Binding and degradation of insulin by human peripheral granulocytes. Demonstration of specific receptors with high affinity.
The interaction of insulin with human circulating granulocytes was studied with the use of 125I-insulin. Human granulocytes, isolated from blood by the Böyum technique, showed high insulin-degrading activity in vitro which almost obscured the presence of specific, high affinity binding sites. Degradation, measured by trichloroacetic acid precipitation and by binding to well characterized insulin receptors on cultured human lymphocytes (IM-9 line), was due to extracellular as well as cell-bound enzymes. Degradation was enhanced by Ca2+ and thiols and inhibited by various protease inhibitors and sulfhydryl-blocking reagents. Phenylmethylsulfonyl fluoride (5 X 10(-4) M), a serine protease inhibitor, was the most potent and inhibited 125I-insulin degradation by 80 to 90%. Tert-butyl hydroperoxide (2 X 10(-3) M), a glutathione-oxidizing reagent, inhibited degradation by 35 to 50%, possibly due to an effect on a glutathione-insulin transhydrogenase. Neither of the inhibitors affected cell viability. In the presence of inhibitors of degradation, binding sites for insulin with high affinity were detected, which by multiple criteria were true insulin receptors. Binding to these sites was rapid, saturable, and reversible with about 1000 sites/cell. The Hill coefficient for binding was 0.7, and the Scatchard plot of B/F versus B was curvilinear, due to site-site interactions of the negative cooperative type; the latter were demonstrated directly by kinetic studies. As shown previously for all other insulin receptors, binding was highly pH-dependent, and insulin analogues had affinities for these sites that closely correlated with their biological potencies.
D001665:Binding Sites; D001667:Binding, Competitive; D002021:Buffers; D006098:Granulocytes / Q000378:metabolism*; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007328:Insulin / Q000378:metabolism*; D007700:Kinetics; D007962:Leukocytes / Q000378:metabolism*; D008214:Lymphocytes / Q000378:metabolism; D011485:Protein Binding; D011956:Receptors, Cell Surface
D002021:Buffers; D007328:Insulin; D011956:Receptors, Cell Surface