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Characterization of a Low Affinity Binding Protein for Growth Hormone in Rat Serum¹

Pituitary Research Unit, Garvan Institute of Medical Research, St. Vincent’s Hospital, Sydney, New South Wales 2010, Australia

Address all correspondence and requests for reprints to: Prof. Ken K. Y. Ho, Garvan Institute of Medical Research, St. Vincent’s Hospital, 384 Victoria Street, Sydney, New South Wales 2010, Australia. E-mail: k.ho{at}garvan.unsw.edu.au

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, ¹²⁵I-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 µM) demonstrated the binding of [¹²⁵I]hGH to be specific, with Scatchard analysis revealing an affinity of 0.88 ± 0.03 x 10⁵ M^-1 (n = 3) and a capacity of 2.46 ± 0.14 µM. Sepharose CL-6B 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.