Rosario Donato

Soluble and membrane-bound S-100 protein in cerebral cortex synaptosomes: Properties of the S-100 receptor

Within cerebral cortex synaptosomes, S-100 protein can be recovered in two forms: soluble and membrane-bound. Synaptosomal S-100 is mainly a soluble protein (85 percent). The membrane-bound S-100 is differently distributed in the synaptosomal membranes, intraterminal mitochondria, and synaptic vesicles. S-100 binds to a specific receptor. The binding is time-dependent, reversible and saturable with respect to S-100. The number of receptors is calculated to be about 9 times 10(12)/mg protein, since saturation is achieved at 31 ng [125I]S-100/0.1 mg protein of disrupted synaptosomes. The rate constant for association of S-100 with its receptor at 37 degrees C, k1, is 4.74 times 10(4) M(-1) sec(-1), and the rate constant for dissociation, k-1, 9.24 times 10(-4) sec(-1).

Further studies on the specific interaction of S-100 protein with synaptosomal particulates.

Abstract— The specific interaction of S‐100 protein with disrupted synaptosomes was further investigated. The specific binding is a saturable and reversible process, and is time, temperature, and strictly Ca2+ ‐dependent. Two affinities affect the interaction (Kins= 7.04 × 10−9 M. 1.28 × 1012 binding sites/ mg protein; Kins2= 3.91 × 10−7M, 2.96 × 1013 binding sites/mg protein). The half‐saturation time is about 5.5 min at 37°C. The half‐life of the complex is 17 min at 37°C. At 0°C the binding is 75% slower than at 37° C, and only one‐third of the binding sites are involved. The binding capacity is decreased by high NaCl concentrations and by pretreating membranes at high temperatures. Digestion of membranes with trypsin practically abolishes the specific binding. Treatment of membranes with phospholipase C decreases the specific binding, while phospholipase D enhances it to some extent. Other lipid extractors decrease significantly the extent of the interaction. Synaptic plasma membranes seem to be the synaptosomal component involved in the high affinity binding. The S‐100 binding activity seems to undergo developmental changes, the adult values of kinetic parameters being reached around the 16th postnatal day in the rat. The results are discussed also in relation to the membrane‐bound fraction of S‐100.

THE SPECIFIC INTERACTION OF S‐100 PROTEIN WITH SYNAPTOSOMAL PARTICULATE FRACTIONS. SITE‐SITE INTERACTIONS AMONG S‐100 BINDING SITES1

Abstract— The Scatchard plot of the specific binding of the brain‐specific S‐100 protein to synaptosomal particulate fractions (SYN) is curvilinear, concave upwards. This could indicate the existence either of multiple classes of sites with different but fixed affinities, or of site‐site interactions of the type defined as negative cooperativity among a single class of sites. To discriminate between these possibilities, the dissociation test described by De Meyts et al. (1976) for demonstrating negative cooperativity among insulin binding sites of human lymphocytes or liver membranes, was applied to the interaction of S‐100 with SYN. The results show that the dissociation of the 125I‐labelled S‐100‐site complex is faster due to an ‘infinite’(100‐fold) dilution of the complex plus an excess of unlabelled S‐100 than due to dilution only, the effect of unlabelled S‐100 being specific and dose‐dependent. 125I‐IabeIIed S‐100 dissociation is time, temperature, and Ca2 +‐dependent. The effect of unlabelled S‐100 is more evident at a low site occupancy than at a high one, suggesting that at high site occupancies 125I‐labelled S‐100 binding sites could be already negatively cooperating. It can be reasonably excluded that the effect of unlabelled S‐100 is due to inhibition of rebinding of the dissociated tracer. Na+ and K+ stimulate the dissociation even at physiological concentrations. At low pH 125I‐labelled S‐100 dissociates very little, while at high pH dissociation is greatly stimulated. Finally, the protein denaturating reagent urea accelerates the dissociation even at concentrations as low as 1m. These data suggest that negative cooperativity occurs among S‐100 binding sites, but do not exclude other possibilities. Together with previously reported findings, they further support the view that S‐100 binds to highly specific sites in nervous membranes.

SOLUBILIZATION AND PARTIAL CHARACTERIZATION OF THE S‐100 PROTEIN BINDING ACTIVITY OF SYNAPTOSOMAL PARTICULATE FRACTIONS

Abstract— Chromatographic evidence is presented that 125I‐labelled S‐100 protein binds to the Triton X‐100 extract of synaptosomal membranes obtained from the cerebral cortex of the adult rat. The interaction is specific and saturable with respect to S‐100 concentration. The binding is time‐ and temperature‐dependent. The affinity of the extract for S‐100 is of the same magnitude as that calculated for whole membranes. Treatment of the extract at high temperatures or with trypsin, phospholipase C and neuraminidase reduces the extent of the interaction, while phospholipase D does not affect it. These results further support the view that S‐100 binds to a highly specific site in nervous membranes.

The Specific Interaction of S-100 Protein with Synaptosomal Particulate Fractions. Evidence for the Formation of a Tight Complex Between S-100 and Its Binding Sites

Abstract: The dissociation of the 125I‐labelled S‐100 specifically bound to synaptosomal particulate fractions (SYN) has been studied under a variety of conditions after different association times. The results indicate that after a critical association time of about 20 min at 37°C, the bound protein becomes progressively less accessible to the dissociating agents or conditions employed. These findings support the view that the partial irreversibility of the 125I‐labelled S‐100 binding to SYN could be due to the formation of a tight complex between the protein and its synaptosomal sites. These data are discussed mainly in relation to the particulate‐bound fraction of native S‐100.

