limiting product release. The existence of a rate-determining step between product formation and release is postulated. Also proposed is a conformational change in P450 2E1 occurring during the course of oxidation and the discrimination of P450 2E1 between acetaldehyde and its hydrated form, the gem-diol. This multistep P450 reaction is characterized by kinetic control of individual reaction steps and by loose binding of all ligands.The microsomal cytochrome P450 1 enzymes are the major enzymes involved in the oxidation of xenobiotic chemicals in eukaryotes (3-5). The P450 monooxygenases catalyze a multitude of oxidation reactions, such as hydroxylation of aliphatic and aromatic carbons, epoxidation of olefins, N-dealkylation of amines, and O-dealkylation of ethers (5, 6). Although a few general catalytic mechanisms appear to be operative for most of the reactions catalyzed by the P450s (Fig. 1) (7-9), certain features such as rate-determining steps and substrate interactions can vary considerably (8,11,12). P450 2E1 is considered to be one of the major human hepatic P450 enzymes (13). Human P450 2E1, as well as the animal orthologs, accepts a broad range of substrates, with apparent preference for small and hydrophobic molecules (4, 14, 15). P450 2E1 is notably active in the oxidation of many low M r volatile solvents with common industrial applications and issues of cancer risk (16,17).Many P450 2E1 reactions have been characterized, at ...

15-deoxy-Δ12,14-PGJ2 (15d-PGJ2) has been identified as an endogenous ligand for PPARγ, inducing adipogenesis in vitro. Additional roles for this molecule in the propagation and resolution of inflammation, ligation of NF-κB, and mediation of apoptosis have been proposed. However, quantitative, physiochemical evidence for the formation of 15d-PGJ2 in vivo is lacking. We report that 15d-PGJ2 is detectable using liquid chromatography–mass spectrometry–mass spectrometry at low picomolar concentrations in the medium of 3T3-L1 preadipocytes. However, despite induction of COX-2, production of PGs, including 15d-PGJ2, does not increase during adipocyte differentiation, a process unaltered by COX inhibition. 15d-PGJ2 is detectable as a minor product of COX-2 in human urine. However, its biosynthesis is unaltered during or after COX activation in vivo by LPS. Furthermore, the biosynthesis of 15d-PGJ2 is not augmented in the joint fluid of patients with arthritis, nor is its urinary excretion increased in patients with diabetes or obesity. 15d-PGJ2 is not the endogenous mediator of PPARγ-dependent adipocyte activation and is unaltered in clinical settings in which PPARγ activation has been implicated

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A key feature of the regulated secretory pathway in neuroendocrine cells is lumenal pH, which decreases between trans-Golgi network and mature secretory granules. Because peptidylglycine ␣-amidating monooxygenase (PAM) is one of the few membrane-spanning proteins concentrated in secretory granules and is a known effector of regulated secretion, we examined its sensitivity to pH. Based on antibody binding experiments, the noncatalytic linker regions between the two enzymatic domains of PAM show pH-dependent conformational changes; these changes occur in the presence or absence of a transmembrane domain. Integral membrane PAM-1 solubilized from rat anterior pituitary or from transfected AtT-20 cells aggregates reversibly at pH 5.5 while retaining enzyme activity. Over 35% of the PAM-1 in anterior pituitary extracts aggregates at pH 5.5, whereas only about 5% aggregates at pH 7.5. PAM-1 recovered from secretory granules and endosomes is highly responsive to low pH-induced aggregation, whereas PAM-1 recovered from a light, intracellular recycling compartment is not. Mutagenesis studies indicate that a transmembrane domain is necessary but not sufficient for low pH-induced aggregation and reveal a short lumenal, juxtamembrane segment that also contributes to pH-dependent aggregation. Taken together, these results demonstrate that several properties of membrane PAM serve as indicators of granule pH in neuroendocrine cells.