Characterization of [125I-Tyr10]human growth hormone-releasing factor (1-44) amide binding to rat pituitary: evidence for high and low affinity classes of sites.
A sensitive binding assay was developed to determine binding characteristics of commercially available [125I-Tyr10]human growth hormone-releasing factor (hGRF) (1-44)NH2 in rat pituitary using 0.1 gland homogenate (70-75 micrograms protein) per incubation tube. Under standard assay conditions, addition of 5 mM EDTA efficiently prevented the degradation of both human and rat GRF for at least 3 h. Association of the ligand was time-dependent: equilibrium was reached within 30 min of incubation at 23 degrees C and remained stable for an additional 150 min (K1 = 5.01 +/- 0.86 nM-1.min-1). Specific binding increased linearly with the amount of protein present in the assay, from 15 to 170 micrograms per incubation tube. This binding was reversible, dissociation occurring almost completely after a 120-min period (K-1 = 8.13 +/- 0.29 x 10(-3) min-1). A concentration of 5-10 mM Mg2+ was required for optimal specific binding whereas 50 mM Mg2+ or monovalent cations such as Na+, K+, Li+ decreased it. Scatchard analysis of cold saturation studies by the Ligand program statistically revealed the presence of two distinct classes of binding sites; the first was of high affinity (0.68 +/- 0.11 nM) and low capacity (140 +/- 22 fmol/pituitary), the second was of lower affinity (590 +/- 347 nM) and higher capacity (38.7 +/- 18.7 pmol/pituitary). Similar values were obtained with various bovine serum albumin (BSA) concentrations and when using crude or washed pituitary homogenates, suggesting that the second low affinity site was not BSA or a soluble protein from the homogenate.(ABSTRACT TRUNCATED AT 250 WORDS)
Abribat
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Neuroendocrinology Laboratory, Notre-Dame Hospital Research Center, University of Montreal, Que., Canada.
Boulanger
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Gaudreau
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D000818:Animals; D002412:Cations / Q000494:pharmacology; D004492:Edetic Acid / Q000494:pharmacology; D013007:Growth Hormone-Releasing Hormone / Q000031:analogs & derivatives* / Q000378:metabolism; D006150:Guanine Nucleotides / Q000494:pharmacology; D066298:In Vitro Techniques; D007457:Iodine Radioisotopes; D007700:Kinetics; D008297:Male; D010446:Peptide Fragments / Q000378:metabolism*; D010903:Pituitary Gland, Anterior / Q000187:drug effects / Q000378:metabolism*; D051381:Rats; D011919:Rats, Inbred Strains; D018013:Receptors, Neuropeptide; D017981:Receptors, Neurotransmitter / Q000145:classification*; D018017:Receptors, Pituitary Hormone-Regulating Hormone; D017337:Sermorelin; D012710:Serum Albumin, Bovine / Q000494:pharmacology
D002412:Cations; D006150:Guanine Nucleotides; D007457:Iodine Radioisotopes; D010446:Peptide Fragments; D018013:Receptors, Neuropeptide; D017981:Receptors, Neurotransmitter; D018017:Receptors, Pituitary Hormone-Regulating Hormone; C040786:somatotropin releasing hormone (1-44)amide; D012710:Serum Albumin, Bovine; D017337:Sermorelin; D013007:Growth Hormone-Releasing Hormone; D004492:Edetic Acid; C048250:somatotropin releasing hormone receptor