GUINEA-PIG SERUM CONTAINS A SPECIFIC HIGH-AFFINITY GROWTH HORMONE-BINDING PROTEIN WITH NOVEL LIGAND SPECIFICITY

Previous workers have suggested that guinea pig serum does not contain a GH-binding protein (GHBP) or that it is defective. The current stud ies, however, have identified and characterized the presence of GH-bin ding activity in guinea pig serum using gel chromatography to separate bound and fi ee hormone. The detection of GH-binding activity is crit ically reliant on the type of radioligand used to measure binding. Cle ar identification of GH-binding activity was demonstrated with [I-125] ovine GH (oGH), but specific binding could not be measured with [I-125 ]human GH. The novel specificity was also shared by guinea pig liver m embrane GH receptor (GHR) and cytosol GHBP, suggesting structural simi larity in the GH-binding domain between the GHR and soluble GHBPs. The binding of oGH was dependent on serum concentration (5 mu l serum pro duced 16.03 +/- 0.5% specific binding; mean +/- SEM; n = II) and incub ation time (equilibrium was reached by similar to 6 h at 21 C) and was completely reversible (t(1/2), similar to 2 h). Scatchard analysis re vealed linear plots with an affinity constant (K-a) of 0.59 +/- 0.09 x 10(9) M-1 and a capacity of 23,181 +/- 4,474 fmol/ml serum. Similar a ssociation constants were obtained for liver membrane GHR (0.79 +/- 0. 22 x 10(9) M-1) and cytosol GHBPs (0.99 +/- 0.15 x 10(9) M-1), but the capacity, when expressed as femtomoles per g tissue, was significantl y increased (4-fold) in cytosol (4,303 +/- 505) over that in membranes (1,071 +/- 257). There was no sex difference in K-a or level of GHBP in guinea pig serum. Surprisingly, the level of GH-binding activity wa s very low to undetectable in pregnant guinea pig serum. Characterizat ion of the native structure of guinea pig GHBPs has indicated the pres ence of several proteins that are structurally distinct. Although the distribution of GH-binding activity covered a large M-r range (similar to 70-350 kDa) the major form of the circulating GHBP identified by g el chromatography had an apparent native M-r of 150-170 kDa. Partially purified GHBP (approximate M-r, 170 kDa) was covalently cross-linked to [I-125]oGH and subjected to nonreducing SDS-PAGE. Specific GHBP com plexes of 158 and 85 kDa were detected, suggesting that the partially purified GHBP complex may be composed of a smaller GHBP associated non covalently with a non-GH-binding protein. ''Pore limit'' native PAGE ( cathodic and anodic) revealed the presence of specific GHBPs of 363, 1 58, 74, and 55 kDa, which cross-hybridized with the rat liver membrane GHR monoclonal antibody mAb 263 but not with the rat serum GHBP-speci fic mAb 4.3. Interestingly, although GH binding was undetectable in pr egnant guinea pig serum, Western immunoblot analysis with mAb 263 demo nstrated the presence of a major immunoreactive GHBP band of 105 kDa i n addition to 158- and 55-kDa GHBPs. The data indicate that the GHBPs are immunologically related to the rat membrane GHR, but provide no ev idence to support the presence of a hydrophilic tail sequence homologo us to that in the rat GHBP. These studies have identified in guinea pi g serum GHBPs that exhibit novel ligand specificity, structural hetero geneity, and an immunological relationship to the rat liver membrane G HR. The identification of serum GHBP and the novel ligand specificity, which is also expressed by the liver membrane GHR, argue against the view that the guinea pig has a defective GHBP.