CHARACTERIZATION OF [I-125-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 the commercially available [125I-Tyr10]human growth hormone-releasing factor (hGRF) (1-44)NH2 in rat pituitary using 0.1 gland homogenate (70-75 μg protein) per incubation tube. Under standard assay conditions, addition of 5 mM EDTA efficiently prevented the degradation of both human and rat GRF for a least 3 h. Association of the ligand was time-dependent: equilibrium was reached within 30 min of incubation at 23 °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 μg per incubation tube. This binding was reversible, dissociation occuring almost completely after a 120-min period (K-1 = 8.13 ± 0.29 × 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. Moreover, since vasocative polypeptide, histidine isoleucine (PHI), secretin or somatostatin (SRIF) could not displace [125I-Tyr10]hGRF(1-44)NH2 binding at a concentration of 2.4 μM, it might be supposed that second site is rather specific for GRF. Finally, a partial inhibitory effect of hyrolysable and non-hydrolysable guanyl nucleotides was observed, the second site is rather specific for GRF. Finally, a partial inhibitory effect of hydrolysable and non-hydrolysable guanyl nucleotides was observed, and the affinity of various GRF analogs and related peptides, as determined by competition studies, correlated with their documented biological activity on GH release in vitro. The high affinity and specificity of [125I-Tyr10]hGRF(1-44)NH2, together with the miniaturization of the assay, allowed us to achieve further characterization of GRF binding to rat pituitaries, in the course of which we have pinpointed the presence of two classes of binding sites for this new radioligand. Whether they represent two different receptor entities and whether they both mediate some physiological actions of GRF remains to be elucidated. © 1990.