Isolation and characterization of thromboxane synthase from human platelets as a cytochrome P-450 enzyme.
Thromboxane synthase from human platelets was purified to apparent homogeneity by conventional chromatographic techniques. A 423-fold enrichment over the specific content in the 100,000 X g sediment from platelet homogenates was obtained. The enzyme gave a single band on sodium dodecyl sulfate-gel electrophoresis corresponding to a monomeric molecular weight of 58,800. One heme per polypeptide chain was present, and by optical and EPR spectroscopy a close analogy to the group of cytochrome P-450 proteins was established. From its substrate prostaglandin H2, the stable end product thromboxane B2 is formed with a specific activity of 24.1 mumol min-1 mg of protein-1 which corresponds to a molecular activity of 1628 min-1. The enzyme formed 12L-hydroxy-5,8,10-heptadecatrienoic acid together with thromboxane B2 in a 1:1 ratio. Both products were identified by gas chromatography-mass spectrometry analysis. As reported previously for platelet microsomes (Ullrich, V., and Haurand, M. (1983) Adv. Prostaglandin Thromboxane Leukotriene Res. 11, 105-110), the pure hemoprotein spectrally interacts with pyridine- or imidazole-based inhibitors and for the potent inhibitor imidazo-(1,5-a)pyridine-5-hexanoic acid a stoichiometric binding to the heme was shown. Substrate analogs with a methylene group replacing the oxygen in either the 9- or 11-position caused difference spectra showing spectral shifts towards 387 and 407 nm, respectively. The identification of thromboxane synthase as a P-450 protein suggests that the heme-thiolate group of the enzyme is required to split and activate the endoperoxide bond of prostaglandin H2.
D001792:Blood Platelets / Q000201:enzymology*; D002855:Chromatography, Thin Layer; D003577:Cytochrome P-450 Enzyme System / Q000097:blood*; D004227:Dithionite / Q000494:pharmacology; D004578:Electron Spin Resonance Spectroscopy; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D008861:Microsomes / Q000201:enzymology; D008970:Molecular Weight; D011450:Prostaglandin Endoperoxides, Synthetic / Q000378:metabolism; D044262:Prostaglandin H2; D011463:Prostaglandins H / Q000378:metabolism; D013056:Spectrophotometry, Ultraviolet; D013929:Thromboxane B2 / Q000378:metabolism; D013930:Thromboxane-A Synthase / Q000302:isolation & purification*
D011450:Prostaglandin Endoperoxides, Synthetic; D011463:Prostaglandins H; D004227:Dithionite; D044262:Prostaglandin H2; D013929:Thromboxane B2; D003577:Cytochrome P-450 Enzyme System; D013930:Thromboxane-A Synthase