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J Biol Chem. 1984 Oct 10;259(19):11739-45.

Properties of the specific binding site for arginine vasopressin in rat hippocampal synaptic membranes.


In a previous paper (Pearlmutter, A. F., Constantini, M. G., and Loeser, B. (1983) Peptides 4, 335-341), we have shown that saturable, high-affinity binding sites for [3H]arginine vasopressin (AVP) are located in rat brain membrane preparations. Binding was dependent upon the presence of Ni2+ and could be dissociated by EDTA. In the hippocampus, [3H]AVP binding could be localized to synaptic membranes. In this paper, we characterize in more depth the specificity of [3H]AVP binding to a crude hippocampal synaptic preparation and the metabolism of [3H]AVP in our synaptic preparation. By means of HPLC analysis we demonstrate that the radioactive material specifically bound to hippocampal synaptic membranes is intact [3H]AVP. The ability of analogues of AVP to displace the high-affinity, specific binding of [3H]AVP parallels closely the potency of these analogues to inhibit the extinction of avoidance behavior. In the presence of membrane and Ni2+, [3H]AVP has a half-life of 7 h. In the absence of Ni2+, the half-life of [3H]AVP is 1.2 h. Fractionation by high-pressure liquid chromatography of the supernatant from the incubation media not containing Ni2+ yields three peaks of radioactivity. Analysis of the biological activity of the [3H]AVP peak and the two non-AVP peaks which represent breakdown products show the following: (a) the [3H]AVP peak (52%) and peak III (8%) bind to fresh membranes and (b) peak II (40%) has no binding activity. Although Ni2+, Co2+, benzamidine, and phenanthroline can prevent [3H]AVP degradation, only Ni2+ and, to a much lesser extent, Co2+, can potentiate specific [3H]AVP binding. The results show that AVP-specific binding has properties which parallel its biological activity in behavioral assays; that, ultimately, proteolysis by membrane-bound peptidases inactivates AVP; and that Ni2+ acts both by preventing AVP breakdown and by potentiating specific binding.

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