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Biochemical pharmacology

Mechanism of deiodination of 125I-human growth hormone in vivo. Relevance to the study of protein disposition.


PMID 1867644

Abstract

Examination of the disposition of proteins employing 125I-labeled tracers can be complicated by the in vivo deiodination of the tracer. The purpose of this study was to characterize the mechanism by which 125I-labeled proteins are deiodinated in vivo using 125I-human growth hormone (hGH) as a model compound. Intravenous (i.v.) administration of 125I-hGH resulted in a biphasic plasma kinetic pattern, with the majority of radioactivity removed from the plasma during the first 15 min. The level of circulating radioactivity at 2 hr was similar to that 15 min after administration. Radioactivity was eliminated from the animals almost exclusively in the urine. The chemical form of radioactivity in the plasma and urine was analyzed by HPLC, and precipitation of radioactivity with silver nitrate or trichloroacetic acid. Fifteen minutes after administration of 125I-hGH, 30% of the circulating radioactivity was present in the form of iodide (125I-). By 2 hr, the majority of radioactivity in the plasma was in the form of 125I-. The radioactivity in the urine was present exclusively in the form of 125I-. In vivo deiodination of 125I-hGH was reflected by the accumulation of radioactivity in the thyroid glands. There was no evidence for the presence of 125I-peptide intermediates in the plasma or urine of treated animals. In vitro, 125I-hGH was degraded to 125I-peptide intermediates by thyroid gland but not liver or kidney homogenates. In the absence of cofactors, 125I- was not observed as an in vitro metabolic product. However, in the presence of dithiothreitol and NADPH as cofactors, the predominant metabolic product formed by thyroid gland homogenates was 125I-. The deiodination of 125I-hGH by thyroid gland homogenates was inhibited by the serine protease inhibitor phenylmethylsulfonyl fluoride (PMSF), indicating that proteolysis of 125I-hGH was required for deiodination to occur. This was supported by the observation that 125I-labeled proteolytic fragments of 125I-hGH, but not 125I-hGH, were deiodinated by liver or kidney homogenates in the presence of these cofactors. Deiodination by thyroid gland homogenates was inhibited by the sulfhydryl-group blocking reagent, iodoacetate, in a concentration-dependent manner. The characteristics of the in vitro deiodination reaction suggest that a form of thyronine 5'-monodeiodinase may be involved in the in vivo deiodination of 125I-hGH and possibly other 125I-proteins. These data suggest that the disposition of proteins may be determined more accurately with 3H-, 14C- or 35S-labeled molecules which better represent the characteristics of the native protein.

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