Effects of cations and temperature on the binding of [3H]spiperone to sheep caudate nucleus

The specific [3H]spiperone binding by sheep caudate nucleus homogenate is increased by divalent cations. The effect of Ca2+ or Mn2+ (5 mM) is temperature-dependent, and it is optimal at about 37 degrees, but is relatively low below 15 degrees and above 50 degrees. In the absence of added Ca2+ or Mn2+, the maximal specific [3H]spiperone binding is observed at about 25 degrees, and the cations shift the optimum to about 37 degrees. Under the experimental conditions used, the KD is about 0.6 nM and is not influenced by Ca2+ or Mn2+, or by temperature (25 and 37 degrees). In addition to Ca2+ and Mn2+, Mg2+ and Zn2+ also increase the specific [3H]spiperone binding, but to a smaller extent. At the concentrations of Ca2+, Mn2+, Mg2+ and Zn2+ which produce a maximal increase in the [3H]spiperone binding, the membranes are nearly saturated with the cations which bind about 100 nmoles of Ca2+ or Mg2+/mg of protein, 170 nmoles Zn2+/mg of protein and at least 300 nmoles Mn2+/mg of protein. It is suggested that the cations increase the [3H]spiperone binding by either exposing more binding sites, by preventing denaturation or by increasing the solubility of [3H]spiperone in the membrane phase, or by a combination of these processes.