Anionic iodotyrosine residues are required for iodothyronine synthesis.
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Biosynthesis of iodothyronines in thyroglobulin occurs by oxidative coupling of two iodotyrosine residues catalyzed by thyroperoxidase. To study the mechanism of iodothyronine formation, iodine-free thyroglobulin was non-enzymatically iodinated and after removal of non-incorporated iodide, incubated with lactoperoxidase and glucose oxidase between pH 4 and 9. The amount of thyroxine (T4). 3,5,3'-tri-iodothyronine (T3), 3,3',5'-tri-iodothyronine (rT3) and 3,3'-di-iodothyronine (T2) formed was measured by radioimmunoassays after hydrolysis of thyroglobulin. T4 is synthesized out of two di-iodotyrosine (DIT) residues in thyroglobulin. The pH dependence of T4 formation fits the dissociation curve of the DIT phenoxy group (pKa 6.5). The formation of T2, synthesized out of two mono-iodotyrosine (MIT) residues, shows a quite different pH dependence. Below pH 6, T2 synthesis could not be observed, while above pH 7.4 a relatively large increase occurred. The values up to pH 8 fitted the dissociation curve of the MIT-phenoxy group with a pKa of 8.7. The gradual loss in enzymatic activity of peroxidase and oxidase in the reaction made the values obtained above pH 8 unreliable. The importance of the ionization of the phenoxy group for the coupling reaction was further consolidated by showing that the pH-dependent oxidation of 2-methoxy-phenol (guaiacol) had 50% maximal product formation at pH 7, a value concordant with pKa 7.0 for the ionization of the phenoxy group of this agent. T3 and rT3 synthesis followed mainly the ionization curve of the inner-ring hydroxyl group, indicating that this ring has the greatest influence on hormonogenesis. Since anion formation facilitates the removal of an electron under oxidative conditions, the pH dependence agrees with the involvement of phenoxy radicals in iodothyronine synthesis, a process that most likely also occurs in vivo since it is mainly T4 that is formed in thyroglobulin. (+info)
The Plasmodium falciparum-CD36 interaction is modified by a single amino acid substitution in CD36.
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CD36 is an 88-kD glycoprotein involved in the cytoadherence of Plasmodium falciparum-parasitized erythrocytes (PE) to endothelial cells. The molecular mechanisms involved in CD36-dependent cytoadherence were examined by expressing three CD36 homologues (human, murine, and rat) in COS-7 cells and observing their PE-binding characteristics over a pH range of 6.0 to 7.4 and following iodination of these receptors. PE binding to human CD36 was pH dependent, with peak binding at pH 6.8 to 7.0, and binding was unaffected by iodination. In contrast, PE adherence to murine and rat CD36 was insensitive to changes in pH, and iodination significantly reduced binding. We further show that the differences observed in the binding phenotype of human and rodent CD36 can be attributed to a single residue. Site-directed mutagenesis of the histidine at position 242 of human CD36 to tyrosine (found in rodent CD36) conferred the binding phenotype of rodent CD36 onto human CD36. Furthermore, substitution of the tyrosine at position 242 of rat CD36 for histidine conferred the binding phenotype of human CD36 onto rat CD36. These findings suggest that residue 242 is part of, or important to the conformation of, the PE-binding domain of CD36. (+info)