HETEROGENEITY OF S-100 PROTEINS OF BRAIN: SUBUNIT COMPOSITIONS

Abstract— Two populations of S-100 proteins (III-IVa-1) and (III-IVb-1) were isolated from bovine brain by a simple three-step procedure involving precipitation with ammonium sulfate, ion exchange chromatography and gel electrophoresis. These two fractions reacted with anti-S-100 serum and showed all the properties of S-100 protein, but differed in the distribution of protein among the four subunits obtained in SDS-gels. These subunits had apparent molecular weights of 7800, 6800, 6100, and 5200. Separations based on charge differences revealed only three subunits. Similar fractions isolated from rat brain contained only two subunits with apparent molecular weights of 7800 and 6500. The two S-100 protein fractions also differed in the specific extinction at 280 nm and in the protein distribution among the separate bands which formed either in a 14% acrylamide-agarose gel-discontinuous buffer system or in 7.5% gels in the presence of calcium. The yield of S-100 protein isolated in the presence of aids (EDTA, EGTA, mercaptoethanol, protease inhibitors) was considerably higher than in their absence (80 mg vs 60 mg/kg tissue), the predominant loss occurring in fraction III-IVa-1. It is concluded that 'S-100 protein'contains a number of molecular species which have different subunit compositions and differences in stability.     

Pyridoxal-5-phosphate levels in rat brain assayed by a modified method using enzymic decarboxylation of l-[14C] tyrosine

Abstract— The formation of a complex between myelin basic protein and S-100 protein was detected from the change in migration of S-100 protein on immunoelectrophoresis. A degree of specificity for the interaction was shown by two observations: (1) two other pure acidic proteins. III-III-2 and bovine serum albumin, did not show it and (2) complex formation was dependent on specific ions, either Ca²⁺ (10 mM) or Mn²⁺ (1 mM). Mg²⁺, Ba²⁺, and Li⁺ had no effect. Non-specific interactions between S-100 protein and other basic molecules (histones. polylysine) are not dependent on specific ions such as Ca²⁺ and Mn²⁺. The complex was stable at physiological salt concentrations and contained 3 mol of basic protein per mol of S-100 protein. Complex formation was also detected from the alteration of migration rate of S-100 protein in polyacrylamide gels. Serological activity (complement-fixation) of S-100 protein with anti-S-100 serum was reduced in the complex by 30%.     

Detection and partial characterization of two bovine pregnancy-specific proteins

Two pregnancy-specific proteins were detected by immunoelectrophoresis using antisera developed to homogenates of bovine extraembryonic membranes. Antisera also reacted to extracts of endometrium from pregnant cows and extraembryonic fluids. However, antisera did not react with a preparation presumed to be bovine placental lactogen, fetuin, extracts of various somatic tissues from pregnant cows or extracts of endometrium from nonpregnant cows. One of the proteins had an estimated molecular weight of 65,000-70,000, an isoelectric point of 4.6-4.8 and yielded a reaction of identity with bovine alpha 1-fetoprotein by immunodiffusion. The second protein yielded a reaction of identity with bovine alpha 1-fetoprotein by immunodiffusion. The second protein had no immunological cross-reactivity with the known proteins or organ extracts which were tested. The molecular weight and isoelectric point was 47,000-53,000 and 4.0-4.4, respectively. These data demonstrate the presence of at least 2 pregnancy-specific proteins in cattle.     

