125I-labelled human TSH was crosslinked to the human thyroid and extraocular eye muscle
membrane and cytosol fractions (which were obtained by centrifugation). Studying crosslinking
of 125I-labelled TSH to the thyroid fractions, TSH binding sites' structures were
demonstrated on the eye muscle membranes and in the cytosol fractions. The binding
of 125I-labelled TSH was inhibited by the addition of 120 mIU/mL of unlabelled TSH
(and not with 12 mIU/mL) which confirmed the presence of TSH binding sites structures
(MW about 66,000 Da) on the eye muscle membrane and in its cytosol. Adding purified
IgG fractions from the sera from controls and Graves' disease (with high titer of
antibodies against TSH receptor) to the thyroid and eye muscle membranes and cytosol
fractions, the binding of 125I-labelled human TSH was inhibited by molecular weight
of about 66,000 Da in the cytosol fractions. The affinity constant of the binding
sites in the human eye muscle cytosol and the number of TSH receptors were found to
be 146 x 10(9) M-1 and 9.8 x 10(10) molecules/mg/mL by Scatchard analysis, respectively.
