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41.
The use of limited proteolysis to probe interdomain and active site regions of beta-galactosidase (Escherichia coli) 总被引:4,自引:0,他引:4
L A Edwards M R Tian R E Huber A V Fowler 《The Journal of biological chemistry》1988,263(4):1848-1854
Limited proteolysis by pancreatic elastase (EC 3.4.21.36) and chymotrypsin (EC 3.4.21.1) was used to study the domain structure and active site of beta-galactosidase (EC 3.2.1.23) (Escherichia coli). Treatment with elastase resulted in a rapid cleavage between residues Ala-732 and Ala-733. No inactivation accompanied this cleavage suggesting that this bond is in a hinge region of the protein. Some slow cleavages beyond the initial one were observed to occur and were accompanied by inactivation. Treatment of beta-galactosidase with chymotrypsin resulted in cleavages first between Trp-585 and Ser-586 and then between Phe-601 and Cys-602. The first of these cleavages resulted in total inactivation of beta-galactosidase. The presence of monovalent ions or isopropyl-beta-D-thiogalactopyranoside protected against the cleavages but when Mg2+ or Mn2+ was present in the reaction mixture, the bond between Trp-585 and Ser-586 was more susceptible to the action of chymotrypsin. These data demonstrate that the conformation of beta-galactosidase around Trp-585 and Ser-586 is dramatically affected by the binding of ions and isopropyl-beta-D-thiogalactopyranoside. The mutant M15 beta-galactosidase, which is missing residues 11 through 41 and is an inactive dimer rather than an active tetramer, was found to be much more labile to proteases than native beta-galactosidase, but the same initial cleavages were found to occur. In addition, trypsin cleaved the M15 protein between Arg-431 and Trp-432 while native beta-galactosidase was stable to trypsin. 相似文献
42.
43.
内毒素(Endotoxin,简称ET)是百日咳全菌苗(Bordetellapertussis vaccine)产生副作用的主要毒素之一,且不易除去。现有的分离方法,如蔗糖密度梯度离心法,较繁琐,成本高。本文采用Sepha-cryl S-300凝胶层折法可以简便有效的去除大部分内毒素。初步毒素试验结果表明:已达到日本生物制品规格的要求。两种保护性抗原FHA和LPF-HA也得到进一步分离纯化,为今后研制高效的百日咳组分菌苗提供了实验条件。 相似文献
44.
Effect of lysosomotropic amines on the secretory pathway and on the recycling of the asialoglycoprotein receptor in human hepatoma cells 总被引:14,自引:10,他引:4
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G J Strous A Du Maine J E Zijderhand-Bleekemolen J W Slot A L Schwartz 《The Journal of cell biology》1985,101(2):531-539
We studied the intracellular transport of secretory and membrane proteins in the human hepatoma cell line HepG-2 infected with vesicular stomatitis virus. Cells were pulse-labeled in the presence of [35S]methionine and chased in the presence of the lysosomotropic agent primaquine. At a concentration of 0.3 mM primaquine effectively inhibited the secretion of albumin and, to a lesser extent, that of orosomucoid and transferrin. The drug also prevented the budding of virus particles at the cell surface. The intracellular transport to the Golgi complex of the membrane protein VSV-G was not affected by primaquine as it acquires resistance to endo-beta-N-acetylglucosaminidase H at the same rate as in control cells. Addition of primaquine at various times after the initiation of the chase period indicates that the effect of primaquine occurs just before secretion. In confirmation of the biochemical data, immunocytochemical localization of albumin in cells treated with NH4Cl demonstrated that albumin accumulated in vesicles at the trans side of the Golgi complex. The effect of primaquine on secretion was also compared with its effect on receptor recycling. The dose-response characteristics of the effect of primaquine on receptor recycling are identical to those of the effects on protein secretion and virus budding. These results indicate that both processes involve the same transport mechanism, and/or that they occur via at least one identical intracellular compartment. 相似文献
45.
46.
