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A scintillation proximity assay for peroxisome proliferator-activated receptor gamma ligand binding domain is described. Scintillation proximity offers an equilibrium method for detecting ligands that is both cost effective and fully automatable. The method described here is the first reported scintillation proximity assay for a peroxisome proliferator-activated receptor. The design of this system is generic in nature, allowing it to be adapted for other ligand binding proteins.

Kynurenine 3-monooxygenase is a flavin-dependent monooxygenase that catalyzes the oxidation of L-kynurenine to 3-hydroxy-L-kynurenine in the kynurenine pathway of tryptophan metabolism. The enzyme requires NADH or NADPH as a cofactor. A discontinuous assay that utilizes L-[3H]kynurenine as substrate is described. The assay offers high precision and a wide range of accessible substrate and cofactor concentrations. The assay was used to measure kinetic isotope effects and the stereospecificity of oxidation of the cofactor. Hydride is transferred from the A-side (pro-R) of NADH and NADPH since primary deuterium isotope effects were observed for both cofactors when they were deuterated on the A-side but not on the B-side. The large isotope effect on Vmax/Km for NADH is sensitive to the concentration of kynurenine, which indicates that NADH can bind before kynurenine.

Human erythrocytes and lymphocytes were isolated from venous blood and subjected to one of two protocols. In one protocol the suspended cells were labeled with fluorophore (fluorescamine or 12(9)AS). This procedure was followed sequentially by cellular lysis, cellular fractionation, and fluorescence and absorption readings. In the other protocol the suspended cells were lysed, and then the cellular homogenate labeled with fluorophore followed by cellular fractionation and spectroscopy readings. The lymphocytes were fractionated into plasma membrane, cytosol, and nuclear-mitochondrial fractions and the erythrocytes into plasma membrane and cytosol fractions. The results demonstrate that under the given labeling conditions, both fluorescamine and 12(9)AS are highly localized to the plasma membrane of intact human erythrocytes and lymphocytes. Furthermore, by P-31 NMR analysis, fluorophore labeling did not alter cellular high energy phosphate metabolism or cellular permeability to Mn2+. Therefore, these fluorophores are potentially powerful probes of human erythrocyte and lymphocyte plasma membrane dynamics in inherited and acquired disease states.

The rate-limiting step of the lipoxygenase reaction involves the abstraction of a hydrogen from the methylene carbon of a 1,4-diene. One possibility for the mechanism of the enzyme is the abstraction of this hydrogen as a proton to generate a carbanionic intermediate or transition state. In order to investigate this possibility, 5-, 8-, 12-, and 15-hydroxy-eicosatetraenoic acid were oxidized to the corresponding ketones and these ketones were assayed as substrates of the 5-, 12-, and 15-lipoxygenases from rat neutrophils, rat platelets, and soybeans, respectively. The ketones were in no case better substrates than arachidonic acid and in some cases the hydroxyeicosatetraenoic acids were equally active as the corresponding ketones. Since no increased rate of oxidation for these electrophilic substrates was observed, it is concluded that no transition state with carbanionic character is generated in the rate-determining step of the lipoxygenase reaction.

A series of substituted 3,4-dihydro-2H-1,4-thiazines inhibit 5-lipoxygenase from rat leukocytes and exhibit submicromolar IC50 values. A novel synthesis of these compounds was developed based on the formation of hydroxymethyleneamine 13 and its cyclization to the title compounds. The dihydrothiazines have low oxidation potentials, typically E1/2 is near 0.3 V, and a representative compound reduces Fe(III)(phen)3, with k = 10(5) M-1s-1. We propose that these lipophilic compounds bind to 5-lipoxygenase and reduce the iron in the active site, thus inactivating the enzyme.