Spermine binding to subsynaptosomal fractions of rat brain cortex

Abstract

Binding sites for [14C]spermine have been identified in rat brain cortex subcellular fractions. The binding, characterized by using synaptosomal membranes, is specific for spermine. It was not detected below 20 degrees C and increased about three/four-fold with a temperature rise of 10 degrees C. Binding occurred only in the presence of -SH reducing agents. It was completely suppressed by metal chelating agents, and was stimulated about four-fold by 1-5 x 10(-5) M Fe2+. Smaller increases were observed in the presence of Mn2+, Ni2+, Ca2+, Mg2+, and Zn2+; in contrast, millimolar concentrations of most divalent cations inhibited the binding differently (Mn2+ = Ni2+ = Zn2+ = Co2+ much greater than Mg2+ greater than Ca2+). Bound radioactive spermine was not displaced by the addition of high concentrations of unlabelled polyamine or chelating agents, nor by precipitation and washing of the membranes with 10 percent trichloroacetic acid, or by boiling of the precipitate in the presence of 1.0 percent SDS and 10 percent beta-mercaptoethanol. The trichloroacetic acid precipitate showed two radioactive bands, corresponding to low Mr (less than 8,000) components, after SDS-polyacrylamide gel electrophoresis and fluorography. The Fe2+-stimulated [14C]spermine binding was neither influenced by a previous heating of the membranes at 100 degrees C for 30 min nor by trypsin or pronase digestion, whereas the heat-treatment increased the binding occurring in the absence of Fe2+ by about two fold. A non-enzymatic formation of a spermine-metal complex tightly bound to some membrane peptide(s) is suggested.