Immunoaffinity purification of human thromboxane synthase.
A recently produced monoclonal antibody against human thromboxane synthase was used to purify the enzyme from platelets in a one-step procedure with good yields. The isolated protein exhibited a single band of about 58 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and contained one heme/mol. Although the visible spectrum of the oxidized enzyme displayed a peak at 418 nm like the previously isolated enzyme after dithionite reduction and CO addition, it shifted to 419 nm but not to 450 nm where only a small shoulder could be detected. Its catalytic activity was only 1-5% of the previous preparations, but with the same Km of about 10 microM and a ratio of thromboxane B2: 12-hydroxyheptadecatrienoic acid of 1:1. Studies with EPR spectrometry and inhibitors confirmed that only a minor part of the enzyme was in its native heme-thiolate conformation, whereas the major part had been converted to the inactive P420 form by the elution procedure. The amino acid analysis revealed 46% hydrophobic residues. According to the sequence of 26 amino acids from the N-terminus and two tryptic peptides no homology to one of the cytochrome P450 monooxygenases, or to cyclooxygenase, or to prostacyclin synthase was detected.
Faculty of Biology, University of Konstanz, Federal Republic of Germany.
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Nüsing
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Schneider-Voss
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Ullrich
D000595:Amino Acid Sequence; D000911:Antibodies, Monoclonal; D001792:Blood Platelets / Q000201:enzymology*; D006801:Humans; D007163:Immunosorbent Techniques; D008969:Molecular Sequence Data; D013930:Thromboxane-A Synthase / Q000032:analysis / Q000302:isolation & purification*
D000911:Antibodies, Monoclonal; D013930:Thromboxane-A Synthase