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Preparation of radio labeled thyroid stimulating immuno globulins by recombining thyroid stimulating immuno globulins heavy chains with iodine 125 labeled light chains direct evidence that the product binds to the membrane thyrotropin receptor and stimulates adenylate cyclase


Endocrinology 103(1): 296-301
Preparation of radio labeled thyroid stimulating immuno globulins by recombining thyroid stimulating immuno globulins heavy chains with iodine 125 labeled light chains direct evidence that the product binds to the membrane thyrotropin receptor and stimulates adenylate cyclase
The biologically active portion of the thyroid-stimulating immunoglobulin (TSI/LATS [long acting thyroid stimulator] molecule is in the Fd portion of the heavy (H) chain. Radioiodination of TSI/LATS by the chloramine T method or contact with the oxidant only results in reduction and/or abolition of its biological activity in vivo. Such treatment also results in a loss of thyroid membrane adenylate cyclase stimulatory activity. A method for labeling TSI was devised which circumvents contact of the H chain with oxidant or radioiodine. The H and L [light] chains of TSI were separated by mild reduction, alkylation and gel filtration in 1 M propionic acid. The separated TSI H chains were then mixed with .kappa.-type Bence-Jones L chains (molar ratio, 1:2) which were previously iodinated by 125I (*L) or 127I (.degree.L), and Ig[immunoglobulin]G was reconstituted by dialysis of the mixture in 10 mM sodium acetate buffer, pH 5.5 IgG was then separated from free H and L chains by gel filtration. The IgG (*L-TSI or .degree.L-TSI) thus contained iodinated L chains, whereas all of the unlabeled H chains were from the original TSI source. After incubation of *L-TSI with human thyroid membranes for 3 h at 37.degree. C in 10 mM Tris-HCl buffer, pH 7.4, containing 30 mM NaCl and 0.5% bovine serum albumin, 1-2% was specifically bound to membranes. This binding could be inhibited by unlabeled TSI and bovine TSH [thyrotropin]. Both .degree.L-TSI and native TSI stimulated thyroid membrane adenylate cyclase and inhibited [125I]bovine TSH binding to membranes in a dose-related fashion; native TSI which was iodinated directly no longer had either property. TSI apparently binds to TSH receptors on thyroid cell membranes and stimulates adenylate cyclase activity. The method offers the potential to obtain membrane-purified and radiolabeled TSI for further biochemical, physiological and clinical studies.


Accession: 006173770



Related references

Preparation of radiolabeled thyroid-stimulating immunoglobulins (TSI) by recombining TSI heavy chains with 125I-labeled light chains: direct evidence that the product binds to the membrane thyrotropin receptor and stimulates adenylate cyclase. Endocrinology 103(1): 296-301, 1978

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