PMID: 3745158Sep 5, 1986Paper

Immunoaffinity purification and characterization of thromboxane synthase from porcine lung.

The Journal of Biological Chemistry
R F Shen, H H Tai

Abstract

Thromboxane synthase has been purified 620-fold from porcine lung microsomes by a three-step purification procedure including Lubrol-PX solubilization, reactive blue-agarose chromatography, and immunoaffinity chromatography. The purified enzyme exhibited a single protein band (53,000 daltons) on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Rabbit antiserum raised against the purified enzyme immunoprecipitated thromboxane synthase activity from crude enzyme preparations of porcine lung, cow lung, and human platelets, indicating the existence of structural homology of the enzyme in these species. Immunoblotting experiment identified the same polypeptide (53,000 daltons) in porcine lung and a polypeptide of 50,000 daltons in human platelets, confirming the identity of the enzyme and the specificity of the antiserum. Purified thromboxane synthase is a hemoprotein with a Soret-like absorption peak at 418 nm. The enzyme reaction has a Km for 15-hydroxy-9 alpha, 11 alpha-peroxidoprosta-5, 13-dienoic acid of 12 microM, an optimal pH of 7.5, and an optimal temperature of reaction at 30 degrees C. Purified thromboxane synthase catalyzed the formation of both thromboxane B2 and 12-hydroxy-5,8,10-heptadecatrienoic acid (HHT)....Continue Reading

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