COMPARATIVE STUDIES ON THE BINDING OF 131I-LABELLED AND UNLABELLED INSULIN TO RAT DIAPHRAGM IN VITRO

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Abstract
Rat hemidiaphragms were incubated with either unlabelled or 131I and 127I-labelled insulin of varying degrees of iodination (0.08–1.4 I/mole). With all types of insulin the percentage disappearance of immunoreactive insulin (IRI) was found to exceed the percentage uptake of radioactive label by the tissue by a factor of 10. This discrepancy is attributed to degradation of labelled hormone taken up by the tissue, degradation of the hormone within the medium and reduced tissue-binding of iodinated insulin molecules. Even after incubation for 15 min, fixation of labelled insulin to the diaphragm appeared to coincide with a return-flow of TCA-soluble labelled fragments into the medium. Washing of the diaphragm for 10 min resulted in a 30% loss of tissue-bound radioactivity. After incubation with unlabelled insulin less than 1% of the tissue-bound IRI could be recovered in the washing medium. After correction for IRI degradation within the medium, the binding of iodo-insulin 1.4 I/mole to the diaphragm amounted to 56% of that of the unlabelled insulin. Since the biological activity at this degree of iodination appeared to be reduced by over 50% of the immunologic activity, it is suggested that the decreased activity of iodo-insulins may be due to the reduced affinity of the hormone to specific receptor sites involved in the initiation of the biological response. The results of this study indicate that accumulation of the radioactive label of 131I-labelled insulin in incubated diaphragms gives no reliable quantitative index for the binding of either labelled or unlabelled insulin to this tissue.

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