Nonsuppressible insulin-like activity in human serum. 3. Differentiation of two distinct molecules with nonsuppressible ILA.

Total nonsuppressible insulin-like activity (ILA) of human plasma (measured by the adipose tissue assay) results from the additive effects of at least two distinct components. They differ in molecular size, solubility in acid-ethanol, and in thermostability. More than 90% of nonsuppressible ILA of human plasma is insoluble in acid-ethanol. Its molecular size of 100,000-150,000 remains unchanged by treatment with acid-ethanol, 5 M acetic acid-0.15 M NaCl, urea, and EDTA. It is inactivated by heat. Approximately 5% of total nonsuppressible serum ILA is soluble in acid-ethanol. The molecular weight is 6000-10,000 after partial purification on Sepadex G-75 (acetic acid-NaCl). This molecule is thermostabile for 3 hr at 80 degrees C. When the acid-ethanol soluble molecule with nonsuppressible ILA is chromatographed on Sephadex G-100 at neutral pH, it is eluted in a broad peak corresponding to a molecular weight of approximately 50,000-70,000. When rechromatographed on Sephadex G-75 (acetic acid-NaCl) its mol wt is irreversibly converted from 70,000 to 6000. Most of the insulin-like activity retained on Dowex-50 ("bound insulin") is eluted off Sephadex G-75 (acetic acid-NaCl) at the same column volume as the small molecular weight nonsuppressible ILA. The latter molecule is retained on Dowex-50, whereas big molecular weight nonsuppressible ILA is not.