Selective sites for polyamine binding to rabbit intestinal brush-border membranes

The intestinal polyamine transporters have not yet been identified. Our aim was to characterize specific polyamine binding sites in rabbit intestinal brush-border membranes (IBBM) as a starting step for identification of polyamine transporters. This was investigated at 4 degrees and at low membrane concentration. Saturation isotherms for [H-3]putrescine (PUT) binding indicated a single population of sites (puT) with a dissociation equilibrium constant K-d of 3.8 mu M and a density of sites B-max of 58 pmol/mg of protein. [H-3]spermidine (SPD) binding also involved only one class of sites (spD), albeit with a lower affinity (K-d = 106 mu M) and higher abundance (B-max = 1240 pmol/mg of protein) than puT. On the contrary, [C-14]spermine (SPM) bound two classes of sites (spM(1) and spM(2)) differing in their affinity (K-d = 2.5 and 31.4 mu M) and abundance (B-max = 467 and 1617 pmol/mg of protein, respectively). Membrane association of SPM at 4 degrees was much faster than that of SPD and PUT, both of which proceeded at a similar rate. In contrast to PUT and SPD dissociation, SPM dissociation at 23 degrees did not follow aiirst order reaction. Specifically bound [H-3]PUT, unlike [H-3]SPD and [C-14]SPM, dissociated at 23 degrees independently of the addition of nonradioactive polyamine. Methylglyoxal-bis-(guanylhydrazone) was an extremely potent inhibitor of PUT binding (K-i = 3.2 +/- 1.5 nM), but as with PUT and cadaverine (CAD), it did not alter [H-3]SPD and [C-14]SPM binding substantially. The intestinal brush-border membrane may contain at least three sites specific for polyamine binding and exhibiting different ligand selectivity. Site puT might be associated with the transport system already described for intestinal uptake of PUT. BIOCHEM PHARMACOL 56;4:517-526, 1998. (C) 1998 Elsevier Science Inc.