Identification of a specific inhibitor of nonsuppressible insulin-like activity in a partially purified human serum fraction.

During purification of low molecular weight (MW) nonsuppressible insulin-like activity (NSILA) from Cohn fraction IV-I of human serum, a fraction from Sephadex G-75 chromatography was gel filtered on Biogel P-30 in 1% formic acid. NSILA activity was all eluted in "fraction III" (Kav 0.4-0.9) with a recovery (compared to applied activity of 216 +/- 21% (mean +/- SE, n = 6). To test the possibility that this increase was due to removal of an inhibitor, a series of mixing experiments was performed. Total inhibition of fraction III occurred on mixing with fraction II (Kav 0.1-0.4), which had no intrinsic activity of its own. Fraction I (Kav 0.01) had no effects. Inhibition by fraction II was dose dependent, nondialysable, partially heat sensitive (boiling, 15 min) and totally destroyed by trypsin. Estimations of the MW of the inhibitor are 16,000-18,000. The inhibitor was shown to be specific for low MW NSILA by inhibiting the stimulatory effects of an acid-ethanol extract of human serum but not insulin or the acid-stable high MW form of nonsuppressible insulin-like activity. Inhibition of NSILA was observed in both rat adipocyte (insulin-like) and costal cartilage (sulfation) bioassays. Lineweaver-Burk analysis suggested the inhibitor acted in a competitive fashion. These studies have demonstrated the presence of a specific inhibitor of NSILA in Cohn fraction UV-I of normal human serum. The identity and physiological role of the inhibitor are as yet unknown.