Neurospecific protein S-100 in the synaptosomes of rat cerebral cortex]

Neurospecific S-100 protein was revealed by the methods of microelectrophoresis in the 15% polyacrylamide gel with a 0.1% sodium dodecylsulfate and by a highly purified S-100 protein "marker" in the composition of low molecular acidic proteins of the rat brain synaptosomes. The S-100 protein conten constitutes about 15-2o% of the low molecular acidic synaptosomal porteins in the rat brain.     

Modulation of brain protein phosphorylation by the S-100 protein

The effects of the nervous system specific protein, S-100, on protein phosphorylation in rat brain is examined. The S-100 protein inhibits the phosphorylation of several soluble brain proteins in a calcium dependent fashion. The most potent effect exhibited by S-100 was on the phosphorylation of a protein having a molecular weight of 73,000. The data suggest that the calcium binding S-100 protein, for which a function has not yet been assigned, may modulate calcium dependent phosphorylation of selected brain proteins.     

S-100 proteins

S-100 is a group of closely related, small, acidic Ca2+-binding proteins (S-100a0, S-100a and S-100b, which are alpha alpha, alpha beta, and beta beta in composition, respectively). S-100 is structurally related to calmodulin and other Ca2+-binding proteins. S-100 is abundant in the brain and is contained in well defined cell types of both neuroectodermal and non-neuroectodermal origin, as well as in their neoplastic counterparts. In the mammalian brain, S-100a and S-100b are confined to glial cells, while S-100a0 is neuronal in localization. Single S-100 isoforms bind Ca2+ with nearly the same affinity. K+ antagonizes the binding of Ca2+ to high affinity sites on S-100. S-100 binds Zn2+ with high affinity. S-100 is found in a soluble and a membrane-bound form and has the ability to interact with artificial and natural membranes. S-100 has no enzymatic activity. S-100 has been involved in several activities including memory processes, regulation of diffusion of monovalent cations across membranes, modulation of the physical state of membranes, regulation of the phosphorylation of several proteins, control of the assembly-disassembly of microtubules. Some of these effects are strictly Ca2+-dependent, while other are not. S-100 is being secreted or released to the extracellular space. In some cases, this event is hormonally regulated. Several S-100 binding proteins are being described.     

Effect of protein S-100 on phosphorylation of nuclear proteins of cells of rat brain and liver

The effect of acidic neurospecific protein S-100 on the phosphorylation of brain and liver nuclear proteins with 1 and 10 microM ATP was investigated. It was shown that protein S-100 increases the phosphorylation of brain nuclear proteins, while antigen D, another acidic neurospecific protein half-identical to 14-3-2 protein, inhibits this process. Ca2+ and cAMP at concentration of 10(-6) M do not affect the phosphorylation of brain nuclear proteins. In control assays the tracer 32P is presumably incorporated into high molecular weight nuclear protein fractions (Mr greater than 40000). After addition of protein S-100 the tracer is mainly incorporated into these proteins as well independently of ATP concentration (1 or 10 microM). The phosphorylation of nuclear proteins with molecular weights above 100000 is mostly increased in this case. At ATP concentration of 1 microM protein S-100 decreases histone phosphorylation 2.3 times but does not affect that of non-histone proteins. However, at 10 microM ATP the inhibitory action of this protein on histone phosphorylation is absent. The possible mechanisms of protein S-100 action on nuclear proteins phosphorylation are discussed.     