Molecular Interaction of S-100 Proteins with Microtubule Proteins In Vitro

Several procedures were employed to examine the in vitro interaction between S-100 proteins and microtubule proteins. Binding of S-100 to tau factors was observed under all experimental conditions. S-100 binding to microtubule-associated protein 2 (MAP2) was best detected by exposing nitrocellulose-immobilized MAP2 or MAPs to either 125I-labeled S-100 or biotinylated S-100. S-100 binding to tubulin was detected when the two protein fractions were first incubated with each other followed by exposure to the bifunctional cross-linker disuccinimidylsuberate, and then separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transfered onto nitrocellulose paper. By this procedure, complex formation between S-100 and tubulin, as well as between S-100 and a relatively low-molecular-weight MAP, was evidenced by immunoblotting using an anti-S-100 antiserum. Alternatively, complex formation between biotinylated S-100 and either tubulin or MAPs was visualized by means of avidin-peroxidase, after SDS-PAGE of the complex mixtures and transfer of the separated proteins onto nitrocellulose. The interaction between S-100 and tubulin was strictly Ca2+ dependent, and resistant to high concentrations of KCl, colchicine, or vinblastine.     

Effect of anti-S-100 serum on36C1− ion permeability across the Deiters' neuron plasma membrane

1. A microtechnique allowing the study of single plasma membranes from the gamma-aminobutyric acid (GABA)-acceptive Deiters' neuron has been utilized in order to assess the effect of both S-100 protein and its antiserum on 36Cl- permeability through such membranes. 2. The results show that both S-100 (in the Ca2+ form) incorporation onto the external side of the membranes and their preincubation with anti-S-100 serum stimulate 36Cl- permeability. 3. These effects are not additive with that of GABA, indicating that both S-100 and anti-S-100 act via the GABAA receptor complexes on Deiters' membranes. 4. When the membranes were incubated first with S-100/Ca2+ and then with anti-S-100, the second treatment resulted in the disappearance of the S-100 effect. However, the anti-S-100 effect was fully displayed.     

Purification and identification of two pertussis-toxin-sensitive GTP-binding proteins of bovine spleen

Two alpha subunits of GTP-binding proteins were purified from bovine spleen membranes. Both proteins were ADP-ribosylated by pertussis toxin in the presence of beta gamma subunits. The major protein had a molecular mass of 40 kDa and its immunological reactivity and fragmentation pattern by limited proteolysis were identical with those of the alpha subunit of Gi2. The minor protein had a molecular mass of 41 kDa and its partial amino acid sequences completely matched with those predicted from human and rat Gi3 alpha cDNAs.     

S-100 protein subunits in bovine oviduct epithelium:In situ distribution and changes during primary cell culture

Summary Cultures of bovine oviduct epithelial cells are widely used in co-culture systems to improve the results ofin vitro fertilization. The aim of the present study was to evaluate the suitability of S-100 protein as a differentiation marker for bovine oviduct epithelial cellsin vitro. The distribution of S-100α and S-100β was examined immunohistochemically in bovine oviduct epitheliumin situ and in primary cell cultures derived from it. Three segments of the Fallopian tube (isthmus, ampulla and fimbriae) were compared and analysed during different stages of the oestrus cycle (luteal phase and follicular phase). Ciliated and non-ciliated cells of the epithelium reacted with anti-S-100α, S-100a(αβ) and S-100β antibodies, except for isthmic non-ciliated cells, which did not bind anti-S-100β or anti-S-100a(αβ). In addition, basal cells never showed immunoreactivity for S-100. In confluent monolayers of cultured oviduct epithelial cells, disappearance of reactivity for S-100 paralleled morphological signs of dedifferentiation (loss of cilia, cytoplasmic vacuolization). Free-floating oviduct epithelial cells, in contrast, retained morphological differentiation and still expressed S-100 antigen even after seven daysin vitro. The immunohistochemical findings were confirmed by polyacrylamide gel electrophoresis and Western blotting. The results indicate that the presence of S-100 is closely connected to morphological differentiation and to the specific functional condition of bovine oviduct epithelial cells.     