The influence of dolichol, dolichol esters, and dolichyl phosphate on phospholipid polymorphism and fluidity in model membranes 总被引:10,自引:0,他引:10
C Valtersson G van Du?n A J Verkleij T Chojnacki B de Kruijff G Dallner 《The Journal of biological chemistry》1985,260(5):2742-2751
The effect of dolichols, polyprenols, dolichol esterified with fatty acids, and dolichyl phosphate on the structure and fluidity of model membranes was studied using 31P NMR, small-angle x-ray scattering, differential scanning calorimetry, and freeze-fracture electron microscopy. These studies suggest that dolichol and dolichol derivatives destabilize unsaturated phosphatidylethanolamine containing bilayer structures and promote hexagonal II phase formation; high concentrations of dolichol induce lipid structures characterized by "isotropic" 31P NMR and particulate fracture faces; dolichol, contrary to cholesterol, has no effect on the thermotropic behavior of membranes consisting of phosphatidylcholine, while dolichyl-P incorporation abolishes the transition from the gel to liquid crystalline phase in 1,2-dimyristoyl-sn-glycero-3-phosphocholine; both dolichol and dolichyl-P increase the fatty acid fluidity in phosphatidylethanolamine mixtures; the effect of dolichol on bilayer structure and fluidity is more pronounced with increasing number of isoprene residues; dolichol esters are only soluble to a limited extent in the bilayer and segregates into domains at low concentrations; the results are consistent with a localization of dolichyl-P in which the phosphate group is oriented to the water interphase. The induction of hexagonal II phase by dolichyl-P may elicit the transmembrane movement of glycosylated lipid intermediate. 相似文献
47.
Nucleoprotein and membrane protein genes are associated with restriction of replication of influenza A/Mallard/NY/78 virus and its reassortants in squirrel monkey respiratory tract. 总被引:15,自引:8,他引:7
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S F Tian A J Buckler-White W T London L J Reck R M Chanock B R Murphy 《Journal of virology》1985,53(3):771-775
An avian influenza A virus, A/Mallard/NY/6750/78(H2N2), was restricted in in replication in the respiratory tract of squirrel monkeys. Avian-human influenza A reassortant viruses possessing the six RNA segments coding for nonsurface proteins (i.e., internal genes) of this avian virus were as restricted in replication in squirrel monkeys as their avian influenza parent. These findings indicated that restriction of replication of the avian influenza virus is a function of one or more of its internal genes. For an investigation of which of the avian influenza genes was responsible for restricted replication in the respiratory tract of primates, reassortant viruses were produced that contained human influenza virus surface antigens from the A/Udorn/72(H3N2) virus and one or more of the internal genes derived from the avian influenza virus parent. Avian-human reassortant influenza A viruses containing only the nucleoprotein or matrix protein RNA segment from the avian influenza virus parent were as restricted in their growth as an avian-human influenza reassortant virus containing each of the six avian influenza internal genes. In addition, an avian-human influenza reassortant virus possessing only the avian RNA 1 and nonstructural genes (which by themselves do not specify restricted replication) manifested a significant reduction of virus replication in squirrel monkey tracheas. Thus, the avian nucleoprotein and matrix genes appear to play a major role in the host range restriction exhibited by the A/Mallard/78 virus and its reassortants, but the combination of RNA 1 and nonstructural genes also contributes to restriction of replication. 相似文献
48.
49.
Individual monosaccharides present in bagasse hemicellulose were determined using HPLC and other chromatographic procedures. The presence of higher oligomers of the monosaccharides could also be determined. No single procedure can separate and identify all the naturally occurring monosaccharides. The pentosan fraction of bagasse wa successfully hydrolyzed and extracted with 5% (m/v)HCl, and the rate of release of individual monosaccharides was determined. Xylose was the main component in the hydrolyzates, while glucose, arabinose, and galactose present in the side chains of the pentosans were initially released at a fast rate. This treatment resulted in obtaining 229 mg/g xylose (85% of theoretical maximum) and 44 mg/g glucose from bagasse. Only arabinose (2.8 mg/g) and galactose (0.75 mg/g) was also present in detectable quantities. A total of 309 mg monosaccharides were obtained from 1 g of bagasse by this treatment. The results indicated that hydrolysis conditions for specific plant materials depend on the composition of the specific material being utilized. A part of the pentosan fraction (77.1%) was hydrolyzed at a high rate, while 22.9% was more stable and hydrolyzed more slowly. Although 39.8% dry bagasse could be obtained in solution by treatment with dilute alkali, only about 72% of the available hemicelluloses could be extracted in this way if the bagasse was not delignified beforehand. Amino acids and peptides or proteins were also extracted to very much the same with the alkali. 相似文献
50.
细菌(Pseudomonas moltophilia)与hCG及LH有特异的亲和力,实验发现,细菌之生长曲线与hCG结合活性成平行关系,96小时达高峰,细菌之培养液中含有可溶性结合蛋白,该蛋白经硫酸铵沉淀(80%饱和度)、Sephadex G-100柱层析、DEAE-纤维素柱0.5mol/L NaCl梯度洗脱,再过Sepharose CL-AB柱,收集之活性部分经SDS电泳测得其分子量为70,000,凝胶层析测Stokes radius为41A,Schiff氏染色未见着色带。 相似文献