Thyrotropin releasing hormone: development of inactivation system during maturation of the rat.

Whereas thyrotropin releasing hormone is rapidly and extensively degraded by plasma of adult rats, no appreciable loss of biological or immunological activity is caused by plasma from rats 4 or 16 days old. The plasma of neonatal rats does not appear to contain an inhibitor of thyrotropin releasing hormone peptidase or a peptidase with altered substrate affinity. The development of an active peptidase in rat plasma suggests a physiological role for inactivation of thyrotropin releasing hormone.

Lower levels of thyrotropin-releasing hormone-degrading activity in human cord and in maternal sera than in the serum of euthyroid, nonpregnant adults.

Thyrotropin-releasing hormone (TRH)-degrading activity was investigated in human cord, maternal, and euthyroid adult sera by measuring (a) the rate of disappearance of TRH and (b) the rate of formation of degradation products. The rate of TRH degradation in cord and maternal sera was 25-33% of that in euthyroid adult serum. Concomitantly, in cord and maternal sera, the rate of formation of proline, a major TRH degradation product in serum, was one-quarter to one-third that in euthyroid adult sera. The differences were highly significant (P less than 0.001). The decreased levels of TRH-degrading activity in cord and maternal sera cannot be explained by (a) the presence of a dialyzable inhibitor, (b) the absence of an activator of TRH degradation, or (c) a reversal of the degradation process. There was no difference in the types of radioactive degradation products formed by cord, maternal, and euthyroid adult sera. The low level of TRH-degrading activity and its possible relationship to high thyrotropin-stimulating hormone levels in cord serum suggest that TRH-degrading activity may be a factor to consider in investigations of the perinatal pituitary-thyroid axis, but further studies are needed to determine the role of serum degradation of TRH in regulating physiological levels of TRH.

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.

The developmental pattern of thyrotropin-releasing hormone-degrading activity in the plasma of rats.

The developmental pattern of TRH-degrading activity in rat plasma was determined by measuring the ability of plasma from rats of various ages to degrade TRH into degradation products. From a rate of 0.519 pmol TRH degraded/microliter-1.h-1 on day 8, plasma TRH-degrading activity increased to 18.1 pmol TRH by day 90 in female rats. The increase in rate of formation of proline, a major plasma degradation product, was in very good agreement with the increase in rate of TRH degradation. The major increase in TRH-degrading activity occurred between the third and seventh week of life. A similar pattern of development was observed in male rats; the only significant difference was that plasma from day 90 male rats was approximately 30% more active than that from female rats (P less than 0.002). Daily T3 administration to rats from days 8--26 resulted in a 2-fold increase in plasma TRH-degrading activity (P less than 0.05). The contribution of plasma degradation to the physiological control of TRH activity is not clear, but the magnitude of the increase in plasma TRH-degrading activity (approximately 30-fold) during the maturation of the rat is suggestive of a mechanism of biological significance.

Thiourea and cyanamide as inhibitors of thyroid peroxidase: the role of iodide.

Thiourea, methylmercaptoimidazole, propylthiouracil, and thiouracil are all potent inhibitors of thyroid peroxidase (TPO)-catalyzed iodination. Unlike the cyclic thioureylenes, thiourea at 5 mM has no effect on guaiacol oxidation. If iodide is added to guaiacol assays containing thiourea, enzyme activity is lost. The latter observation may be explained as follows. In the presence of iodide, the iodinating species [TPO.Ioxid], oxidizes thiourea to formamidine disulfide. This product decomposes to cyanamide at neutral pH. We have shown cyanamide to be an inhibitor of the peroxidative and iodinating functions of TPO. Studies in rats demonstrate that doses of thiourea which completely inhibit in vivo protein-bound iodine formation have no irreversible effect on TPO, as measured by guaiacol peroxidation after removal of the thyroids. The major in vivo action of cyanamide is similar to that of thiourea. The data suggest that the primary in vivo and in vitro mode of action of thiourea is the reversible Ioxid-trapping mechanism. The anomalous inhibition of guaiacol peroxidation seen in the presence of thiourea plus iodide derives from the formation of formamide disulfide, followed by its nonenzymic decomposition to cyanamide.

The irreversible inactivation of thyroid peroxidase by methylmercaptoimidazole, thiouracil, and propylthiouracil in vitro and its relationship to in vivo findings.

A reinvestigation of the mechanism of action of methylmercaptoimidazole, propylthiouracil, and thiouracil on thyroid peroxidase (TPO) was undertaken. A preliminary incubation of TPO and H2O2 with methylmercaptoimidazole, propylthiouracil, or thiouracil was carried out in the absence of oxidizable substrates (i.e. I- or guaiacol). This incubation resulted in irreversible inactivation of TPO. The extent of inactivation could be determined after removal of the drug by gel filtration or by dilution into the assay mixture. Preincubation, as above, in the presence of iodide or thiocyanate prevented the irreversible inactivation of TPO. Rats receiving doses of these drugs which completely inhibited protein-bound iodine formation showed normal levels of TPO in their thyroid glands 30 min after drug administration. These findings suggest that the initial in vivo action of these drugs is to block iodination by trapping oxidized iodide, not by acting as "general inhibitors" of the TPO.

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.

The reaction of N-iodosuccinimide with the goitrogens: a model for the active iodine of the thyroid gland.

The reactivities of N-iodosuccinimide and iodine with various goitrogenic compounds were studied with dimethylsulfoxide through which nitrogen had been bubbled as a solvent. Although iodine does not react with all of the compounds, N-iodosuccinimide does react with all of them. N-Iodosuccinimide may, therefore, serve as an interesting model for the "active iodine" of the thyroid.

Interaction of thyroid peroxidase with concanavalin A covalently coupled to agarose.

We have investigated the interaction between concanavalin A-agarose (Con A-agarose) and thyroid peroxidase, an integral membrane protein found in the 105,000 X g, 1-h particulate fraction of thyroid tissue. An intact form of porcine thyroid peroxidase was obtained by solubilization with the nonionic detergent Triton X-100 and two fragmented, hydrophilic forms of the enzyme were prepared by trypsin treatment of the membrane. The three types of thyroid peroxidase bind to Con A-agarose and can be eluted with alpha-methyl-D-mannoside. The alpha-methyl-D-mannoside eluate of the most purified thyroid peroxidase preparation has been analyzed by polyacrylamide gel electrophoresis. Peroxidase activity corresponds with a glycoprotein band. The binding of thyroid peroxidase to Con A-agarose can be inhibited by sugars in the following order: alpha-methyl-D-mannoside greater than D-mannose greater than alpha-methyl-D-glucoside greater than D-glucose greater than D-galactose. This order of specificity is typical of Con A-sugar interactions. Furthermore, inactivation of the carbohydrate binding site of Con A by demetallization greatly reduces the extent of thyroid peroxidase binding. Reactivation of the carbohydrate binding site by the addition of Ca2+ and Mn2+ to demetallized Con A-agarose restores thyroid peroxidase binding. These and other experiments suggest that htyroid peroxidase is, like several other peroxidases, a glycoprotein. In addition, the interaction between thyroid peroxidase and Con A-agarose may provide a new purification tool for thyroid peroxidase.