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  • 1
  • 2
    ISSN: 1471-4159
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Medicine
    Notes: A sialoglycoprotein has been isolated from the cortical grey matter of calf brain after homogenization in 0.32 M-sucrose or in 0.15 M-NaCl. The sialoglycoprotein is present in the supernatant obtained after centrifugation at 100,000 g for 60 min. It is designated GP-350 on account of its elution with 350 mM-NaCl on a DEAE-cellulose column. From DEAE-cellulose chromatography it is evident that compounds comparable to GP-350 occur in the brain of calf and sheep, whereas they seem to be absent in calf liver and kidney. After purification, with polyacrylamide gel electrophoresis only one band can be shown both at pH 8.9 and 7.5. GP-350 consists of about 83 percent of protein and about 17 per cent of carbohydrate. The polypeptide core has an acidic character: amino acid analysis gives 26 per cent for glutamic acid plus aspartic acid and their amides, with a ratio of acidic to basic amino acids of 3.3. The carbohydrate moiety contains 2.4% sialic acid, 5.5 % hexosamine and 9.4% hexose. It is remarkable that this brain sialoglycoprotein comprises 4% glucose. Care was taken to prevent contamination with glucose-containing materials during the purification procedure of GP-350. The complete absence of other glucose-containing compounds which occur in brain, Le. glycogen and gangliosides, was demonstrated. GP-350 accounts for at least 3 per cent of the total saline-extractable protein and about 20 per cent of the total saline-extractable protein-bound sialic acid of the cortical grey matter of calf brain. These percentages correspond to 390 pg of protein and to 14 μg of sialic acid per g wet weight. GP-350 remains soluble when the pH is brought to 3.9 or when ethanol is added to 70 % (v/v).
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  • 3
    ISSN: 1365-2958
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Biology , Medicine
    Notes: Abf1p and Rap1p are global regulatory factors which play an essential role in the transcription activation of yeast ribosomal protein genes. This functional link prompted us to investigate whether these factors may be functionally interchangeable. We focused on the indispensable C-terminal portions of both factors and performed mutual domain swaps. The functional capacity of the resulting hybrid proteins was subsequently examined using yeast strains conditionally expressing either the ABF1 or the RAP1 gene. Both the Abf1p–Rap1p and the Rap1p–Abf1p fusion proteins were found to be able to complement the growth defect of the respective strains. Furthermore, Abf1p and Rap1p are both able to promote transcription of a reporter gene through a combination of the respective binding site and a T-rich promoter element. These data strongly suggest that the C-terminal domains of Abf1p and Rap1p have, at least partially, identical functions. Finally, a deletion analysis of the so far largely uncharacterized C-terminal domain of Abf1p was performed, which revealed that two regions of 50 amino acids can perform all essential Abf1p functions.
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  • 4
    ISSN: 1471-4159
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Medicine
    Notes: The apparent molecular weight of GP-350, a sialoglycoprotein from calf and rat brain, has been determined by SDS-polyacrylamide gel electrophoresis. The electrophoretic mobility corresponds to the mobility of a polypeptide with a molecular weight of 11,600 ± 200. On this basis it can be calculated that only one sialic acid residue is present/GP-350 molecule.From isoelectric focusing experiments it appeared that the isoelectric point of GP-350 is about 2. The determination of the amide content of the polypeptide chain showed that out of 22.0 acidic amino acid residues of glutamic acid and aspartic acid, only 4.9 residues are amidated. The total amount of the basic amino acid residues lysine and arginine, is 6.5. So, per molecule GP-350 10.6 acidic amino acid residues are not counteracted by basic amino acid residues. The surplus of the acidic amino acid residues as well as sialic acid result in the pronounced acidic character of GP-350. This fact is supported by the electrophoretic experiments.The carbohydrate-polypeptide linkage type has been studied by alkaline sodium borohydride treatment. Two thirds of all the galactosamine was destroyed, whereas the amount of glucosamine remained the same. Amino acid analysis indicated a decrease in serine and threonine with a concomitant small increase in alanine. These data point to the occurrence of linkages between the carbohydrate chain and the polypeptide core of the galactosamineserine or –threonine type. Per molecule GP-350 about two residues of galactosamine are destroyed, indicating that two carbohydrate chains of this binding type are present. Only one of these chains can be terminated by a sialic acid residue. The other carbohydrate chain may be terminated by fucose.Regional distribution studies showed the presence of GP-350 in all brain areas studied; in relatively large amounts in the regions rich in ganglia such as caudate nucleus, cerebellar grey matter, pons and medulla oblongata, and in relatively small amounts in the regions poor in ganglia such as corpus callosum, cerebral white, cerebral grey and cerebellar white matter.GP-350 is also present in the pituitary gland. In the cerebrospinal fluid a glycoprotein is present with the same electrophoretic mobility as GP-350. However, this glycoprotein gave no precipitin reaction with GP-350 specific antiserum. Moreover, the amino acid composition was quite different from that of GP-350.Subcellular distribution study revealed that GP-350 is present in the soluble cell fraction and in the synaptosomal membrane fraction, whereas it is absent from the purified nuclei, mitochondria, myelin, and also from the microsomal fraction.
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  • 5
    Electronic Resource
    Electronic Resource
    Oxford, UK : Blackwell Publishing Ltd
    ISSN: 1365-3083
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Medicine
    Type of Medium: Electronic Resource
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  • 6
    ISSN: 1471-4159
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Medicine
    Notes: —The incorporation of d-[3H]glucosamine into the nervous specific sialoglycoprotein GP-350 has been studied in adult rat brain. Both the 100,000 g supernatant fluid and the 12,500 g pellet were used for the investigation, since GP-350 could only be detected in the soluble cell fraction (Van Nieuw Amerongenet al., 1972) and in the synaptosomal membranes, sedimenting in the crude mitochondrial fraction (Van Nieuw Amerongen & Roukema, 1973, 1974). GP-350 was separated from the other proteins by polyacrylamide gel electrophoresis at pH 7.5 and the incorporation of radioactivity into GP-350 was measured at different time intervals, ranging from 1 to 96 h after the administration of the radioisotope. The maximal incorporation of radioactivity into the soluble GP-350 was obtained after about 2 h and into the membrane-bound GP-350 after about 3 h. After 2 h there is a very rapid decrease of the radioactivity of GP-350 from the soluble cell fraction (up to 70 per cent within 2 h). Thereafter the disappearance is more gradual and of the same order as that found for the membrane-bound fraction of GP-350. The half-life of the soluble GP-350 was estimated to be 19 h and for the membrane-bound GP-350 a value of 18 h was calculated. Compared to the total pool of brain (glyco) proteins and specific nervous (glyco) proteins GP-350 has a very rapid turnover. The rapid initial decrease of the radioactivity from the soluble GP-350 may be interpreted in terms of a rapid transport of the newly-synthesized GP-350 from the cytoplasma of the perikaryon to the membranes of the synaptic region by an axoplasmic flow.
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