Inhibition of growth hormone bioactivity by recombinant human growth hormone-binding protein in the eluted stain assay system

Abstract

The effects of a recombinant human GH-binding protein (rhGHBP; amino acids 1-238) on GH stimulation of rat Nb2 lymphoma cells were examined with an eluted stain assay system (ESTA). This precise bioassay utilizes the colorimetric reduction by stimulated Nb2 cells of a yellow tetrazolium 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 lactogenic bioassay allowed the investigation of hGHBP specificity for human GH (hGH) as opposed to prolactin. rhGHBP inhibited pituitary hGH bioactivity in a dose-dependent manner. No significant inhibition of prolactin or ACTH bioactivity occurred. It was confirmed that recombinant 20 kDa hGH also stimulated the Nb2 cells and that its relative potency was approximately 10% of that of pituitary-derived hGH. Stimulation by 20 kDa hGH was also inhibited by rhGHBP. The highly quantitative ESTA system demonstrated that the binding protein inhibited in a competitive manner. hGH activation of the Nb2 cells did not appear to be governed by a Michaelian first-order reaction. As might then be anticipated, the concentration of rhGHBP required for 50% inhibition of GH bioactivity (IC50) changed with agonist concentrations for both 20 kDa and 22 kDa hGH. However, with equimolar concentrations of these two isohormones, the IC50 of the binding protein was virtually identical. Potentiation of hGH bioactivity in vivo by low concentrations of hGHBP has been reported but was not observed in our in vitro system when tested over a wide range of binding protein concentrations.(ABSTRACT TRUNCATED AT 250 WORDS)

Physiology and pathophysiology of growth hormone-binding protein: Methodological and clinical aspects

“…The level of GHBP necessary for 50% inhibition (IC 50 ) of 22 kDa activity (0.156 mU/l) was 0.9 ± 0.25 nmol/l, which is comparable with circulating levels of GHBP, whereas the the IC 50 for 20 kDa GH (1 ng/ml) was 4.6 nmol/l [107].…”

Clinical pharmacological aspects of growth hormone administration

“…Furthermore, GHBP may serve as a reservoir of GH, or it could play a role as transport protein presenting the GH molecule to the tissues. Alternatively, GHBP may compete with receptors for GH, and thereby inhibit GH actions [118][119][120], and determine the responsiveness to GH [121].…”

Factors Modifying Growth Hormone Estimates in Immunoassays

“…In the circulation, approximately 50% of GH is complexed to GHBP, which corresponds to the extracellular domain of the GH receptor in rabbit and humans [20][21][22], The function of this high-affinity GHBP and the pre cise effect on GH bioavailability are unknown [23][24][25]. In addition to this high-affinity binding protein, a low-affinitv binding protein is present in serum [26].…”