Binding of nonsuppressible insulinlike activity to human serum. Evidence for a carrier protein.

When ¹²⁵I-labeled nonsuppressible insulinlike activity—soluble in acid/ethanol (NSILA-S) is incubated with human serum between 10 and 20% of the radioactivity are bound to serum proteins and can be displaced specifically by cold NSILA-S. Chromatography of the incubation mixture on Sephadex G-200 at pH 7.5 reveals three peaks of radioactivity in the large molecular weight region and a fourth one corresponding to low molecular unbound labeled NSILA-S. An excess of cold NSILA-S during preincubation leads to the disappearance of the two major large molecular weight peaks and to a concomitant increase of the peak eluting in the low molecular weight range. Binding of ¹²⁵I-labeled NSILA-S is highly sensitive to small concentrations of cold NSILA-S, whereas insulin, ACTH and human growth hormone are completely ineffective in displacing bound ¹²⁵I-labeled NSILA-S. NSILA-S preparations of different purity show displacement according to their specific biological activities. Furthermore, binding of ¹²⁵I-labeled NSILA-S to serum pH- and time-dependent and displays saturation characteristics. Chromatography of serum on Sephadex G-200 with 0.15 m acetic acid/0.15 m NaCl localizes the binding fraction in the 50,000–70,000 molecular weight range. Boiling of serum for 5 min abolishes binding completely.These studies help explain why the molecular weight of NSILA varied considerably from one group of investigators to the other.