PURIFICATION AND SOME PROPERTIES OF S-100 PROTEIN FRACTIONS FROM SHEEP AND PIG BRAINS

Abstract— The S-100 protein fraction of pig and sheep brain was purified in 40 per cent yield by a modification of the procedure of M oore (1965), which avoided selective loss of S-100 components. The S-100 fraction of both pig and sheep is a mixture of proteins as indicated by acrylamide gel electrophoresis and N -terminal amino acid analysis. Differences in amino acid composition, electrophoretic heterogenity and N -terminal analysis were found.
One fraction (fraction A) was isolated by DEAE-Sephadex chromatography from pig brain S-100 protein fraction. It was considered to be a single protein since it migrated as a single band on acrylamide gel electrophoresis and showed a single symmetrical peak during ultracentrifugal analysis. Only one N -terminal amino acid was detected in fraction A. The amino acid composition of this fraction showed minor but significant differences from that of the complete S-100 protein fraction from pig brain. The S-100 protein fraction of both species, as well as fraction A, had similar s _{20, w} values and similar molecular weights (about 20,000) as indicated by gel filtration. These results, together with the immunological data obtained by other authors, suggest that the proteins of the S-100 fraction are closely related; the heterogeneity of the S-100 protein fraction may be of the same type as the lactate dehydrogenase isoenzymes.     

PURIFICATION OF SOLUBLE GLYCOPROTEINS FROM HUMAN NERVOUS TISSUE AND LIVER

—The isolation of some water-soluble, 50% methanol-soluble glycoproteins from human normal brain, human ependymoma and human liver is reported. One of them (AM protein) has a similar, or even identical, electrophoretic mobility to brain-specific protein S-100 under certain electrophoretic conditions. Although bands of identical mobility are found in both brain and liver samples, we lack experimental evidence, at the present stage of this work, to draw conclusions on the identity (or relatedness) of such proteins. On the other hand, the brain-soluble glycoproteins appear to be antigenically different from brain protein S-100, from brain α₂-glycoprotein and from GP 350 glycoprotein.     

PROTEIN SYNTHESIS IN VITRO IN NUCLEI OF HUMAN NERVOUS TISSUE

Abstract—

• 1 In vitro incorporation of tritiated leucine into nuclear proteins of normal human brain, astrocytoma I and II and glioblastoma multiforme was investigated. The distribution of radioactivity among various protein fractions of nuclei was determined.
• 2 The results demonstrate that the isolated nuclei of astrocytoma I and II incorporate radioactivity into proteins at least 40 times more actively than nuclei of normal human brain or glioblastoma multiforme.
• 3 The residual protein fraction was the most highly labelled fraction among the nuclear pioteins. This fraction from astrocytomas incorporates relatively more radioactivity than the similar fraction of normal human brain or glioblastoma multiforme. The buffered-saline soluble protein fraction of astrocytoma nuclei contained a relatively lower amount of radioactivity than the similar fraction of normal human brain or glioblastoma multiforme. The total radioactivity incorporated into the deoxyribonucleoproteins seemed to increase with the malignancy of the tissue investigated. The significance of the results with respect to malignant transformation is discussed.

     

Human chromosome structure as revealed by an immunoperoxidase staining procedure

Morphological alterations accompanied by an increase of the glia-specific protein S-100 have been shown to occur in a glial cell line (138 MG) of a human brain tumour if serum is removed from the culture medium. The glial S-100 protein was immunologically indistinguishable from S-100 present in human brain.     

Morphological alterations and increase of S-100 protein in cultured human glioma cells deprived of serum

Morphological alterations accompanied by an increase of the glia-specific protein S-100 have been shown to occur in a glial cell line (138 MG) of a human brain tumour if serum is removed from the culture medium. The glial S-100 protein was immunologically indistinguishable from S-100 present in human brain.     

DISTRIBUTION OF POLYSOMES IN MOUSE BRAIN TISSUE

Abstract— Polysomes were isolated from several different fractions of mouse brain tissue. After homogenization, the extract was centrifuged to yield a post-mitochondrial supernatant fraction and a pellet fraction. Sucrose gradient analysis of the material in the post-mitochondrial supernatant fraction indicated that 80 per cent of the ribosomes were present in polysomes and that little, if any, of the pellet fraction was present. Sucrose gradient analysis of the solution obtained after washing the pellet showed that very little polysomal material was present. The remaining pellet fraction was resuspended in a detergent mixture of deoxycholate-Tween 40. Sucrose gradient analysis of the resulting detergent-soluble solution indicated that large amounts of ribosomal material, in which 60–70 per cent of the ribosomes were associated in polysomes, were present.
In brain tissue from young animals, 20 per cent of the polysomes were found in the post-mitochondrial supernatant fraction whereas 80 per cent of the polysomes were released from the pellet fraction by detergent treatment. In contrast, in brain tissue from adult animals, 40 per cent of the polysomes were found in the post-mitochondrial supernatant fraction, whereas 60 per cent of the polysomes were released from the pellet fraction by detergent treatment.     

