Characterization of receptors for immune interferon in U937 cells with 32P-labeled human recombinant immune interferon.

Recombinant human immune interferon (HuIFN-gamma) was labeled with [gamma-32P]ATP and cyclic-AMP-dependent protein kinase from bovine heart to a specific radioactivity of 11,000 Ci/mmol. At least two molecules of phosphate were incorporated per molecule of interferon. The binding of [32P]HuIFN-gamma to human U937 histiocytic lymphoma cells was time dependent, and displaceable by HuIFN-gamma but not by HuIFN-alpha A or HuIFN-beta. The specific binding was saturable with less than 10% nonspecific binding. The dissociation constant of [32P]HuIFN-gamma for U937 interferon receptors was calculated to be 1.5 X 10(-10) M with a total of 1,800 binding sites/cell. Dissociation of bound [32P]IFN-gamma at 24 degrees C exhibited two distinct rates. A fast dissociation with a specific rate constant of 0.141 min-1, and a slow dissociation with a specific rate constant of 0.0027 min-1. The Kd for [32P]HuIFN-gamma was calculated from kinetic constants to be 5.4 X 10(-10) M.