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Binding of Rat and Bovine Thyrotropin to Specific Sites in Rat Thyroid Tissue^*

Surgical Service at Veterans Administration Hospital and Department of Surgery, University of California, San Francisco, California 94121; and the Thorndike Laboratory of Harvard Medical,School and the Department of Medicine Beth Israel Hospital, Boston, Massachusetts

Address requests for reprints to: Dr. Orlo H. Clark,Surgical Service (112), Veterans Administration Hospital, 4150 Clement Street, San Francisco, California 94121.

Abstract

Both ¹²⁵I-labeled bovine TSH (bTSH) and rat TSH (rTSH) bind rapidly and saturably to a articulate fraction of thysroid glands of rats. Unlabeled bTSH and rTSH displaced ¹²⁵I-labeled rTSH from binding sites in the thyroid particulate in an analogous manner. Specific binding of tracer concentrations of [¹²⁵TJbTSH to the rat thyroid particulate fraction was several times higher than that of [¹²⁵I]rTSH. Rat GH failed to displace ¹²⁵I-labeled bTSH, rat FSH had only a slight inhibitory effect at the concentrations used, but rat LH displaced labeled bTSH better than bTSH did. Specific binding of [¹²⁵I]bTSH to thyroid (21.6%) was several times greater than that to spleen (5.6%), liver (3.6%), or kidney (2.4%). At 37 C, maximum binding was achieved within 30 min but quickly decreased to lower levels. Incubation at temperatures above 4 C for 3 h resulted in a reduction in binding. At 4 C, binding oi [¹²⁵I]bTSH to the particulate fraction w s rapid, saturable, and remained stable for 4.5 h but decreased within 21 h. Optimum binding occurred at pH 6.0, but experiments were conducted at pH 7.45. An excellent correlation coefficient (0.994) was apparent between maximum binding capacity, as determined from analyses of Scatchard plots, and particulate protein concentration. Scatchard analysis of bTSH binding was usually most consistent with the presence of a single binding site, although in some experiments a lower affinity, higher capacity site also seemed to be present. The mean association constant of the single binding site when one was found or the high affinity low capacity site when two sites were evident was 2.2 ± 0.5 10* M (mean ±SD) in four experiments. In the same experiments, the maximum binding capacity averaged 117.5 ± 43.0 juUµg, 2.9 ± 1.1 ng/µg, or 6.3± 2.3 x 10 ' sitesµi.g particulate protein. The system here described, which makes use of a particulate fraction from the rat thyroid, may prove a valuable model for studying the role of alterations in TSH receptors in the response of the thyroid to a variety of stimuli

Footnotes

^* This work was supported in part by the Medical Research Service of the Veterans Administration, by the University of California Academic Senate Grant 2-5-5515- 19900-3-4, and by Grant AM-18416 from the NIAMDD,NIH.

Received February 6, 1978.