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.     

TRITON SOLUBILIZED ACETYLCHOLINESTERASE OF BRAIN

—The total AChE of brain can be readily extracted into aqueous Triton X-100. By column chromatography of these extracts a preparation was obtained at least six times as active as the original material and in yields of 60-80 per cent of the original amounts. The molecular weight of this material was estimated to be over 200,000. When brain tissue was treated with venom or bacterial protease, a water-soluble AChE was obtained with an overall purification of 150-fold. The most active preparation of AChE hydrolysed 880 μ-moles of acetylthiocholine/hr/mg of protein. The molecular weight of this preparation was estimated to be about 100,000. The enzyme which was extracted by Triton X-100 is probably the precursor of the more water-soluble enzyme that can be prepared from brain tissue by treatment with venom or protease.     

IMMUNOCHEMICAL STUDIES ON THE BRAIN SPECIFIC PROTEIN

Abstract— In the soluble brain proteins of various species-man, ox, cat, rabbit, rat, mouse, hen, snake, frog and fish–there is a protein group which migrates more slowly than Moore's S-100 protein and faster than the albumin fraction on disc electrophoresis. The protein group is absent from any organs other than brain, and has a different pattern and number of fractions in different species. Immunochemically, the protein fraction group of the mammalian brains shows some common and identical distinctive antigenic determinants compared with the brain protein of the other animals-hen, snake, frog and fish. The protein group was designated the 'SPR' proteins, which were separated to 'P_II,', 'P_III', 'P_IV' and 'P_v' fractions. Common antigenic determinants are found in these fractions. The protein group is found in human brain in larger amounts in grey matter than in white matter and in small amounts in the cellular nuclei of human and bovine brain.     

INCORPORATION IN VIVO OF RADIOACTIVE LEUCINE INTO NEURONAL AND GOAL NUCLEAR PROTEINS OF RAT BRAIN

Purified neuronal and glial nuclei were separated from rat brain cells. The fraction rich in neuronal nuclei contained 68 ± 9 per cent neuronal nuclei and the fraction rich in glial nuclei contained 89 ± 6 per cent glial nuclei. The fraction rich in neuronal nuclei isolated from cells of adult rat brain incorporated l -[4,5-³H]leucine into TCA-insoluble material at a rate comparable to those of the microsomal and the soluble fractions of the brain, and at a much higher rate than the fraction rich in glial nuclei. The proteins soluble in buffered-saline, the acid-soluble deoxyribonucleoproteins, and the residual proteins of the neuronal nuclei are apparently the proteins which account for the higher specific activity of neuronal proteins compared with glial nuclear proteins. In liver and kidney, the incorporation of [³H]leucine into nuclear proteins was lower than into other subcellular fractions from the same organs.     

人脑醛糖还原酶的纯化和一些基本性质

使用DEAE纤维素柱层析、PBE-94层析聚焦、NADP~+-Sepharose 4B亲合层析及SephadexG-100凝胶过滤分离纯化了人脑醛糖还原酶。在DEAE层析中,用咪唑-HCI缓冲液替代了磷酸缓冲液,改善了分离效果。在聚丙烯酰胺及SDS聚丙烯酰胺凝胶电泳中,纯化的人脑醛糖还原酶均呈一条区带。它的pI为5.6,最适pH为6.5,分子量为36,000,底物特异性和氨基酸组成与其它哺乳动物的醛糖还原酶有相似性。开链式醛糖是醛糖还原酶的真正底物,它在开链式和半缩醛的平衡体系中占比例极小,因而推知醛糖还原酶对此底物有很高的K_(cat)和K_(cat)/K_m值,能有效地将它们还原成相应的醇。     

NUCLEAR LOCALIZATION OF S-100 PROTEIN

Abstract— S-100 protein has been found in the nuclei isolated from the brain cortex of rabbit. The nuclear S-100 constitutes a small portion (0.55 per cent) of the S-100 present in the cytosol. Most of the large and pale nuclei appear to contain much more S-100 than the small and dark ones. The nuclear membrane is permeable in vitro to S-100 in presence of divalent cations. Three forms of S-100 occur in subnuclear fractions: free S-100, present in the soluble protein fraction; labile-bound S-100, present in the deoxyribonucleoprotein fraction and stable-bound S-100, present in the residual or‘nucleolar’fraction. The localization of the S-100 in those regions of the nucleus that are most active in RNA synthesis provides basic information for further studies on the possible role of this protein on genomic expression in nervous tissue.     

