Characterization of a low affinity binding protein for growth hormone in rat serum

GH forms a high M-r complex in rat serum distinct from that with GH-binding protein (GHBP). The present study investigates the nature of this complex. When subjected to AcA44 filtration chromatography, I-125-labeled human GH (hGH) in rat serum eluted in four peaks. Peak 1 eluted at the void volume, whereas peaks 2, 3, and 4 corresponded to the GHBP complex, free hGH, and iodide, respectively. Stripping of GHBP in serum by immunoaffinity chromatography depleted peak 2 but did not affect peak 1. Peak 1 accounted for 11.4 +/- 1.2% of the total radioactivity (mean +/- SEM; n = 6) in stripped serum. Addition of unlabeled hGH (0.9-9 mu M) demonstrated the binding of [I-125]hGH to be specific, with Scatchard analysis revealing an affinity of 0.88 +/- 0.03 x 10(5) M-1 (n = 3) and a capacity of 2.46 +/- 0.14 mu M Sepharose CL-GB filtration chromatography showed the complex to be 260 kDa in size. The distribution of GH binding to GHBP and this high M-r serum factor was investigated by incubating [125I]hGH in sera containing a low (5 nM) and a high (35 nM) concentration of GHBP over a range of physiological GH concentrations. In sera containing a low concentration of GHBP, the proportion of GH complexed in peak 1 increased with increasing GH concentrations. In sera with a high concentration of GHBP, GH was complexed mainly in peak 2. Studies with normal rat sera revealed that more GH was complexed in peak 1 in male than in female rats (3.4 +/- 0.4% and 1.4 +/- 0.1%, respectively; P < 0.006), in contrast to that of peak 2 (1.1 +/- 0.2% and 7.6 +/- 0.4%, respectively; P < 0.002). In summary, we provide strong evidence for the existence of a factor in rat serum that binds GH with low affinity and high capacity. It has a M-r of approximately 240 kDa, assuming a 1:1 binding stoichiometry, and is immunologically distinct from GHBP. This factor may provide supplementary capacity for GH binding when binding to GHBP is saturated.