S-100 and Other Acidic Proteins Promote Ca2+-Independent Phosphorylation of Protamine Catalyzed by a New Protein Kinase from Brain

Abstract: A new protein kinase modulated by S-100 (tentatively referred to as protein kinase X) was partially purified from pig brain extracts. The activity of protein kinase X, which was independent of Ca²⁺, was demonstrated when protamine (free base), but not protamine sulfate and other proteins (including histone), was used as substrate. The enzyme activity, found to distribute in both soluble and particulate fractions and to occur at the highest level in brain compared with other tissues (heart, kidney, liver, skeletal muscle, spleen, and testis) of rats, was also modulated by other acidic proteins (calmodulin, troponin C, and stimulatory modulator) in a Ca²⁺-independent manner. S-100 and other acidic proteins appeared to function as "substrate modifiers" by interacting with protamine (a highly basic protein), but not with the enzyme, thus rendering protamine in the complex a superior phosphate acceptor. The two isoforms of S-100 (i.e., a and b) were equally effective. Although the enzyme was not inhibited by many agents (trifluoperazine, melittin, cytotoxin I, polymyxin B, and spermine) shown to inhibit markedly phospholipid/Ca²⁺- or calmodulin/Ca²⁺-stimulated protein kinase, gossypol was found to inhibit specifically protein kinase X. The present findings suggest that S-100, a major acidic protein specific to nervous system, may promote phosphorylation by protein kinase X of certain neural proteins resembling protamine or containing protamine-like domains, in addition to its presumed role of a low-affinity Ca²⁺-binding protein.     

The Amino Acid Sequence of the Tryptophan-Containing Subunit (α-Subunit) of Bovine Brain S-100 Protein

Abstract: The tryptophan-containing subunit (α-subunit) of bovine brain S-100 protein was purified from a S -aminoethyl derivative of S-100a protein, and its amino acid sequence was determined. The α-subunit contained 93 residues, including one tryptophan, and had a molecular weight of 10,400. The sequence shows an extensive homology (58% identity) to the sequence of another "tryptophan-free" subunit (β-subunit) found in both S-100a and S-100b protein, and has a calcium binding site characteristic of the "E-F hand" proteins, such as calmodulin or troponin C. The tryptophan residue is located at position 90 which is presumably adjacent to the C-terminal end of the α-helix following the calcium binding loop, and thus appears likely to serve as a specific probe in structure-function studies of S-100a protein.     

Inhaled carbon monoxide provides cerebral cytoprotection in pigs

Carbon monoxide (CO) at low concentrations imparts protective effects in numerous preclinical small animal models of brain injury. Evidence of protection in large animal models of cerebral injury, however, has not been tested. Neurologic deficits following open heart surgery are likely related in part to ischemia reperfusion injury that occurs during cardiopulmonary bypass surgery. Using a model of deep hypothermic circulatory arrest (DHCA) in piglets, we evaluated the effects of CO to reduce cerebral injury. DHCA and cardiopulmonary bypass (CPB) induced significant alterations in metabolic demands, including a decrease in the oxygen/glucose index (OGI), an increase in lactate/glucose index (LGI) and a rise in cerebral blood pressure that ultimately resulted in increased cell death in the neocortex and hippocampus that was completely abrogated in piglets preconditioned with a low, safe dose of CO. Moreover CO-treated animals maintained normal, pre-CPB OGI and LGI and corresponding cerebral sinus pressures with no change in systemic hemodynamics or metabolic intermediates. Collectively, our data demonstrate that inhaled CO may be beneficial in preventing cerebral injury resulting from DHCA and offer important therapeutic options in newborns undergoing DHCA for open heart surgery.     

Quantitative analysis of the interaction between S-100 proteins and brain tubulin

