INHIBITION OF GROWTH-HORMONE BIOACTIVITY BY RECOMBINANT HUMAN GROWTH HORMONE-BINDING PROTEIN IN THE ELUTED STAIN ASSAY SYSTEM

The effects of a recombinant human GH-binding protein (rhGHBP; amino a cids 1-238) on GH stimulation of rat Nb2 lymphoma cells were examined with an eluted stain assay system (ESTA). This precise bioassay utiliz es the colorimetric reduction by stimulated Nb2 cells of a yellow tetr azolium salt (3-[4, 5-dimethyl-thiazol-2-yl]-2,5-diphenyl-tetrazolium bromide) to a purple formazan as its end-point. The use of a lactogeni c bioassay allowed the investigation of hGHBP specificity for human GH (hGH) as opposed to prolactin. rhGHBP inhibited pituitary hGH bioacti vity in a dose-dependent manner. No significant inhibition of prolacti n or ACTH bioactivity occurred. It was confirmed that recombinant 20 k Da hGH also stimulated the Nb2 cells and that its relative potency was similar to 10% of that of pituitary-derived hGH. Stimulation by 20 kD a hGH was also inhibited by rhGHBP. The highly quantitative ESTA syste m demonstrated that the binding protein inhibited in a competitive man ner. hGH activation of the Nb2 cells did not appear to be governed by a Michaelian first-order reaction. As might then be anticipated, the c oncentration of rhGHBP required for 50% inhibition of GH bioactivity ( IC50) changed with agonist concentrations for both 20 kDa and 22 kDa h GH. However, with equimolar concentrations of these two isohormones, t he IC50 of the binding protein was virtually identical. Potentiation o f hGH bioactivity in vivo by low concentrations of hGHBP has been repo rted but was not observed in our in vitro system when tested over awid e range of binding protein concentrations. In conclusion, the ESTA bio assay system permitted a detailed characterizationof the inhibition of hGH bioactivity by rhGHBP. The hormonal specificity confirms earlier radioligand binding studies, except that we found that the 20 kDahGH v ariant interacts with the rhGHBP.