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Proteomic analysis of human neutrophil granules.
Lominadze G, Powell DW, Luerman GC, Link AJ, Ward RA, McLeish KR
(2005) Mol Cell Proteomics 4: 1503-21
MeSH Terms: Actins, Ammonium Sulfate, Cytoplasmic Granules, Electrophoresis, Gel, Two-Dimensional, Gelatinases, Humans, Neutrophils, Proteome, Proteomics, Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
Show Abstract · Added February 20, 2015
Stimulated exocytosis of intracellular granules plays a critical role in conversion of inactive, circulating neutrophils to fully activated cells capable of chemotaxis, phagocytosis, and bacterial killing. The functional changes induced by exocytosis of each of the granule subsets, gelatinase (tertiary) granules, specific (secondary) granules, and azurophil (primary) granules, are poorly defined. To improve the understanding of the role of exocytosis of these granule subsets, a proteomic analysis of the azurophil, specific, and gelatinase granules from human neutrophils was performed. Two different methods for granule protein identification were applied. First, two-dimensional (2D) gel electrophoresis followed by MALDI-TOF MS analysis of peptides obtained by in-gel trypsin digestion of proteins was performed. Second, peptides from tryptic digests of granule membrane proteins were separated by two-dimensional microcapillary chromatography using strong cation exchange and reverse phase microcapillary high pressure liquid chromatography and analyzed with electrospray ionization tandem mass spectrometry (2D HLPC ESI-MS/MS). Our analysis identified 286 proteins on the three granule subsets, 87 of which were identified by MALDI MS and 247 were identified by 2D HPLC ESI-MS/MS. The increased sensitivity of 2D HPLC ESI-MS/MS, however, resulted in identification of over 500 proteins from subcellular organelles contaminating isolated granules. Defining the proteome of neutrophil granule subsets provides a basis for understanding the role of exocytosis in neutrophil biology. Additionally, the described methods may be applied to mobilizable compartments of other secretory cells.
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10 MeSH Terms
Leukocyte 12-lipoxygenase: expression, purification, and investigation of the role of methionine residues in turnover-dependent inactivation and 5,8,11,14-eicosatetraynoic acid inhibition.
Richards KM, Marnett LJ
(1997) Biochemistry 36: 6692-9
MeSH Terms: Amino Acid Sequence, Ammonium Sulfate, Animals, Arachidonate 12-Lipoxygenase, Catalase, Chemical Precipitation, Chromatography, Ion Exchange, DNA, Complementary, Dithiothreitol, Escherichia coli, Gene Expression, Humans, Leukocytes, Lipoxygenase Inhibitors, Methionine, Molecular Sequence Data, Mutagenesis, Site-Directed, Recombinant Proteins, Sequence Alignment, Structure-Activity Relationship, Swine
Show Abstract · Added March 5, 2014
Porcine leukocyte 12-lipoxygenase cDNA was cloned into the expression vectors pSE280, pSE380, and pSE420. pSE380 yielded the highest level of 12-lipoxygenase activity when these vectors were tested for expression in Escherichia coli Top10 cells. Optimal expression of the protein from this vector occurred in cells cultured at 30 degrees C and harvested 18 h following induction of expression by 0.5 mM isopropyl thiogalactoside (IPTG). The enzyme was purified from the 100000 g supernatant to 98% homogeneity by a combination of ammonium sulfate precipitation, anion exchange chromatography, and chromatofocusing. Addition of dithiothreitol and catalase to buffers at various steps in the purification protocol enabled the isolation of enzyme having a specific activity of 12 micromol min(-1) mg(-1). The recovery of purified protein from this expression system was 56%, resulting in a 109-fold purification. On the basis of amino acid sequence comparisons between mammalian 15- and 12-lipoxygenases, three methionine residues in the porcine leukocyte 12-lipoxygenase (M338L, M367V, and M562L) were targeted for mutation to assess their potential role in turnover-dependent inactivation and inhibition by 5,8,11,14-eicosatetraynoic acid (ETYA). The mutants were expressed and purified by the same procedure used for the wild-type enzyme. These amino acid changes did not significantly alter enzyme catalysis as judged by the kinetic constants Km and k(cat)/Km, nor did they affect the rate of turnover-dependent inactivation or inhibition by ETYA. The results indicate that these methionine residues do not play a pivotal role in catalysis, autoinactivation, or sensitivity to inhibition by acetylenic compounds.
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21 MeSH Terms
Detection, assay, and isolation of allene oxide synthase.
Brash AR, Song W
(1996) Methods Enzymol 272: 250-9
MeSH Terms: Ammonium Sulfate, Chemical Precipitation, Chromatography, Chromatography, High Pressure Liquid, Intramolecular Oxidoreductases, Isomerases, Lipid Peroxides, Plants, Solubility, Spectrophotometry, Ultraviolet, Substrate Specificity
Added December 10, 2013
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11 MeSH Terms
Noncovalent modification of deoxyhemoglobin S solubility and erythrocyte sickling.