S-100 was shown to regulate the in vitro assembly of brain microtubule proteins (MTPs) in a Ca2+-mediated way by acting on both the nucleation and the elongation of microtubules (MTs). Here data will be shown suggesting that S-100 binds to tubulin. The binding is time-, temperature-, Ca2+-, and pH-dependent, and saturable with respect to S-100. At pH 6.75, the saturation curve is biphasic, displaying a high affinity component (dissociation constant, Kd1, approximately 0.1 microM) and a low affinity component (Kd2 approximately 3.8 microM). At pH 6.75, as the free Ca2+ concentration raises from 0 to 100 microM, the overall binding capacity increases from 0.065 to 0.66 mol S-100/mol tubulin dimer. This finding, together with the observation that the S-100 effect on MTP assembly is Ca2+-dependent at that pH, suggests that the S-100-induced inhibition of MTP assembly depends on S-100 binding to the low affinity sites on the tubulin molecule. The S-100 binding to tubulin is pH-dependent; as the pH raises from 6.75 to 8.3, both binding components are affected, the major changes consisting of an increase in the binding capacity and a decrease in the overall affinity. Moreover, as the pH raises, Ca2+ is no longer required for S-100 to bind to tubulin. S-100 also interacts with a component of whole MTPs (probably tubulin, on the basis of the above results). No S-100 binding to microtubule-associated proteins (MAPs) could be evidenced by the techniques employed in this study. On the contrary, some competition between S-100 and MAPs for binding sites or tubulin seems to occur.     

S-100-Mediated Inhibition of Brain Protein Phosphorylation

The effects of the glial-specific, calcium-binding, S-100 protein on brain membrane and supernatant protein phosphorylation were assessed. S-100 concentrations as low as 5 micrograms/ml caused a marked inhibition of the phosphorylation of a soluble brain protein having a molecular weight of 73,000 daltons (73K). This protein was designated the S-100 protein-modulated phosphoprotein (SMP). Half-maximal inhibition of the phosphorylation of SMP by S-100 was obtained at concentrations of 12 micrograms/ml (0.57 microM). The inhibition of SMP phosphorylation by S-100 was calcium-dependent, with a calculated calcium Ka of 2.0 +/- 0.3 microM. SMP phosphorylation was also inhibited by calmodulin, but only partially and with a much lower potency. The inhibition of SMP phosphorylation by S-100 was not inhibited by fluphenazine, whereas the effect of calmodulin was. SMP was found in many brain areas, with the highest levels seen in the corpus callosum. Various peripheral tissues, such as kidney; liver; and pineal, pituitary, and adrenal glands, did not contain detectable SMP levels. At higher S-100 concentrations, greater than 10 micrograms/ml, the phosphorylation of several other soluble proteins was markedly inhibited. These proteins have molecular weights of 56K, 50K, and 47K. The phosphorylation of these proteins was enhanced by calmodulin. These data suggest that the S-100 protein may function to modulate the phosphorylation of brain proteins in a manner analogous to (although in a reciprocal fashion) that of calmodulin.     

S-100B蛋白在新生儿缺氧缺血性脑病中的临床应用进展

S-100B蛋白是一类钙离子结合蛋白,由神经系统胶质细胞分泌,广泛分布于神经组织中。在正常情况下发挥重要的生理作用,但分泌过高具有神经毒性。血清中S-100B蛋白含量与新生儿缺氧缺血性脑病(HIE)呈正相关,可作为早期诊断HIE脑损伤及判断损伤程度及预后的有效指标。通过动态监测S-100B蛋白含量,了解脑损伤后血清中S-100B蛋白水平变化的时间规律性,S-100B蛋白可作为脑损伤后神经生化新标志物。本文对血清S-100B蛋白在新生儿缺氧缺血性脑病诊断中的价值作一综述。     

Immunochemical and immunohistochemical detection of S-100-like immunoreactivity in spinach tissues.

S-100 proteins represent a group of closely related acidic, calcium binding proteins originally isolated from the mammalian nervous system and later detected in non-neural cell types and in a wide variety of vertebrate and invertebrate species. The present study used immunochemical and immunohistochemical methods to extend the investigation of S-100 during phylogenesis to plant tissues. The presence of S-100-like immunoreactive material was detected in extracts of spinach (Spinacia oleracea L.) terminal buds and young leaves by the ELISA method and by Western blotting using different anti-S-100 rabbit antisera. Using the PAP method, serial sections of young spinach leaves treated with the same antisera exhibited an immunoreaction product that was confined to the cytoplasm and nucleus (but absent from the vacuoles) in meristematic, epidermal, and parenchymal cells. The present data enlarge the field of investigation of S-100 proteins in the search of the function(s) of S-100 in biological organisms.     