Biospecific adsorption of hepatic DT-diaphorase on immobilized dicoumarol. I. Purification of cytosolic DT-diaphorase from control and 3-methylcholanthrene-treated rats

Liver cytosolic DT-diaphorase has been purified from both control and 3-methylcholanthrene (MC)-treated rats, employing a new efficient affinity chromatography gel azodicoumarol coupled to divinyl sulfone cross-linked Sepharose 6B. A subsequent gel filtration on Sephacryl S-200 results in a homogeneous enzyme preparation with a yield of 50–55%. The enhanced DT-diaphorase activity observed in MC-treated rats is most probably due to an increase in enzyme concentration. This conclusion is based on the following results: (a) cycloheximide, an inhibitor of protein synthesis, prevents the increase in DT-diaphorase activity normally caused by MC; (b) purifications of DT-diaphorase from control and MC-treated rats result in enzyme preparations exhibiting closely similar specific activities; 15 times higher amounts of enzyme are obtained from MC-treated rats as compared to controls; (c) immunological identity between DT-diaphorase isolated from control and MC-treated rats are found with the Ouchterlony immunodiffusion method employing antisera raised against either type of enzyme preparation; (d) rocket immunoelectrophoresis reveals a severalfold higher specific content of DT-diaphorase in cytosol from MC-induced rats as compared to controls. The investigated physicochemical properties of DT-diaphorase are not altered after MC treatment of rats. The minimum molecular weight based on the flavin content of the enzyme is close to that obtained with SDS-slab gel electrophoresis, i.e., approximately 28,000, indicating that the dimer of DT-diaphorase contains two molecules of FAD. The molecular activities of DT-diaphorase with various electron acceptors have been investigated; no significant differences between DT-diaphorase isolated from control and MC-treated rats are found. However, the molecular activity of the enzyme with 2,6-dichlorophenolindophenol and menadione varies considerably from one preparation to another, irrespective of source. Employing fused rocket immunoelectrophoresis, it has been possible to detect at least three antigenic forms of DT-diaphorase with different reactivities toward electron acceptors such as 2,6-dichlorophenolindophenol and menadione. The possible existence of several forms of DT-diaphorase is discussed.     

Studies on species cross-reactivity of hemopexin by use of monoclonal and polyclonal antibodies

The extent of immunological cross-reactivity between hemopexins of four species (rat, human, rabbit and chicken) was assessed with four affinity purified polyclonal antibodies and three monoclonal antibodies using RIA, Western blotting and rocket immunoelectrophoresis. Neither the two monoclonal antibodies to rabbit hemopexin (Rb3D11 and Rb3H9), the monoclonal antibody (R4B3) to rat hemopexin nor any of the polyclonal antibodies showed shared antigenic determinants between avian and mammalian hemopexins as judged by RIA or rocket immunoelectrophoresis. Western blotting with polyclonal antibodies revealed some reactivity raising the possibility of a few shared, though distantly related, epitopes. Polyclonal antibodies, raised to the mammalian hemopexins cross-reacted to variable extents with the respective antigens by RIA, results paralleled by data obtained by Western blotting. Anti-rat monoclonal antibodies reacted only with rat hemopexin in Western blots and minimally with rabbit hemopexin in RIA. The anti-rabbit monoclonal antibodies recognized two distinct epitopes one of which is shared with human hemopexin and presumably highly conserved.     

Characterization of glycoconjugates found in granular cell tumors, with special reference to keratan sulfate

Nine granular cell tumors (GCTs) were studied using the immunoperoxidase technique with a mouse monoclonal antibody to keratan sulfate and a polyclonal antibody to S-100 protein. Various lectins and basic dye stains were also employed. Schwannomas benign and a malignant, a neurofibroma, a leiomyoma, two examples of nevus pigmentosus and a congenital epulis were similarly examined to compare the histochemical reactivities. Tumor cells of all the GCTs reacted intensely with the antibodies to keratan sulfate and S-100 protein. Peripheral nerve bundles and other neurogenic tumors showed stained for S-100 protein but not for keratan sulfate. Basic dye stain indicated the presence of sulfated glycoconjugates in GCTs. Lectin stains demonstrated that GCTs were rich in glycoconjugates but the reactivity patterns for 14 lectins differed between GCTs and normal tissue components. None of the lectins used in this study was specific for GCTs. These results indicate that GCTs contain abundant glycoconjugates and that the monoclonal antibody to keratan sulfate may be an immunohistochemical marker for GCT.     

