| Budget Amount *help |
¥1,700,000 (Direct Cost: ¥1,700,000)
Fiscal Year 1987: ¥500,000 (Direct Cost: ¥500,000)
Fiscal Year 1986: ¥300,000 (Direct Cost: ¥300,000)
Fiscal Year 1985: ¥900,000 (Direct Cost: ¥900,000)
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| Research Abstract |
The crude plasma membrane of porcine and rat anterior pituitaries was homogenized in 40mM Tris HCl buffer containing 1% digitonin, incubated at 0 C for 30 min, and centrifuged at 100,000xg for 60 min. The resulting supernatant was incubated with (3h-MetHis)TRH at 0 C for 3 h, and the bound labeled TRH was separated by means of a rapid gel filtration. One% digitonin yielded to 40% solubilization of the original TRH receptor density. The peak of (3h) TRH-labeled TRH receptor was observed to correspond to that of (14C)myosin on a HPLC using Sorbax column, the data suggesting that a molecular weight of TRH receptor in the porcine anterior pituitary is approximately 200xK. The solubilized porcine TRH receptor was adsorbed by a wheat germ agglutinin column, and the adsorbed receptor eluted by 0.3M N-acetyl glucosamine. The data indicate that TRH receptor possesses N-acetyl-D-glucosaminyl residues. The solubilized porcine TRH receptor was observed to be Kd=30 nM and Bmax=62 fmol/mg protein. The sera from 18 patients with Hashimoto's thyroiditis were incubated with the solubilized porcine TRH receptor and (3H)TRH, to investigate the possible existence of an anti-TRH receptor antibody. However, those sera did not change TRH bound to the porcine anterior pituitary. The solubilized TRH receptor was applied to a AH-sephadex 4B coulumn coupled with TRH, and eluted by unlabeled TRH. This TRH-affinity chromatography system could result in an approximately 200- fold concentration of porcine TRH receptor. Further studies are in progress to obtain the 1,000-fold concentrated TRH receptor and determine an oligonucleotide probe corresponding to amino-acid sequence of porcine TRH receptor.
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