Quantitative assay of S-100 protein in mouse brain cortex synaptosomes.

1. Data on the presence of S-100 protein in synaptic endings are revised, and evidence is given in favor of its localization inside mouse brain cortex synaptosomes and on the surface of their external membrane. 2. For identification of the S-100-specific polypeptide, proteins of external synaptosomal membranes were iodinated with lactoperoxidase fixed on cyanogen bromide (CNBr)-Sepharose, and after synaptosome lysis S-100-positive material was isolated by means of affinity chromatography antibodies to S-100 protein (a-S-100)-Sepharose. The molecular weight of the polypeptide obtained corresponded to that of S-100 subunits (10 kD), and iodine incorporation pointed to its localization on the surface of synaptosomal membranes. 3. With the help of antibodies labeled with horseradish peroxidase (a-S-100-HP) or 125I (a-S-100-125I), which do not penetrate into noninjured synaptosomes, the amount of S-100 protein on synaptosomal membranes was found to be 18.5 ng/mg total protein (as assayed with a-S-100-HP) or 95.33 ng/mg (as assayed with a-S-100-125I). 4. At the same time, the total S-100 protein content in synaptosomes measured by means of radioimmune analysis after their complete lysis turned out to be 284 +/- 0.84 ng/mg, i.e., a part of S-100 seemed to be inside synaptosomes. 5. Cosedimentation of water-soluble S-100 protein with the synaptosomal fraction during isolation was insignificant. Prefixation with glutaraldehyde or paraformaldehyde decreased the amount of material reacting with antibodies, possibly due to steric effects or denaturation of active centers. This could have influenced the earlier attempts to detect S-100 protein in synapses. Treatment of nonfixed synaptosomes with a conjugate of a-S-100 with colloidal gold made it possible to detect S-100-positive material on pre- and postsynaptic membranes, which confirms the biochemical data.

[Measurement of the albumin content in individual cells]. / Izmerenie soderzhaniia al'bumina v otdel'nykh kletkakh.

A cytophotometric method is proposed for albumin determination in particular cells based on the immunoenzymatic detection of this protein with immuno-peroxidase complexes. The relative contents of albumin in hepatocytes of mouse in two inbred stocks were measured by two independent methods, and the results obtained well compared.

[Albumin synthesis by mouse liver cells]. / Sintez al'bumina kletkami pecheni myshi.

The presence of albumin in mouse liver cells was examined using the immunoperoxidase method with the help of monospecific antibodies to mouse serum albumin. All the experiments were carried out on the cell suspension. The rat cells were used as a control in the mixture of rat and mouse liver cells. All mouse hepatocytes were shown to contain serum albumin. Our results are compared with those reported by other authors. The advantages of our method over the routine histological technique are discussed.

[Quantitative determination of neurospecific protein S-100 in mouse brain cortical synaptosomes]. / Kolichestvennoe opredelenie neirospetsificheskogo belka S-100 v sinaptosomakh kory golovnogo mozga myshei.

Using monospecific antibodies against protein S-100 labelled with horseradish peroxidase (a-S-100-HP) or iodine (a-S-100-125I) two types of protein S-100 bound to the synaptosomal membrane were obtained. The first type accessible for a-S-100-HP (6.4% of total protein S-100 in the synaptosomes) and the second type accessible for a-S-100-125I (22.4%) differ in their location on the membrane surface. The total content of protein S-100 in the synaptosomes determined by solid phase radioimmune analysis is 284 +/- 0.84 ng per mg of total synaptosomal protein. Coprecipitation of the protein with the synaptosomal fraction is insignificant. After treatment of the synaptosomal membranes with 1% glutaric aldehyde or 4% paraformaldehyde, 20 and 50% of membrane-bound protein S-100 are found in the synaptosomal membrane, respectively. The immunohistochemical localization of protein S-100 in synaptosomes was established.

[Immunochemical isolation and characteristics of polyribosomes synthesizing rat serum albumin]. / Immunokhimicheskoe vydelenie i kharakteristika poliribosom, sinteziruiushchikh krysinyi syvorotochnyi al'bumin.

A novel one-step procedure is described for purification of a specific class of polysomes. The analysis of optimal conditions for immunoadsorption of albumin-synthesizing polysomes was carried out. The native albumin-synthesizing polysomes were isolated. This preparation has the sedimentation coefficient similar to that of the class revealed amongst polysomes from rat liver with 125I- and peroxidase-labeled antibodies. We translated albumin-synthesizing polysomes in oocytes. The analysis of the product of translation of the preparation of albumin-synthesizing polysomes shows that the albumin makes up 90% of the exogenous synthesis.

Studies on the interaction of Ca2+ ions with some fractions of the neurospecific S-100 protein.

Fractions of neurospecific S-100 protein were purified from bovine brain and their physicochemical properties were studied. Conformational changes caused by the binding of calcium to S-100 protein fractions were detected by means of differential and fluorescence spectroscopy. Fractions demonstrating opposite shifts of their spectra also differ in the distribution in double-phase system. The number of calcium-binding centers and their association. The nature of the differences in the interaction of various S-100 protein fractions with calcium is discussed.

