Characterization of a low affinity binding protein for growth hormone in rat serum.
GH forms a high Mr 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 microM) demonstrated the binding of [125I]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 microM. Sepharose CL-6B filtration chromatography showed the complex to be 260 kDa in size. The distribution of GH binding to GHBP and this high Mr 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 Mr 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.
Leung
K C
KC
Pituitary Research Unit, Garvan Institute of Medical Research, St. Vincent's Hospital, Sydney, New South Wales, Australia.
Doyle
N
N
Ho
K K
KK
D000465:Algorithms; D000818:Animals; D001798:Blood Proteins / Q000737:chemistry / Q000378:metabolism*; D002352:Carrier Proteins / Q000097:blood* / Q000737:chemistry; D002850:Chromatography, Gel; D003432:Cross-Linking Reagents; D013006:Growth Hormone / Q000737:chemistry / Q000378:metabolism*; D019382:Human Growth Hormone / Q000378:metabolism; D006801:Humans; D066298:In Vitro Techniques; D007457:Iodine Radioisotopes; D008970:Molecular Weight; D011485:Protein Binding; D051381:Rats
D001798:Blood Proteins; D002352:Carrier Proteins; D003432:Cross-Linking Reagents; D007457:Iodine Radioisotopes; D019382:Human Growth Hormone; D013006:Growth Hormone; C051815:somatotropin-binding protein