Immunoelectrophoretic analysis of major component proteins in cystic fluid of Taenia solium metacestodes.

When cystic fluid of Taenia solium metacestodes (CF) was filtrated through Sephacryl S-300 Superfine, major proteins were in fractions III and IV. Major protein in fraction III was Band C protein of 150 kDa and that in fraction IV was Band N protein (Choi et al., 1990). When CF was electrophoresed in 0.9% agarose gel and reacted with anti-CF rabbit serum (RACF), two main bands, a long outer and a short inner band, were precipitated, together with 8 minor bands. RACF reacted with fraction III forming the long outer band whereas RACF formed the short inner band with fraction IV in immunoelectrophoresis (IEP). The long outer precipitin band of CF fraction III was similar to antigen B in hydatid fluid (HF) of Oriol et al. (1971), while the short inner band of CF fraction IV was similar to HF antigen 5 of Capron et al. (1967). When HF was reacted with RACF, the short inner band was immunoprecipitated without forming the long outer band. Common antigenicity between CF and HF seemed to exist in fraction IV rather than in fraction III of CF. Patient sera of neurocysticercosis reacted more frequently with fraction III than with fraction IV.     

IMMUNOHISTOCHEMICAL STUDIES OF S-100 PROTEIN DURING POSTNATAL ONTOGENESIS OF THE BRAIN OF TWO STRAINS OF RATS

Abstract— We have studied the dynamics of the appearance of cells reacting positively with anti-S-100 protein antiserum, during postnatal neurocytogenesis in the brain of rats of two strains differing in their susceptibility to sound stimuli. The postnatal time of appearance of cells reacting positively with anti-S-100 protein antiserum was somewhat later in rats susceptible to sound-induced seizures than in sound-resistant rats. These differences concerned mainly the cerebral cortex of 12-day-old rats. By day 21 of postnatal life these differences had disappeared. In subcortical structures of the brain, S-100 protein was first found on the 4th to the 5th day of life and the rate of appearance of cells containing this protein was similar in the two strains.     

Identification of immunoreactive antigens of human papillomavirus type 6b by using Escherichia coli-expressed fusion proteins.

Human papillomavirus (HPVs) infect the genital epithelium and are found in proliferative lesions ranging from benign condylomata to invasive carcinomas. The immunological response to these infections is poorly understood because of the lack of purified viral antigens. In this study, bacterially derived fusion proteins expressing segments of all the major open reading frames (ORFs) of HPV type 6b (HPV-6b) have been used in Western blot (immunoblot) assays to detect antibodies directed against HPV-encoded proteins. The most striking reactivities present in sera from patients with genital warts were to the HPV-6b L1 ORF protein and, to a lesser extent, to the HPV-6b L2 ORF protein. Two cases of reactivity to HPV-6b E2 ORF were observed, but no reactivities were seen with other HPV-6b constructs. Two sera reacted with the HPV-16 L2 fusion protein, and two sera reacted with the HPV-16 E4 protein. The antibodies directed against the HPV-6b fusion proteins showed no cross-reactivity with comparable regions of the HPV-16 ORFs. This assay provides a useful approach for further studies of HPV serology.     

脊髓和周围神经可溶性酸性蛋白质的研究

 利用微型双向电泳、SDS电泳、免疫印迹法、DEAE-Sephadex色谱、高效液相色谱及氨基酸分析等方法,对牛脊髓(中枢神经)和马尾神经(周围神经)的可溶性酸性蛋白质进行了研究。结果表明在牛脊髓和马尾神经中有钙调素(CaM)、S-100蛋白和神经元特异烯醇化酶(NSE)等可溶性酸性蛋白质存在;脊髓中这些酸性蛋白质的含量远较马尾神经为高。     

Comparison of Two Human Papovaviruses with Simian Virus 40 by Structural Protein and Antigenic Analysis

