Effects of Divalent Cations and Disulfide Bond Reducing Agents on Specific Binding of Growth Hormone to Liver Membrane Receptors from Snakehead Fish (Ophiocephalus argus, Cantor).

Divalent cations, Ca(2 ), Mg(2 ) and Mn(2 ) enhance the binding of bream growth hormone (brGH) to snakehead fish liver membrane, and their optimum concentration was found to be 8 12 mmol/L, at which Ca(2 ), Mg(2 ) and Mn(2 ) could increase, respectively, the specific binding to 230%, 180%, and 200%, compared with the binding in the absence of ions. The Eadie-Scatchard plot was used for the dynamic analysis of the Ca(2 ) binding site. A low affinity Ca(2 ) binding site was found in the GH-receptor complex with K(m)=0.384 mmol/L, and the affinity constant (K(a)) was increased from 1.045x10(9) L.mol(-1) to 1.295x10(9) L.mol(-1) by the addition of 10 mmol/L CaCl(2). The effects of disulfide bond reducing agents, DTT and ME, on (125)I-brGH binding to growth hormone receptor (GHR) on snakehead fish liver memebrane were also analyzed. The addition of 0.1 20 mmol/L DTT or 0.01% 1% ME to the radioreceptor assay system caused a significant dose dependent increase in the specific binding for (125)I-brGH. In the presence of 0.8 mmol/L DTT or 0.08% ME, the specific binding of (125)I-brGH was increased from 10.2% to 15.5% and 13.2% respectively, and the affinity constant was also increased from 1.265x10(9) L.mol(-1) to 2.185x10(9) L.mol(-1) and 1.625x10(9) L.mol(-1), respectively but no changes in the binding capacity were observed. Further studies showed that the effects of reductants on the specific binding of brGH were due in part to the ligand itself and in part to GHR. In addition, it was observed that one of the three disulfide bonds of brGH could be reduced by 0.8 mmol/L DTT.