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.
Abribat. T T; Boulanger. L L; Gaudreau. P P
Key Findings
- Two distinct binding site classes for GRF were identified: high‑affinity/low‑capacity and low‑affinity/high‑capacity.
- Optimal binding required 5‑10 mM Mg2+; higher Mg2+ or monovalent cations reduced binding.
- EDTA (5 mM) prevented peptide degradation for at least 3 hours, allowing reliable measurements.
Practical Outcomes
- For DIY users, the study suggests that magnesium levels can influence how well sermorelin interacts with its target receptors, so formulations should avoid excessive Mg2+. Adding a small amount of EDTA may help keep the peptide stable in solutions. However, the work is mainly a basic binding assay and does not directly translate into dosing or performance protocols.
Summary
Researchers measured how a radioactive version of human growth hormone‑releasing factor (GRF, also called sermorelin) sticks to rat pituitary tissue. They found two kinds of binding spots: one that grabs the peptide tightly but in small numbers, and another that holds it more loosely but in larger numbers. The test works best with a modest amount of magnesium, and the binding can be stopped with EDTA to keep the peptide stable.
Abstract
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)
Study Information
pubmed
1990
1990-10-01T00:00:00.000Z
10.1016/0006-8993(90)91670-c