Joseph T. Neary

Evidence for a thyroid peroxidase associated “active iodine” species

Abstract The role of thyroid peroxidase (donor:hydrogen-peroxide oxidoreductase, EC 1.11.1.7) in tyrosyl iodination has been investigated using a tripeptide substrate, α- l -glutamyl- l -tyrosyl- l -glutamic acid (Glu-Tyr-Glu). Competition experiments performed with thyroid peroxidase and several iodide acceptors indicate that there is a site for these substrates on the enzyme. The rates of three reactions, peroxidase catalyzed iodination of Glu-Tyr-Glu, chemical iodination of the same tripeptide and peroxidase catalyzed production of iodine were measured as a function of pH, and were found to be different from one another, indicating that the enzymic iodinating species is not free iodine. An iodinating species was demonstrated to be peroxidase-associated: This species was generated by incubation of the enzyme with iodide-125 and hydrogen peroxide. The labeled intermediate was separated from low molecular weight reaction components by gel filtration and was shown capable of iodinating Glu-Tyr-Glu in the absence of added iodide or peroxide. The iodinated product, Glu-Tyr(I)-Glu was identified by thin layer chromatography and radio-autography. These results support the idea that thyroid peroxidase plays a direct catalytic role in iodination. Iodination does not occur by way of free iodine generated by the enzyme and released into the medium, but rather via an iodinating species associated with the enzyme.

Iodination by thyroid peroxidase

Publisher Summary The Iodination of tyrosyl moieties in thyroglobulin and other proteins is catalyzed by the thyroid peroxidase (TPO), an integral membrane, heme glycoprotein. Tyrosine and tyrosyl peptides can also be iodinated by TPO. The iodination reaction requires H 2 O 2 , I - , an enzyme-associated iodinating intermediate (TPO-I oxid ), and an iodide acceptor, such as free or protein-bound tyrosine. The TPO-catalyzed reaction produces both free and protein-bound mono and diiodotyrosine. TPO can also catalyze the formation of tetraiodothyronine by a coupling mechanism. In the presence of I - and H 2 O 2 , TPO catalyzes the iodination of tyrosyl moieties on proteins such as thyroglobulin and bovine serum albumin (BSA). For routine assays during purification, BSA is used as an I - acceptor. The protein-bound, iodinated tyrosines and thyronines can be identified by paper chromatography after Pronase digestion in the presence of 4m M 1-methyl-2-mercaptoimidazole (MMI). The tripeptide Glu-Tyr-Glu has also been used in TPO assays and the iodinated product has been identified by thin-layer chromatography and autoradiography.

Partial Purification and Some Properties of Thyroid Peroxidase Solubilized by Extraction with n-Butanol

Abstract Thyroid peroxidase has been extracted In 100% yield from suspensions of frozen pig thyroid “microsomes” by treatment with aqueous n-hutanol at pH 8.9. The solubilized enzyme does not sediment at 105, 000 × g in 1 hour, is retarded on a Sephadex G-200 gel and contains no membrane-like structures detectable by electron microscopy. Additional purification of the aqueous butanol extract is achieved by isoelectric precipitation and ion-exchange chromatography on DEAE cellulose. The partially purified TPO is recovered in 90% yield, with a 55 fold increase in specific TPO activity over the homogenate and a final nucleic acid content of about 4%. The phospholipid content is reduced to 10–15% of the 105.000 × g particles. The partially purified preparations catalyze the peroxidation of guaiacol and the iodination of moniodotyrosine. The preparation solubilized by butanol is subject to aggregation as either protein or ionic concentration is increased. Aggregation is partialis reversed by dilution of protei...

Thyroid microsomal membrane proteins. Effects of solubilization on molecular size.

The molecular size of microsomal membrane proteins from frozen porcine thyroids before and after solubilization by proteolytic and non-proteolytic techniques has been investigated by means of polyacrylamide-gel electrophoresis in the presence of 1% sodium dodecylsulfate. When thyroid microsomal membrane proteins are solubilized by non-proteolytic methods such as high pH, n-butanol, or deoxycholate, no major change in the electrophoretic pattern compared to untreated microsomes has been observed, thereby suggesting that these non-proteolytic methods are capable of extracting membrane proteins from thyroid microsomes without altering their molecular size. However, treatment of microsomes with protein-solubilizing levels of trypsin (1-5 mug trypsin per mg thyroid protein) results in degradation of all major proteins with a molecular weight greater than 30 000. The high-molecular-weight proteins are particularly susceptible to attack by trypsin. Thus, these experiments indicate that the use of trypsin to solubilize thyroid microsomal membrane proteins, particularly thyroid peroxidase, will result in fragmented proteins and should be avoided if intact membrane proteins are desired.

Serum thyrotropin releasing hormone (TRH)-degrading activity: A sensitive and rapid radiochemical assay procedure

A rapid and sensitive method has been developed to assay thyrotropin releasing hormone (TRH)-degrading activity in serum. In this assay, the formation of proline, a major serum degradation product of TRH (pGlu-His-Pro-NH2), is measured. The procedure is based on the finding that proline can be readily separated from TRH with Dowex 50 by a batchwise procedure in a test tube.