The proteins of simian virus 40 (SV40) and two human papovaviruses, the hemagglutinating BK virus and the non-hemagglutinating DAR virus, were analyzed and compared by sodium dodecyl sulfate polyacrylamide gel electrophoresis. The virions of SV40 and DAR contain seven proteins. By molecular weight analysis the constituent proteins of SV40 and DAR are identical. Approximately 84% of the viral protein has a molceular weight of 45,000. The major protein of BK virus is 3,000 to 5,000 daltons lighter than the major proteins of SV40 and DAR viruses. The five most rapidly migrating proteins of BK virus are indistinguishable by molecular weight analysis from the corresponding proteins of SV40 and DAR viruses. Radial immunodiffusion and immunoelectrophoresis of whole virus gave lines of identity between SV40 and DAR when reacted with SV40 antibody. SV40 antiserum tested against BK virus and BK antiserum tested against SV40 virus showed no reactivity by complement fixation, immunodiffusion, or immunoelectrophoresis.     

Significance of S-100 protein immunostaining in the immunohistological analysis of normal and neoplastic lymphoid tissues--an appraisal

S-100 protein is a heterogeneous fraction of dimeric polypeptides (alpha and beta subunits) that can exist in different combination forms within the various tissues. Concerning the S-100 protein immunodetection within lymphoid tissue, the heterogeneity of the S-100 antigen, the tissue quality (frozen or paraffin-embedded after treatment with different fixatives) and the treatment of the tissue with different immunostaining methods and antibodies of different nature, all make for inconsistent results obtained in the immunohistological studies reported in the literature. Most of the S-100-positive cells of the lymphoreticular system are dendritic cells involved in the immune response (interdigitating reticulum cells, Langerhans cells, and follicular dendritic reticulum cells), other S-100-positive cells belonging to the mononuclear/phagocytic system. S-100 protein immunostaining may be used as a helpful immunohistological diagnostic clue to certain malignancies of the immune system (follicular center cell lymphomas) on the basis of their specifically related dendritic cell microenvironment. In addition to monoclonal antibodies for the immunophenotypic characterization of dendritic cells and macrophages and to enzyme reactions, the combined use of anti-S-100 antibodies specific for each of the S-100 protein subunits, tested with sensitive procedures, would be a very useful tool in the attempt to classify the proliferative disorders of dendritic cells and macrophages.     

Biochemical and immunological heterogeneity of 100 A filament subunits from different chick cell types.

The 100 A filament subunit proteins of chick fibroblasts and gizzard smooth muscle were compared. These proteins are major cellular components in these cell types, constituting up to 98% of the cell's total protein. Co-electrophoresis of cytoskeletal fractions of fibroblasts and smooth muscle revealed that the subunit proteins differed in their molecular weights: 58,000 daltons in fibroblasts and 55,000 daltons in smooth muscle. Cytoskeletal fractions from other cell types were also examined: chondroblasts contained the 58,000 dalton subunit, and cytoskeletons of skeletal muscle and cardiac muscle contained both 55,000 and 58,000 dalton proteins. Chick skin and rat kangaroo Pt K2 cells had more complex subunit patterns which resemble prekeratin. The peptide patterns resulting from proteolytic digestion of the 58,000 dalton protein of fibroblasts, the 55,000 dalton proteins of smooth muscle and PT K2 cells, and chick brain tubulin differed from one another. Two-dimensional electrophoresis of reconstituted gizzard smooth muscle 100 A filaments showed the 55,000 dalton subunit to be composed of two major components, differing in their isoelectric points. Antibodies prepared against electrophoretically purified 55,000 dalton subunit protein reacted in immunodiffusion against the original smooth muscle antigen and cytoskeletal fractions from skeletal and cardiac muscle, but not from fibroblasts, brain, liver, or skin cells. A specific antigenic determinant common to subunit proteins in smooth, skeletal, and cardiac muscle, is therefore indicated. A previously described antibody against fibroblast subunit protein reacted weakly against smooth muscle filament protein in immunodiffusion revealing the presence of a common antigenic determinant between the two subunit proteins. These data demonstrate striking antigenic and primary structural differences in 100 A filament subunits from even such closely related cell types as fibroblasts on the one hand and muscle cells on the other.     

