Heterogeneity of the S‐100 Protein Specific Binding Sites in Synaptosomal Particulate Fractions and Subfractions

Abstract

The specific interaction of S-100 protein with synaptosomal particulate fractions (SYN) was further investigated with special reference to the number of binding components and their localization in synaptosomal subfractions. Binding studies were conducted on SYN from various CNS regions, on synaptosomal subfractions from the cerebral cortex, and on cerebral cortex SYN under various conditions. The results suggest that S-100 binds to two populations of membrane sites: high -affinity sites, which seem to be confined to neuronal membranes (synaptosomal plasma membranes and synaptic vesicles), and low-affinity sites, which are also detected in other membranes. The data are consistent with the view that the biphasic profile of S-100 binding to SYN does not result from heterogeneity of the S-100 molecule, and that the Ca2+ conformation of the protein is as important as the proper conformation of the binding site for full expression of high-affinity binding.

The Specific Interaction of S‐100 Protein with Synaptosomal Particulate Fractions. Modulation of Binding by Fractional Occupancy of Sites and by the Physical State of Membranes

“…The results (not shown) indicated that the protein bound in the presence'of KC1 completely dissociated in approximately 5 min after any association time, and to the same extent whether or not an excess of unlabeled S-100 was present in the dissociation medium; in addition, protein bound to phospholipase C-or D-treated SYN dissociated to a much greater extent than that bound to untreated SYN, both in the absence and presence of unlabeled s-100. Together, these data indicate that S-100 forms a tight complex with high-affinity sites and confirm that S-100 must be in its Caz+ conformation (Calissano et al, 1976) for high-affinity binding to occur (Donato, 1982).…”

“…Dissociation experiments were also conducted after incubation of 12sI-labeled S-100 with SYN in the presence of 0.2M KCI or with SYN pretreated with either phospholipase C (EC 3.1.4.3) or D (EC 3.1.4.4). Under all these conditions high-affinity S-100 binding appeared to be abolished (Donato, 1982). The results (not shown) indicated that the protein bound in the presence'of KC1 completely dissociated in approximately 5 min after any association time, and to the same extent whether or not an excess of unlabeled S-100 was present in the dissociation medium; in addition, protein bound to phospholipase C-or D-treated SYN dissociated to a much greater extent than that bound to untreated SYN, both in the absence and presence of unlabeled s-100.…”

“…3). When the preincubation was conducted in the presence of 415 nM unlabeled S-100-a concentration that inhibits the high-affinity binding of 12"I-labeled S-100 (Donato, 1982)-the bound tracer dissociated completely and mono- or 415 (0) nM unlabeled S-100. After centrifugation to remove the unbound S-100, the pellet was resuspended in 0.18 ml of buffer medium, and aliquots of 10 pi were either counted for bound radioactivity or diluted 100 times with the buffer medium in the absence (open symbols) or in the presence (closed symbols) of 100 p g of unlabeled S-100.…”

“…Procedures were as described in the accompanying report (Donato, 1982). Details of experiments reported here are given in the legends to figures.…”

“…4 and 5). If the radiolabeled ligand and the unlabeled ligand have the same binding properties and the complex is in nonrapid equilibrium with the medium (Donato, 1982), then preincubation of SYN with unlabeled S-100 should result in changes both in the Scatchard plot of data obtained from the subsequent interaction of 1251-labeled S-100 and in the dissociation profile, depending on the concentration of unlabeled S-100 used in the preincubation experiment. SYN were thus incubated at 37°C for 60 min with various concentrations of unlabeled S-100.…”