Biosynthesis of thromboxane B2: assay, isolation, and properties of the enzyme system in human platelets.
The microsomal fraction of human platelets catalyzed the conversion of arachidonic acid to an unstable platelet-aggregating factor and a hydrolyzed product on the thin-layer chromatography (TLC). This product was isolated on TLC, purified by silica gel column chromatography and identified by combined gas chromatography-mass spectrometry as the hemiacetal derivative of 8-(1-hydroxy-3-oxopropyl)-9, 12L-dihydroxy-5, 10-heptadecatrienoic acid (thromboxane B2). The enzymatic activity was dependent upon methemoglobin and tryptophan as cofactors. Reduced glutathione had no effect either alone or in combination with other cofactors. Methemoglobin could be replaced by hematin or hemin; and tryptophan by 3-indolacetic acid or catecholamines. The apparent requirement for methemoglobin is due to the reductive activity of ferriprotoporphyrin IX. The reaction, however, catalyzed by the ferriprotoporphyrin IX in the thromboxane synthesizing system is different from that described for the decomposition of lipid peroxides. Certain transition metals and hydrogen donors, such as hydroquinone and ascorbate, which have been shown to stimulate the catalytic activity of ferriproroporphyrin IX in the decomposition of 15-hydroperoxy-prostaglandin E1 are inhibitors of thromboxane B2 formation. This enzyme preparation also transformed eicosa-8. 11, 14-trienoic acid to an unknown product on TLC. The enzyme system was rapidly inactivated upon incubation in the reaction mixture.
D015126:8,11,14-Eicosatrienoic Acid / Q000378:metabolism; D001095:Arachidonic Acids / Q000097:blood; D001205:Ascorbic Acid / Q000494:pharmacology; D001779:Blood Coagulation Factors / Q000032:analysis / Q000096:biosynthesis* / Q000302:isolation & purification; D001792:Blood Platelets / Q000378:metabolism*; D002395:Catecholamines / Q000494:pharmacology; D002849:Chromatography, Gas; D002855:Chromatography, Thin Layer; D005978:Glutathione / Q000494:pharmacology; D006427:Hemin / Q000494:pharmacology; D006801:Humans; D006863:Hydrogen-Ion Concentration; D006873:Hydroquinones / Q000494:pharmacology; D006880:Hydroxy Acids / Q000032:analysis / Q000096:biosynthesis* / Q000302:isolation & purification; D007210:Indoleacetic Acids / Q000494:pharmacology; D013058:Mass Spectrometry; D008706:Methemoglobin / Q000494:pharmacology; D008861:Microsomes / Q000378:metabolism; D011166:Porphyrins / Q000378:metabolism; D011458:Prostaglandins E / Q000494:pharmacology; D011714:Pyrans / Q000032:analysis / Q000096:biosynthesis* / Q000302:isolation & purification; D014364:Tryptophan / Q000494:pharmacology
D001095:Arachidonic Acids; D001779:Blood Coagulation Factors; D002395:Catecholamines; D006873:Hydroquinones; D006880:Hydroxy Acids; D007210:Indoleacetic Acids; D011166:Porphyrins; D011458:Prostaglandins E; D011714:Pyrans; D006427:Hemin; D014364:Tryptophan; D008706:Methemoglobin; D015126:8,11,14-Eicosatrienoic Acid; D005978:Glutathione; D001205:Ascorbic Acid