Immunochemical evidence for a transmembrane orientation of both the (Na+, K+)-ATPase subunits

Antibodies were raised against the large catalytic subunit (apparent Mr 96000) and the glycoprotein (apparent Mr 60000) of the sodium- and potassium-dependent adenosine triphosphatase [(Na+, K+)-ATPase] from Bufo marinus. The specificity of each antiserum was assessed by two-dimensional immunoelectrophoresis using toad kidney microsomes or the purified holoenzyme as a source of antigen and by indirect immunoprecipitation of detergent-solubilized (Na+, K+)-ATPase subunits from radioiodinated or biosynthetically labeled kidney holoenzyme, microsomes, or postnuclear supernatant. The anticatalytic subunit serum reacted exclusively with a 96000-dalton protein. The antiserum to the glycoprotein was rendered specific to this subunit by absorption with purified catalytic subunit. The two antisera were agglutinating and lytic in the presence of complement when toad erythrocytes were used as targets, indicating that antigenic determinants of both subunits were exposed on the cell surface. The specific reactivities with surface-exposed antigenic determinants of both subunits could be absorbed with toad red blood cells. Such absorbed antisera still reacted with detergent-treated or untreated kidney microsomes, revealing the presence of cytoplasmic and/or intramembranous antigenic sites. Our immunochemical data demonstrate that the glycoprotein subunit of (Na+, K+)-ATPase spans the lipid bilayer and confirm the transmembrane orientation of the catalytic subunit postulated from functional studies.     

Immunological analysis of plant mitochondrial NADH dehydrogenases.

Plant mitochondrial NADH dehydrogenases were analysed by two immunological strategies. The first exploited an antiserum raised to a preparation of SDS-solubilized mitochondrial-inner-membrane particles. By using a combination of activity-immunoprecipitation and crossed immunoelectrophoresis, it was shown that Triton X-100-solubilized membranes contain at least three immunologically distinct NADH dehydrogenases. Two of these were subsequently isolated by line immunoelectrophoresis and analysed for polypeptide composition: one contained three polypeptides with molecular masses of 75, 62 and 41 kDa; the other was a single polypeptide with a molecular mass of 53 kDa. The other approach was to probe plant mitochondrial membranes with antibodies raised to a purified preparation of ox heart rotenone-sensitive NADH dehydrogenase and subunits thereof. Cross-reactions were observed with the subunit-specific antisera against the 30 and 49 kDa ox heart proteins. However, the molecular masses of the equivalent polypeptides in plant mitochondria are slightly lower, at 27 and 46 kDa respectively.     

Serological characterization of a ribonuclease from Hordeum vulgare

Specific rabbit antisera against purified Hordeum vulgare seedling RNase I from two winter barley cultivars each formed a single precipitin band when reacted with the homologous crude tissue extract. RNase antigen from either cultivar was equally reactive with both antisera when evaluated by immunodiffusion and immunoelectrophoresis. A small but consistent difference in anti-RNase specificity between cultivars was shown by passive hemagglutination inhibition, suggesting that molecular differences may exist between the two RNase antigens. Immunodiffusion and rocket immunoelectrophoresis were used to qualitatively test the cross-reactivity of protein preparations from various members of the genus Hordeum and species from other related grass genera. Neither antiserum showed cross-reactivity with soluble protein preparation from species outside the genus Hordeum. A few species within the genus Hordeum were cross-reactive. A modification of rocket immunoelectrophoresis was developed to determine the amount of RNase in unpurified tissue extracts. The technique involved a template-reservoir which allowed detection of 250 ng RNase in tissue extract volumes of 50 μl. The amount of RNase in unpurified protein extracts from the two cultivars of barley was similar.