A Second, Lower Affinity Growth Hormone-Binding Protein in Human Plasma*

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Abstract
In our previous description of the circulating GH-binding protein (GH-BP) we observed, in addition to the main GH-BP complex, a second macromolecular component designated peak I during gel filtration of [125I]GH-plasma mixtures. This component was not further characterized because of its small magnitude, seeming nonsaturability, and suspected artifactual nature. We have now characterized peak I as the complex of a low affinity BP with GH. To gain a better knowledge of the nature of peak I, whole plasma or plasma fractions containing isolated peak I-BP (ammonium sulfate precipitated or prepared by gel filtration of plasma) were incubated with monomeric [125I]GH and varying concentrations of unlabeled GH. The mixtures were then analyzed by Sephadex G-100 chromatography to separate free from protein-bound GH. Peak I-associated radioactivity was saturable at high concentrations of human GH, but not with animal GHs. Saturation/Scatchard analysis yielded an association constant of 105 M-1 and a maximum binding capacity of 15 mg/L plasma. Chemically cross-linked complexes of [125I]GH with isolated peak I BP were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, isoelectric focusing, and two-dimensional electrophoresis. These experiments yielded a mol wt of 124 kD and a pI of 7 for the cross-linked complex. This is in contradistinction to the previously reported high affinity GH-BP complex, which when cross-linked has a mol wt of 76 kD and a pI of 5. Peak I BP appears to be a single chain protein that cannot be converted to the high affinity BP upon dissociation and sulfhydryl reduction. Under physiological conditions, about 10-15% of complexed GH are associated with this BP. We conclude that a second specific BP for GH exists in human plasma. This BP is structurally distinct from the previously described high affinity BP, has lower affinity, but higher binding capacity, and contributes to the protein-bound GH fraction in plasma. It appears to be unrelated to the GH receptor.