[Genetic-biochemical analysis of the shuttle box behavior of 2 inbred strains of mice differing in their brain protein S-100 content. II. Genes determining the animals coat color and their learning]. / Genitiko-biokhimicheskii analiz povedeniia v chelnochnoi kamere myshei dvukh inbrednykh linii, kontrastno razlichaiushchikhsia po soderzhaniiu belka S-100 v golovnom mozge. Soobshchenie II. Geny, opredeliaiushchie ikrasku mekha u zhivotnykh, i obuchenie.

The influence of genes, determining the coat colour in mice, on active avoidance conditioned reflex formation in a shuttle box was studied. Negative correlation was found to exist between theoretically calculated mean number of dominant genes determining the coat colour and the level of aviodance in a shuttle box. Among F2 hybrids and backcrosses the groups of mice characterized by different coat colour phenotype have similar concentration of neurospecific S-100 protein in cerebral cortex and hippocampus.

[Genetic-biochemical analysis of the shuttle-box behavior of 2 inbred strains of mice differing in brain protein S-100 content. I. Neurospecific protein S-100 and learning]. / Genetiko-biokhimicheskii analiz povedeniia v chelnochnoi kamere myshei dvukh inbrednykh linii, kontrastno razlichaiushchikhsia po soderzhaniiu belka S-100 v golovnom mozge. Soobshchenie I. Neirospetsificheskii belok S-100 i obuchenie.

A hybridological analysis of a mode of inheritance of two characters: biochemical (brain S-100 protein level) and behavioral (rate of active avoidance conditioned reflex formation in a shuttle box), in mice of inbred strains DBA/2J and AKR/J characterized by respectively high and low levels of these characters. The high concentration of water soluble S-100 protein in whole brain is a recessive character. The inheritance of total S-100 protein in whole brain and of the level of its water soluble fraction in brain cortex and hippocampus is complex. In all hybrids of a given couple of parents from different strains, dominance and over-dominance of a low rate of the formation of the conditioned reflex takes place. No sex differences in the avoidance level were found. Neurospecific S-100 protein content correlates positively with the rate of active avoidance conditioned reflex formation.

[Reaction between calcium and different protein S-100 fractions]. / Vzaimodeistvie razlichnykh fraktsii belka S-100 s kal'tsiem.

The interaction between various fractions of neurospecific S-100 protein and calcium was studied by means of differential spectrophotometry. Fractions demonstrating "blue" and "red" shifts of the spectrum were detected. The change of conformation, occurring when calcium is bound, results in a redistribution of the protein in the double-phase system (polyetylenglycol-dextran), and the fractions, demonstrating the "blue" shift are transferred to the more hydrophobic phase (PEG), while fractions with the "red" shift move to the less hydrophobic phase (dextran). The existence of fractions with different reaction to calcium binding is discussed considering their participation in controling ionic permeability of neuronal membranes.

Neurospecific S-100 protein content in brains of different mouse strains.

Total whole brain concentrations of S-100 protein and of its water-soluble fraction were determined in 11 inbred mouse straine: DBA/2J, AKR/J, CBA/Lac, C57BL/6J, C57BL/6J-Ay, C3H/He, C3H/f, DD, A/He, BALB/cLac, CC57BR/Mv, and in cerebral cortex, cerebellum and hippocampus in DBA/2J, AKR/J and CBA/Lac strains. Highly significant differences in the concentrations of the water-soluble S-100 protein were found between some strains. Slight differences were found in total S-100 protein content in whole brains between the strains (0.01 less that P less than 0.05). The DBA/2J mice had the highest brain S-100 protein content, and were characterized by a higher learning rate in shuttle-box as compared to CBA/Lac and AKR/J mice, who had a low content of this neurospecific protein.

[Dynamics of neurospecific protein S-100 accumulation in the brain of AKR/J and DBA/2J strain mice in the process of postnatal development]. / Dinamika nakopleniia neirospetsificheskogo belka S-100 v golovnom mozge myshei linii AKR/J i DBA/2J v protsesse postnatal'nogo razvitiia.

The content of total S-100 protein and its water-soluble fraction was determined in the brain of DBA/2J and AKR/J mice during postnatal development. Reliable differences between the two strains were found in the content of total S-100 protein (from the 27th day after birth on) and its water-soluble fraction (from the 35th day).

[The heterogeneity of neurospecific S-100 protein]. / K voprosu o geterogennosti neirospetsificheskogo S-100 belka

A modified method is described of the isolation of neuro-specific S-100 protein from bovine brain tissue by means of thermal denaturation, ammonium sulphate fractionation, chromatography on DEAE-Sephadex A-50 and gel filtration through Sephadex G-100. Considerable heterogeneity of S-100 protein is discovered: it produces 4 fractions (f1--f4) under fractionation on DEAE-Sephadex A-50. The constant elution from the column of the main fraction, eluted with 0.325--0.35 M NaCl, and minor S-100 protein fractions, eluted with more high NaCl concentrations, is registered. At the same time, considerable variation in the quantitative yield of the main component of S-100 protein and in the relative content of main and minor components of S-100 protein is developed. Fast- (in f1 and f4 fractions) and slow-migrating (in f2 and f3 fractions) components are revealed under polyacrylamide gel electrophoresis. The f1 fraction is found to consist mainly of the component with molecular weight of 20 000 while fz, f3 and f4 fractions contain two components having molecular weight of 32 000 and 71 000. Antiserum to S-100 protein produced a positive immunodiffusion reaction with f1 and f2, and not with f3 and f4 fractions.