The resulting microsomal fraction pellets were resuspended in 50 mM Tris-HCl, 5 mM MgCl2 (pH 7.6) and then diluted based on the original tissue weights to a final dilution of 1: 10. Diluted tissue suspensions were used immediately in the radioreceptor assay. Protein concentrations of the resuspended pellets were measured by the dye-binding method of Bradford using BSA as the protein standard. Assay protein concentrations were in the range of 5-100 mg/tube, which was within the range where specific binding is linearly correlated with protein concentration. Radioreceptor assays were carried out as described by Parry et al. using 125I-d(CH2)5 [Tyr(Me)2, Tyr4, Orn8, Tyr-NH29]-vasotocin (125I-OTA) as the labeled ligand. ampicillin antibiotic
The receptor assay mixture consisted of 0.1 ml diluted tissue suspension, 0.1 ml 125I-OTA (150 000 cpm/ml), and 0.1 ml assay buffer (50 mM Tris-HCl, 5 mM MgCl2, 0.2% BSA, pH 7.6) containing a range of 0.005-1 pmol/tube unlabeled OTA (kindly provided by Dr. Maurice Manning, Medical College of Ohio, Toledo, OH). Nonspecific binding was determined by the addition of 40 pmol/tube OTA. The data were examined by nonlinear regression analysis using the Ligand computer program (Bethesda, MD) to obtain the binding affinity (Ka) and the receptor content (Rq) for radiolabeled ligand binding. Competitive binding radioreceptor assays determined ligand specificity using MT, OT, arginine vasopressin (AVP), lysine vasopressin (LVP) (all from Bachem UK Ltd., Saffron Walden, Essex, UK), and a vasopressin V1 receptor antagonist (d(CH2)5, Tyr (Me)2-AVP; MC); kindly provided by Dr. Maurice Manning, Medical College of Ohio, Toledo, OH). The interaction of each peptide with 125I-OTA was expressed as relative displacement curves B/ Bo versus log concentration and fitted with sigmoid curves.