Spermine Binding To Subsynaptosomal Fractions of Rat-brain Cortex

Binding sites for [14C]spermine have been identified in rat brain cortex subcellular frac¬tions. The binding, characterized by using synaptosomal membranes, is specific for sperm¬ine. It was not detected below 20°C and increased about three/four-fold with a temperature rise of 10°C. Binding occurred only in the presence of -SH reducing agents. It was com¬pletely suppressed by metal chelating agents, and was stimulated about four-fold by 1-5 x 10-5 M Fe 2+. Smaller increases were observed in the presence of Mn 2+ , Ni2+, Ca2+, Mg2+ , and Zn2+; in contrast, millimolar concentrations of most divalent cations inhibited the binding differently (Mn2+ = Ni2+ = Zn2+ = Co2+ > Mg2+ > 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 mem¬branes with 10 percent trichloroacetic acid, or by boiling of the precipitate in the presence of 1.0 percent SDS and 10 percent -mercaptoethanol. The trichloroacetic acid precipitate showed two radioactive bands, corresponding to low Mr (< 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°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.