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Saturated Ammonium (saturated + ammonium)
Selected AbstractsNitrilase of Rhodococcus rhodochrous J1FEBS JOURNAL, Issue 1 2000Conversion into the active form by subunit association Nitrilase-containing resting cells of Rhodococcus rhodochrous J1 converted acrylonitrile and benzonitrile to the corresponding acids, but the purified nitrilase hydrolyzed only benzonitrile, and not acrylonitrile. The activity of the purified enzyme towards acrylonitrile was recovered by preincubation with 10 mm benzonitrile, but not by preincubation with aliphatic nitriles such as acrylonitrile. It was shown by light-scattering experiments, that preincubation with benzonitrile led to the assembly of the inactive, purified and homodimeric 80-kDa enzyme to its active 410-kDa aggregate, which was proposed to be a decamer. Furthermore, the association concomitant with the activation was reached after dialysis of the enzyme against various salts and organic solvents, with the highest recovery reached at 10% saturated ammonium sulfate and 50% (v/v) glycerol, and by preincubation at increased temperatures or enzyme concentrations. [source] Expression and characterization of ,-glucosidase III in the dwarf honeybee, Apis florea (Hymenoptera: Apoidea: Apidae)INSECT SCIENCE, Issue 4 2007CHANPEN CHANCHAO Abstract Alpha-glucosidase is synthesized in the hypopharyngeal glands located in the head of worker bees including Apis florea. To analyze the developmental stage-specific expression of the ,-glucosidase gene in A. florea, total RNA was isolated from eggs, and the heads of nurse and forager bees. By reverse transcription polymerase chain reaction (RT-PCR), it was shown that the highest expression levels of the ,-glucosidase III gene, in the three examined developmental stadia, were found in forager bees, with much lower expression levels in nurse bees and no detectable expression in eggs. A complete ,-glucosidase III cDNA was obtained by RT-PCR and sequenced. The 1 701 bp cDNA nucleotide sequence and the predicted 567 amino acids it encodes were assayed by BLASTn, BLASTp and BLASTx programs and revealed a 95% and 94% similarity to the A. mellifera,-glucosidase III gene at the DNA and amino acid sequence levels, respectively. For purification of the active encoded enzyme, forager bee heads were homogenized in sodium phosphate buffer solution and the crude extract (0.30 U/mg) sequentially precipitated with 95% saturated ammonium sulfate (0.18 U/mg), and purified by DEAE cellulose ion exchange chromatography (0.17 U/mg), and gel filtration on Superdex 200 (0.52 U/mg). After resolution through sodium dodecyl sulfate-polyacrylamide gel electrophoresis, a single enzymically active band (73 kDa) was identified from renatured substrate gels. Excision of this band, elution of the protein and tryptic peptide digestives identified by matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) revealed six matching masses to the A. mellifera (Q17958) and predicted A. florea,-glucosidase III protein with 12% coverage, supporting the probable purification of the same ,-glucosidase III protein as that encoded by the cloned cDNA. [source] Improved crystals of Thermus thermophilus prolyl-tRNA synthetase complexed with cognate tRNA obtained by crystallization from precipitateACTA CRYSTALLOGRAPHICA SECTION D, Issue 2 2000Anna Yaremchuk The complex between Thermus thermophilus prolyl-tRNA synthetase (ProRSTT) and its cognate tRNA has been crystallized using two different isoacceptors of tRNAPro. Similar bipyramidal crystals of the complexes of ProRSTT with the two different tRNAPro isoacceptors grow within two weeks from 32% saturated ammonium sulfate solution. They belong to space group P43212, with unit-cell parameters a = 143.1, b = 143.1, c = 228.6,Å. The crystals diffract weakly to a maximum resolution of 3.1,Å. Superior quality crystals were obtained by growing slowly from precipitate over 5,6 months. These are of the same space group but have slightly altered unit-cell parameters, a = 140.8, b = 140.8, c = 237.0,Å. These crystals diffract more strongly to at least 2.8,Å resolution and a complete data set to 2.85,Å resolution has been collected from a single crystal. Comparison of the packing in the two crystal forms shows that domain flexibility contributes to the presence of different crystal contacts in the two forms. [source] Preliminary X-ray data analysis of crystalline hibiscus chlorotic ringspot virusACTA CRYSTALLOGRAPHICA SECTION F (ELECTRONIC), Issue 6 2009Ao Cheng Hibiscus chlorotic ringspot virus (HCRSV) is a positive-sense monopartite single-stranded RNA virus that belongs to the Carmovirus genus of the Tombusviridae family, which includes carnation mottle virus (CarMV). The HCRSV virion has a 30,nm diameter icosahedral capsid with T = 3 quasi-symmetry containing 180 copies of a 38,kDa coat protein (CP) and encapsidates a full-length 3.9,kb genomic RNA. Authentic virus was harvested from infected host kenaf leaves and was purified by saturated ammonium sulfate precipitation, sucrose density-gradient centrifugation and anion-exchange chromatography. Virus crystals were grown in multiple conditions; one of the crystals diffracted to 3.2,Å resolution and allowed the collection of a partial data set. The crystal belonged to space group R32, with unit-cell parameters a = b = 336.4, c = 798.5,Å. Packing considerations and rotation-function analysis determined that there were three particles per unit cell, all of which have the same orientation and fixed positions, and resulted in tenfold noncrystallography symmetry for real-space averaging. The crystals used for the structure determination of southern bean mosaic virus (SBMV) have nearly identical characteristics. Together, these findings will greatly aid the high-resolution structure determination of HCRSV. [source] | ||||||||||