Purification of branched chain aminotransferase from rat heart mitochondria

This paper presents the first purification of the branched chain aminotransferase (EC 2.6.1.42) from rat heart mitochondria. The enzyme has been purified from the 100,000 x g supernatant obtained after sonication and ultracentrifugation of rat heart mitochondria. A combination of open column chromatography, high pressure liquid chromatography (HPLC), and discontinuous polyacrylamide disc gel electrophoresis was used. The key step in the procedure was hydrophobic interaction chromatography on HPLC. The final purification step was polyacrylamide disc gel electrophoresis where the enzyme appeared as a doublet. When electroeluted from the gel, each of these bands had the same specific activity demonstrating that there are two forms of the purified enzyme which differ slightly in electrical charge. On sodium dodecyl sulfate-polyacrylamide gel electrophoresis, these two enzyme forms appeared as a single band with a molecular mass of 43 kDa. Size exclusion chromatography on Sephacryl S-100 identified the enzyme as a 50-kDa protein. These experiments argue against the existence of a dimeric form of this enzyme. The ratio of enzyme activity with leucine (0.84), valine (0.88), or glutamate (0.66) as amino acid substrate versus isoleucine remained constant throughout the purification procedure. Specific activity of the final preparation was 66 units/mg of enzyme protein. Polyclonal antibodies against the purified enzyme were raised in rabbits. On an immunoblot the antiserum recognized a 43-kDa protein in the 100,000 x g supernatant from a rat heart mitochondrial sonicate but did not recognize any proteins in rat brain cytosol. Quantitative immunodot assay resulted in an estimated enzyme content of about 100 micrograms of branched chain aminotransferase protein/g of heart, wet weight. Finally, 97% of the heart branched chain aminotransferase activity could be neutralized by the antiserum, but the antiserum would not neutralize aminotransferase activity in brain cytosol. These data suggest that close sequence homology does not exist between the two proteins.     

Interaction Between S-100 Proteins and Steady-State and Taxol-Stabilized Microtubules In Vitro

S-100 proteins are a group of three 21-kilodalton, acidic, Ca2+-binding proteins of the "E-F hand" type shown to regulate several cell activities, including microtubule (MT) assembly-disassembly. We show here that S-100 proteins interact with MTs assembled from either whole microtubule protein or purified tubulin, both in the absence and in the presence of the MT-stabilizing drug taxol. Evidence for the binding of S-100 to MTs comes from both kinetic (turbidimetric) and binding studies. Kinetically, S-100 enhances the disassembly of steady-state MTs in the presence of high concentrations of colchicine or vinblastine at 10 microM free Ca2+ and disassembles taxol-stabilized MTs at high Ca2+ concentrations. Experiments performed using 125I-labeled S-100 show that S-100 binds Ca2+ independently to a single set of sites on taxol-stabilized MTs assembled from pure tubulin with an affinity of 6 x 10(-5) M and a stoichiometry of 0.15 mol of S-100/mol of polymerized tubulin. Under certain conditions, S-100 proteins also cosediment with MTs prepared by coassembly of S-100 with MTs, probably in the form of an S-100-tubulin complex. Because S-100 binds to MTs under conditions where this protein fraction does not produce observable effects on the kinetics of assembly-disassembly, e.g., in the absence of Ca2+ at pH 6.7, we conclude that the S-100 binding to MTs does not affect the stability of MTs per se, but rather creates conditions for increased sensitivity of MTs to Ca2+.     