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.

Specific binding sites for S-100 protein in isolated brain nuclei.

Abstract: Isolated brain nuclei possess binding sites for S‐100 protein. The interaction of S‐100 with these sites is specific and time‐, temperature‐, and Ca+‐dependent. The profile of the 125I‐labelled S‐100 binding inhibition is biphasic, displaying a high‐affinity component and a low‐affinity component. The S‐100 binding to brain nuclei is largely irreversible, probably owing to the formation of a tight complex between the protein and its nuclear binding sites. The S‐100 binding to brain nuclei is in most aspects similar to that to synaptosomal membranes. Several lines of evidence indicate, however, that the S‐100 binding to nuclei is not due to contamination of these structures with plasma membranes. Isolated liver nuclei do not possess the high‐affinity component of S‐100 binding.

Subnuclear Distribution of the S‐100 Protein Specific Binding Sites in Rat Brain

Abstract: Fractionation of isolated brain nuclei previously reacted with 125I‐labelled S‐100 showed that most of the specifically bound radioactivity associated with the nuclear membranes and the nucleoli. Labelling of nucleoli, which indicates the entrance of 125I‐labelled S‐100 into the nucleus, was observed at 37°C, but not at 0–4°C. When tested separately for 125I‐labelled S‐100 specific binding, both the nuclear membranes and the nucleoli were found to bind 125I‐labelled S‐100 in a biphasic manner, the binding displaying a high affinity and a low affinity component, as observed with intact nuclei. However, the binding to nuclear membranes was largely irreversible, while that to nucleoli was fully reversible after any association time.

Brain-specific effect of the S-100 protein on the RNA-polymerase / activity in isolated nuclei

The brain-specific S-100 protein 19 is a component both of glial 2,5,21 and neuronal cytoplasm6,7As,z°,26; additional aliquots of the protein have also been extracted from particulate matterT,10, 2z or found in synapses 6,7,9. An interesting localization of the S-100 is in the nucleus, where the protein has been detected by immunohistological procedures13, 25 and has been studied in detail in subnuclear fractions by immunochemical assays 17. To gain additional information about the nuclear S-100, we demonstrated that the protein stimulates the RNA-polymerase I activity in isolated brain nuclei 16. Since S-100 is a brain-specific protein, an organ-specificity may be hypothesized for this action. The present study verifies this property of the protein. Nuclei from various sources were isolated according to the procedure described by Lovtrup-Rein and McEwen 14 with minor modifications, as indicated elsewherO 7. The RNA-polymerase I activity was measured according to the method described by Montanaro et al. 18. In a final volume of 0.5 ml the reaction mixture contained: 100 mM Tris.HCl buffer pH 8.0, 4 mM MgCI2, 14 mM 2-mercaptoethanol, 0.6 mM each of ATP, CTP and UTP, and 0.06 mM of [3H]GTP (sp. act. I00 Ci/mole). Nuclear suspensions were preincubated for l0 min at 37 °C in 100 mM Tris.HCI buffer, pH 8.0 containing 4 mM MgC12, in the presence or in the absence of S-100 protein. In the reaction tubes the DNA/S-100 ratio (2:1) was comparable to that which occurs in brain tissue of adult rat 16. The assay was started in the reaction mixture at 37 °C with 0.1 ml of preincubated suspension, and was stopped after 15 min with 5 ml of ice-cold perchloric acid (0.5 N) in 1 ~ of sodium pyrophosphate. One milligram of bovine serum albumin was added as a carrier. The precipitate was washed twice more with 6 ml of perchloric acid (0.2 N) in 1 o/,~ of sodium pyrophosphate. The final precipitate was solubilized in 0.5 ml of NCS reagent (Nuclear Chicago Solubilizer), and 10 ml of toluene-based scintillation liquid were added for counting the radioactivity in a Nucclear Chicago scintillation spectrometer. Counting efliciencv~ was about 35 .... ,o. The activity was expressed in disint./min/mg DNA. DNA concentrations were determined

Immunocytochemical localization of S-100 protein binding sites in synaptosomal fractions and subfractions

Summary1.Immunocytochemical evidence is presented of the ultrastructural localization of binding sites for S-100 protein in synaptosomal fractions and subfractions. Synaptosomes or isolated synaptosomal subfractions were first incubated with S-100, then centrifuged to remove unbound S-100, and finally fixed before treatment with anti-S-100 antiserum, using the unlabeled antibody peroxidase-antiperoxidase (PAP) method.2.When intact synaptosomes were used, the immunoreaction product was localized to the postsynaptic density including the postsynaptic membrane. In some reactive synaptosomes, the presynaptic membrane was labeled as well, in the region of synaptic contact. No reaction deposit was found in the synaptic cleft or on intrasynaptosomal structures. Divalent cations (Ca2+ and Mg2+) were essential for S-100 to interact with synaptosomes. Of the synaptosomal subfractions tested, i.e., synaptic vesicles and intraterminal mitochondria, only synaptic vesicles showed immunoreactivity when treated with S-100 and anti-S-100 antiserum as described above.3.The S-100 immunoreactivity in synaptic structures documented in this report parallels the distribution of the high-affinity binding sites for radiolabeled S-100 in synaptosomal fractions and subfractions.