SOLUBLE PROTEINS IN NORMAL and DISEASED HUMAN BRAIN

Abstract– Six brain regions (frontal cortex, parts of the basal ganglia, thalamus and substantia nigra) were examined from over 80 human brains obtained at post-mortem. After elimination of patients with evidence of either 'cerebral hypoxia', lingering modes of death or abnormal brain morphology brain extracts were found to contain a characteristic pattern of 6 major soluble-acidic protein bands (neuronin-type proteins). As judged by studies using cortical biopsy specimens these proteins are relatively unaffected by post-mortem changes. Moreover, in adulthood the pattern is not noticeably age-dependant. Two of the protein bands have been identified as S-100 (neuronin S-1 and 2) while a third (neuronin S-5) is similar in most respects to antigen α (14-3-2). S-100 is increased in brains with evidence of marked gliosis. The other protein bands have not been identified. Two of them (neuronin S-3 and 4) are rarely depleted while the concentration of neuronin S-6 is affected particularly in extracortical regions in controls with either lingering modes of death and/or 'cerebral hypoxia' and in all regions in most patients with Alzheimer's disease, senile dementia and mixed senile and vascular dementia.     

SUBCELLULAR DISTRIBUTION AND STRUCTURAL POLYMORPHISM OF MYELIN BASIC PROTEIN IN NORMAL AND JIMPY MOUSE BRAIN

Abstract— Brains from 20 day old normal and 20 day old Jimpy mice were fractionated by a modification of the procedure described by Eichberg et al. (1964). Each of the fractions obtained was subjected to radioimmunoassay (RIA) for myelin basic protein (MBP). From both the normal brain and the Jimpy brain MBP was recovered in three separate membrane fractions designated P1A. P2A. and P3A. which differed in their sedimentation properties but which had similar densities (less than 1.08 g'ml). In the Jimpy brain compared to normal brain the amounts of P1A and P2A were greatly reduced but the amount of P3A was increased. During development in the normal brain the amount of MBP in the PIA fraction increased in parallel with the accumulation of myelin. The amount of MBP in P2A increased gradually during active myelination and decreased slightly in the adult. The amount of MBP in P3A increased sharply during the period of most active myelination and decreased approx 10-fold as the rate of myelination in the brain declined. Electron microscopic examination revealed that the P1A and P2A fractions from normal brain contained myelin fragments while the P1A and P2A fractions from Jimpy brain contained numerous vesicular membranous structures with little if any identifiable myelin. The P3A fraction from both normal and Jimpy brain contained small vesicles of uniform size, some with polyribosomes attached. Each of the fractions was analyzed by a technique combining sodium dodecyl sulfate polyacrylamide gel electrophoresis with RIA for MBP in order to identify and quantitate the four different forms of MBP with molecular weights of 21.5 K. 18.5 K. 17 K and 14 K dalton. The proportions of the four MBPs were characteristic for each fraction. The relative proportions of the four proteins were 14 K > 18.5 K > 17 K > 21.5 K daltons in all the fractions except P1A Jimpy in which 21.5 K dalton protein was the predominant form of MBP present. The cellular origin of the MBP containing fractions from normal and Jimpy brain is discussed.     

GP-350, A SIALOGLYCOPROTEIN FROM CALF BRAIN: ITS SUBCELLULAR LOCALIZATION AND OCCURRENCE IN VARIOUS BRAIN AREAS

Abstract— A membrane-bound form of GP-350, a sialoglycoprotein from calf brain has been shown to occur in the crude mitochondrial fraction (P₂). After subfractionation on a discontinuous sucrose gradient, consisting of 0.8 and 1.2 m -sucrose, GP-350 was immunologically only detectable in the synaptosomal fraction. After further separation of the lysed synaptosomal fraction on a discontinuous sucrose gradient (0.4, 0.8 and 1.2 m ) GP-350 could be detected only in the 0.8–1.2 m -sucrose interface.
The membrane-bound GP-350 from the cerebellar grey matter was purified and analysed. The amino acid composition and the data obtained for galactose, galactosamine, glucosamine and sialic acid were quite similar to those of the soluble GP-350, indicating their identity, which was sustained by immunodif-fusion, immunoelectrophoresis and polyacrylamide gel electrophoresis.
The membrane-bound GP-350 was isolated from 15 different brain areas. Per g wet wt at least 60 μg ( corpora quadrigemina inferior ) and at most 470 μg ( thalamus ) of GP-350 protein were obtained. Compared to the soluble GP-350 protein 10–52 per cent could be isolated from the crude mitochondrial fraction. Our results on GP-350 indicate an association with membranes of nerve endings.     

