Evaluation of the growth hormone-binding proteins in human plasma using high pressure liquid chromatography gel filtration.
A technique using high pressure liquid chromatography gel filtration was used to evaluate GH-binding proteins (BP) in human plasma; eluate was monitored for radioactivity in a gamma-detection system connected to a computer. Plasma (200 microL) was incubated with [125I]human (h) GH (200,000 cpm) at 4 C for 20 h. The main GH-BP (peak II) was well separated from free [125I]hGH (peak III) and from a higher mol wt complex (peak I), which was minor. In our control plasma, the specific binding of [125I]hGH to peak II BP (II-BP) was 32.2 +/- 0.6% of the radioactivity. Scatchard analyses indicate an association constant of 3.6-7.4 X 10(8) M-1 and a binding capacity ranging from 24-86 ng/mL for peak II-BP in five normal adult plasma samples. Peak I material, separated from plasma of boys with pubertal delay, bound hGH with a low affinity (3 x 10(6) M-1) and a very high capacity (2 micrograms/mL). In cross-linking experiments, peak I appeared as two proteins of 165 and 174 kD; these mol wt were much higher than that of peak II-BP, previously estimated at 53,000. hGH complexed to peak II-BP remained fully immunoreactive with use of the anti-hGH antibodies of our assay. In plasma containing 10-20 micrograms/L hGH, the proportion of bound hormone (peak II) was 44.5 +/- 2.3%, whereas the amount of hGH in peak I was very low or undetectable. Specific binding of hGH to II-BP was lowest during the first year of life and highest in adulthood. No sex difference was found. I-BP is differentially regulated, since its binding activity was significantly lower in adults than in prepubertal children. Normal values for age should be taken into account to interpret GH-binding activity, particularly in children 2 yr of age or younger. Our GH binding assay offers important gains in terms of rapidity and resolution; it has permitted a clear separation and characterization of the two GH-binding components present in human plasma.
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Unité de Biologie et Pathologie de la Croissance et du Développement, INSERM Unité 30, Hôpital des Enfants-Malades, Paris, France.
Hocquette
J F
JF
Souberbielle
J C
JC
Clot
J P
JP
Brauner
R
R
Postel-Vinay
M C
MC
D000180:Acrylic Resins; D000328:Adult; D000375:Aging / Q000097:blood; D001345:Autoradiography; D001665:Binding Sites; D002352:Carrier Proteins / Q000097:blood*; D002850:Chromatography, Gel; D002851:Chromatography, High Pressure Liquid / Q000379:methods; D005260:Female; D013006:Growth Hormone / Q000097:blood*; D006801:Humans; D011863:Radioimmunoassay
D000180:Acrylic Resins; D002352:Carrier Proteins; C016680:polyacrylamide gels; D013006:Growth Hormone; C051815:somatotropin-binding protein