ACIDIC PROTEINS IN RAT BRAIN NUCLEI: DISC ELECTROPHORESIS

—Disc electrophoretic patterns of soluble, acidic proteins in brain and liver nuclei and in the respective tissue homogenates were studied. In brain nuclei, a fast component moving with the electrophoretic front constitutes a relatively large amount of the acidic proteins which migrate toward the anode. Electrophoretic patterns obtained with fractionated brain nuclei (neuronal, astrocytic and glial) were similar to those obtained with total brain nuclei. The S-100 protein could not be detected in any of the nuclei examined. Protein patterns in brain and liver nuclei markedly differed both quantitatively and qualitatively.     

Zinc and copper in fish from natural waters and rearing ponds in Northern Italy

Zinc and copper were detected in several tissues of fresh and saltwater fish. Liver concentrations varied widely, with respect to the storage and detoxication functions of the organ. In muscular tissues the two metals are linked to aerobic metabolism being higher in the heart and lower in the white muscle. High levels of zinc were found in the female gonad, while in the brain zinc has been shown to be more constant and possibly regulated better than copper. In sea bass supplemented with artificial diets no correlation was found between the metal content in the diet and that of the tissue. In goldfish attempts using gel filtration to isolate specific metal binding proteins of low molecular weight gave negative results, the metals were mostly bound to ligands excluded from the gel.     

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.     

Chlorpromazine inhibits the calcium-mediated effects of S-100 protein(s) on assembled brain microtubule proteins, but not those on microtubule protein assembly

We have examined the S-100-chlorpromazine interplay at the level of brain microtubule proteins in vitro. The results indicate that in the presence of 0.12 M KCl and 10 microM free Ca2+ the inhibitory effect of S-100 on microtubule assembly is additive to that of chlorpromazine, but S-100 fails to potentiate the disassembling effect of 0.1 mM Ca2+ if added to assembled microtubule proteins after chlorpromazine and Ca2+, probably because of inhibition of S-100 by the phenothiazine. Chlorpromazine does not compete with S-100 for binding to purified tubulin.     

Modulation of tyrosine phosphorylation of p36 and other substrates by the S-100 protein

The inhibitory effects of Ca2+-binding proteins on tyrosine phosphorylation of p36 protein isolated from bovine intestinal epithelium by immunoprecipitated p130fps were investigated. S-100 protein dose dependently inhibited the p36 phosphorylation, and calmodulin weakly depressed the phosphorylation, whereas parvalbumin and troponin C had no significant effects. The S-100 preparation purified from bovine brain did not contain phosphatase activity or ATPase activity. The concentration of ATP did not affect the S-100-mediated inhibition of phosphorylation but the substrate protein, p36, reversed the inhibition. S-100 similarly inhibited the tyrosine phosphorylation of p36 by p60src but did not affect the p36 phosphorylation by protein kinase C. S-100 inhibited the tyrosine kinase activity of p130fps using the other substrates tested as well. These results suggest that S-100 interacts with the substrate binding site of retroviral tyrosine-specific protein kinases and may play a regulatory role in the tyrosine phosphorylation.     

Calcium-independent, pH-regulated effects of S-100 proteins on assembly-disassembly of brain microtubule protein in vitro

At alkaline pH, Ca2+ is no longer required for S-100 proteins to inhibit the assembly and to promote the disassembly of brain microtubules in vitro, though the presence of Ca2+ significantly favors the S-100 effects. These effects are inversely related to the microtubule protein concentration and directly related to the S-100 concentration and the pH. Ca2+-independent, pH-regulated inhibition of assembly of phosphocellulose-purified tubulin by S-100 is also described. The microtubule disassembling effect of S-100 is additive to that of alkali (used to raise the pH), and S-100 further disassembles microtubules after alkalinization. Thus the larger inhibitory effect of S-100 on microtubule assembly at alkaline versus acid pH depends on both a decrease in the assembly rate and an increase in the disassembly rate. Together with previous data on this topic, the present findings indicate that S-100 proteins act on microtubule protein in vitro primarily by binding to tubulin, this event being Ca2+-regulated at a given pH, and pH-regulated at a given free Ca2+ concentration.