Abstract
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.
Highlights
From the Roche Institute of Molecular Biology, Roche Research Center, Nutley, New Jersey 07110 and the $Department of Molecular Genetics, Hoffmann-La Roche Inc., Nutley, New Jersey 07110
[12sI]IFN-rT. he high specific activity andspecificity of binding permitaccurate equilibrium andkineticstudies.This paper reports initial resultswith this novel labeled ligand
Beyond receptor binding are not known. To characterize the This observation, in agreement with the findings of Orchanmolecular mechanisms by which different interferonsproduce sky et al (8), Sarkar and Gupta (9), andO'Rourke et al (12), certain biological activities,itisessentialtoidentifyand suggest that IFN-y binds atosite differentfrom that towhich characterize the IFN-binding sites
Summary
Characterization of f'P]HuIFN-y-Recombinant human interferon y was phosphorylated with [y-"'P]ATP andbovine heart muscle protein kinaseas described under "Experimental Procedures." The specific radioactivity of [32P]IFN-y was measured to be 11,000 Ci/mmol. To measure the rate of dissociation of [3ZP]HuIFN-yfrom the U937 receptors, the binding reaction was allowed to reach equilibrium for 70 min at 24 "C. The equilibrium dissociation constant was determined from Scatchard analysis (Fig. 2, inset) as well as from the ratios of the fast dissociation rate (k-1) to the associate rate, k l. These values are not corrected for 20% binding capability of [32P]HuIFN-y(see text). Specific binding of [3ZP]HuIFN-.ywas measured as described under "Experimental Procedures." A , the IFN concentrations arecalculated two rates of dissociation to theobserved data, a fast ratwe ith by amino acid analysis and given as mole/liter. Internalization of IFN-a (6, 33-35) and IFN-y (36) receptors has been reported
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