Waterman MR, Yamaoka K, Dahm L, Taylor J, Cottam GL
(1974) Proc Natl Acad Sci U S A 71: 2222-5
MeSH Terms: Ammonium Sulfate, Anemia, Sickle Cell, Buffers, Dimethyl Sulfoxide, Dimethylformamide, Dioxins, Ethanol, Guanidines, Hemoglobin C, Hemoglobins, Abnormal, Humans, Hydrogen-Ion Concentration, Osmolar Concentration, Phosphates, Potassium, Solubility, Solvents, Urea
Show Abstract · Added February 12, 2015
Ammonium sulfate, as well as potassium phosphate, can be used to measure solubility differences between hemoglobin S and hemoglobin A. In addation, the solubility of deoxyhemoglobin C(Harlem) in 1.96 M phosphate has a markedly different temperature dependence from that of deoxyhemoglobin S. This observation indicates that the solubility measurement is quite sensitive to changes in protein structure. Because of the large degree of comparability between the solubility and the aggregation of deoxyhemoglobin S, solubility was used to measure the effectiveness of organic compounds as noncovalent modifiers of deoxyhemoglobin S aggregation. Organic solvents (ethanol, dimethylsulfoxide, 1,4-dioxane, dimethylformamide) alter the solubility characteristics of deoxyhemoglobin S in 1.96 M phosphate buffer, pH 7.0. The concentrations of solvent necessary to provide a half-maximal effect are remarkably similar (about 0.5 M), although the chemical nature of these compounds is quite different. The effect of these solvents must be to prevent the noncovalent bond formation necessary to produce the insoluble hemoglobin precipitate, perhaps by altering the water structure around the deoxyhemoglobin S molecules. In addition to these organic solvents, guanidine hydrochloride and urea, two well-known protein denaturants, were studied. Guanidine hydrochloride was as effective as the best organic solvent in increasing the solubility of deoxyhemoglobin S; urea was far less effective. Studies in vitro with intact erythrocytes from individuals homozygous for hemoglobin S showed that sickling is decreased up to 50% by treatment with ethanol. This offers further evidence that solubility is monitoring a phenomenon similar to the aggregation of deoxyhemoglobin S inside erythrocytes. While use of these particular compounds in vitro would seem to have no clinical implications, these studies do suggest that the use of chemicals that do not modify hemoglobin S covalently should be explored in efforts to prevent deoxyhemoglobin S aggregation.
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18 MeSH Terms
Kynurenine formamidase. Purification and characterization of the adult chicken liver enzyme and immunochemical analyses of the enzyme of developing chicks.
Bailey CG, Wagner C
(1974) J Biol Chem 249: 4439-44
MeSH Terms: Age Factors, Aminohydrolases, Ammonium Sulfate, Animals, Arylformamidase, Centrifugation, Chick Embryo, Chickens, Chromatography, DEAE-Cellulose, Chromatography, Gel, Electrophoresis, Disc, Female, Hot Temperature, Hydrogen-Ion Concentration, Immunodiffusion, Liver, Molecular Weight, Precipitin Tests, Rabbits, Structure-Activity Relationship
Added January 20, 2015
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20 MeSH Terms
Shape analysis of the histone octamer in solution.
Uberbacher EC, Harp JM, Wilkinson-Singley E, Bunick GJ
(1986) Science 232: 1247-9
MeSH Terms: Ammonium Sulfate, Histones, Molecular Conformation, Nucleosomes, Sodium Chloride, Solutions
Show Abstract · Added January 30, 2015
The conformation of the histone octamer is shown to depend upon the specific salt used to solubilize it. In 2M sodium chloride the octamer is similar in size and shape to the histone component of crystallized core nucleosomes. In contrast, in 3.5M ammonium sulfate the octamer is elongated, resembling an ellipsoid with approximate dimensions of 114 by 62 by 62 angstroms. These results indicate that the elongated conformation seen in the 3.3 angstroms electron density map of the histone octamer crystallized in ammonium sulfate is due to the particular salt conditions used.
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6 MeSH Terms
Partial purification and properties of calf thymus deoxyribonucleic acid dependent RNA polymerase III.
Weil PA, Blatti SP
(1975) Biochemistry 14: 1636-42
MeSH Terms: Amanitins, Ammonium Sulfate, Animals, Cattle, Centrifugation, Density Gradient, Chromatography, Ion Exchange, DNA, DNA-Directed RNA Polymerases, Dactinomycin, Electrophoresis, Polyacrylamide Gel, Isoenzymes, Kinetics, Solubility, Templates, Genetic, Thymus Gland
Show Abstract · Added March 5, 2014
DNA-dependent RNA polymerase III (nucleosidetriphosphate: RNA nucleotidyltransferase, EC 2.7.-7.6) has been isolated and partially purified from calf thymus tissue. Significant amounts of enzyme III are present in this tissue (up to 15% of the total activity of thymus homogenates). This enzyme has been characterized with respect to its chromatographic properties, broad ammonium sulfate optimum (0.04-0.2 M), template requirements, divalent metal optima, and its unique alpha-amanitin sensitivity (50% inhibition of activity occurring at an alpha-amanitin concentration of 10 mug/ml).
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15 MeSH Terms