Products, kinetics, and substrate specificity of homogeneous thromboxane synthase from human platelets: development of a novel enzyme assay.
Homogeneous thromboxane synthase from human platelets converted prostaglandin H2 (PGH2) to thromboxane A2 (measured as thromboxane B2, TxB2), 12(L)-hydroxy-5,8,10-heptadecatrienoic acid (HHT), and malondialdehyde (MDA) in equimolar amounts under a variety of experimental conditions. PGG2 was transformed to MDA and corresponding 15- and 12-hydroperoxy products. PGH1 was enzymatically transformed into 12(L)-hydroxy-8,10-heptadecadienoic acid (HHD) and PGH3 into TxB3 and 12(L)-hydroxy-5,8,10,14-heptadecatetraenoic acid (delta 14-HHT) as earlier reported for solubilized and partially purified thromboxane synthase preparations. The ratio of thromboxane to C17 hydroxy fatty acid formation was 1:1 with PGG2, PGH2, and PGH3 as substrates. These results confirm and extend earlier observations with partially purified enzyme that the three products are formed in a common enzymatic pathway (Diczfalusy, U., Falardeau, P., and Hammarström, S. (1977) FEBS Lett. 84, 271-274). A convenient spectrophotometric assay for thromboxane synthase activity measuring the ultraviolet light absorption of the C17 hydroxy acid formed (e.g., HHT) was developed. The validity of the assay was determined employing specific inhibitors for thromboxane synthase. The substrate specificity of thromboxane synthase was determined using this assay. PGG2 and PGH3 showed Vmax and KM values similar to those of PGH2. The KM value of PGH1 was also identical to that of PGH2 but the Vmax value PGH1 was more than twice as high as that of PGH2.
D001792:Blood Platelets / Q000201:enzymology*; D005231:Fatty Acids, Unsaturated / Q000032:analysis; D006801:Humans; D066298:In Vitro Techniques; D007700:Kinetics; D008315:Malondialdehyde / Q000032:analysis; D011449:Prostaglandin Endoperoxides / Q000302:isolation & purification; D011462:Prostaglandins G; D011463:Prostaglandins H; D013379:Substrate Specificity; D013930:Thromboxane-A Synthase / Q000097:blood*
D005231:Fatty Acids, Unsaturated; D011449:Prostaglandin Endoperoxides; D011462:Prostaglandins G; D011463:Prostaglandins H; C025560:12-hydroxy-8,10-heptadecadienoic acid; D008315:Malondialdehyde; C038291:prostaglandin G2; D013930:Thromboxane-A Synthase