PROTEIN SYNTHESIS IN FRACTIONS FROM ISOLATED BRAIN CELL NUCLEI

Abstract— 1. The incorporation in vivo and in vitro of isotopically labelled leucine into fractions of nuclear proteins from young and adult rat brain was investigated.
2. During post-natal cerebral maturation, the ability of nuclei from brain cells to synthesize proteins decreased. The specific activities of all the fractions of nuclear protein were highest in 3-day-old rats and declined thereafter. Nuclei from adult brain cells exhibited only 10 per cent of the activity found in nuclei from brain cells of 3-day-old rats.
3. The 'residual protein' fraction was most rapidly labelled, peak activity being reached within 30 min after injection. In vitro , the 'residual protein' fraction attained maximum activity within 40 min.
4. The specific activity of the chromatin acidic proteins (HCl-insoluble) was considerably higher than that of the histones both in vivo and in vitro. Histones were the most inert of all the nuclear protein fractions studied.
The possible functional significance of the various protein fractions during the process of cerebral maturation and in the adult brain is discussed.     

Dynamics of neurospecific protein S-100 accumulation in the brain of AKR/J and DBA/2J strain mice in the process of postnatal development

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).     

PROTEINS OF HUMAN BRAIN TISSUE OBTAINED DURING SURGICAL PROCEDURES

Abstract— A series of normal and abnormal brain tissues were obtained during surgical procedures; adjacent portions were evaluated histologically or extracted with dilute buffer. The proteins were separated in acrylamide gels using electrophoresis and isoelectric focusing. Twelve of the patients showed a very similar distribution of the proteins on electrophoretic separation referred to as the 'typical' pattern. This group includes all of the histologically normal specimens, and in addition, one mild gliosis, two cases of juvenile lipidosis, one mucopolysaccharidosis and one Unverricht epilepsy. Remarkably little variability in the electrophoresis gel pattern was found in a series of five pairs of histologically normal gray and white matter samples. An increased density of several bands was apparent in extracts of gray matter when compared with corresponding white. No differences which could be correlated with the different areas of the cortex were seen.
Six of the patients showed gel patterns different from the 'typical' pattern. One of these (glioblastoma) differed only in the increase in band 9. Another glioblastoma showed a virtual absence of nearly all of the brain proteins usually found in the gels. Two astrocytomas and one surgical scar specimen showed a very dense protein band (No. 15) and a novel minor protein component, band 20, while one case of SSPE showed only the heavy band in the area of band 15. The content of band 5 was reduced in those specimens which presented a dense band 15. The pronounced increase in band 15 and band 20 appears to correlate with the marked increase in astrocytic elements in these three patients.     

SUBCELLULAR FRACTIONATION OF GANGLIOSIDE SIALIDASE FROM HUMAN BRAIN

—A subcellular fractionation was performed on forebrain cortex from three human brains and the fractions obtained were assayed for ganglioside sialidase and four p-nitrophenyl glycohydrolases. Differences in the sedimentation patterns of the enzymes were observed. From 53 to 77 per cent of the recovered sialidase activity was found in the synaptosomal fraction, while the p-nitrophenyl glycosidases were mainly recovered in the lysosome-enriched fraction. Three possible interpretations of the sialidase sedimentation pattern are suggested: (1) The ganglioside sialidase is bound to the limiting membrane structure of the nerve ending. (2) The ganglioside sialidase is lysosomal, although bound to lysosomes of low density. (3) The enzyme occurs mainly in lysosomes primarily located in the nerve endings, being trapped under the formation of the synaptosomes.     

PROTEIN SYNTHESIS BY HIGHLY AGGREGATED AND PURIFIED POLYSOMES FROM YOUNG AND ADULT RAT BRAIN

The state of aggregation and the activity of polyribosomes as well as the activity of the pH 5 enzyme fraction were studied at two stages of postnatal brain development, 9 and 50 days after birth. When the polyribosomes were prepared at 0°C in the presence of 5 mm -Mg²⁺, more than 85 per cent of the polyribosome material exhibited a sedimentation coefficient higher than 110 S. High Mg²⁺ concentrations are, therefore, unnecessary to obtain highly aggregated brain polyribosomes. The basal amino acid incorporating activity of both 9- and 50-day-old rat brain preparations is at least equal to that of rat liver. When prepared by the same procedure as above, 9-day-old rat brain polyribosomes seem to be more active (20 per cent) than those of adult brain. However, this difference in activity depends on the presence of a non-ribosomal inactive contaminant which is always present in higher amounts in adult brain preparations. When purified from this contaminant, the preparations do not differ in activity. High Mg²⁺ concentrations are also not necessary for optimal protein synthetic activity and, in fact, are inhibitory. When assayed with both types of highly aggregated polyribosomes, the pH 5 enzyme fraction from adult brain is clearly less active than that of 9-day-old rats. These results suggest that the loss of brain protein synthesis during development does not depend on the stability of the messenger RNA-ribosome complex but only on the soluble pH 5 enzyme fraction.