The estimation of ammonia using the indophenol blue reaction.
ABSTRACT The addition of sodium hypochlorite solution of pH 12.5 to an ammonia solution before the addition of sodium phenate has been shown to increase the sensitivity and accuracy of the indophenol blue reaction. This reaction is not temperature-dependent over the range −5° to +25°. The pH of the phenate solution is much less critical when it is added after the hypochlorite; a reagent with a pH in the range 10.6–11.2 is quite suitable.This modified method may be applied to the later stages of Fenton's1 method of estimating plasma ammonia and while it increases the sensitivity and accuracy it does not impair the specificity.
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ABSTRACT: We studied the effects of the application of organic matter (OM) and chemical fertilizer (CF) on soil alkaline phosphatase (ALP) activity and ALP-harboring bacterial communities in the rhizosphere and bulk soil in an experimental lettuce field in Hokkaido, Japan. The ALP activity was higher in soils with OM than in soils with CF, and activity was higher in the rhizosphere for OM than in the bulk soil. Biomass P and available P in the soil were positively related to the ALP activity of the soil. As a result, the P concentration of lettuce was higher in OM soil than in CF soil. We analyzed the ALP-harboring bacterial communities using polymerase chain reaction based denaturing gradient gel electrophoresis (DGGE) on the ALP genes. Numerous ALP genes were detected in the DGGE profile, regardless of sampling time, fertilizer treatment or sampled soil area, which indicated a large diversity in ALP-harboring bacteria in the soil. Several ALP gene fragments were closely related to the ALP genes of Mesorhizobium loti and Pseudomonas fluorescens. The community structures of the ALP-harboring bacteria were assessed using principal component analysis of the DGGE profiles. Fertilizer treatment and sampled soil area significantly affected the community structures of ALP-harboring bacteria. As the DGGE bands contributing to the principal component were different from sampling time, it is suggested that the major bacteria harboring the ALP gene shifted. Furthermore, there was, in part, a significant correlation between ALP activity and the community structure of the ALP-harboring bacteria. These results raise the possibility that different ALP-harboring bacteria release different amounts and/or activity of ALP, and that the structure of ALP-harboring bacterial communities may play a major role in determining overall soil ALP activity.Soil Science and Plant Nutrition 01/2008; 54(1):62 - 71. · 0.89 Impact Factor
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ABSTRACT: All known nitrilase superfamily amidase and carbamoylase structures have an additional glutamate that is hydrogen bonded to the catalytic lysine, in addition to the Glu, Lys, Cys "catalytic triad". In the amidase from Geobacillus pallidus, mutating this glutamate (E142) to a leucine or aspartate renders the enzyme inactive. X-ray crystal structure determination shows that the structural integrity of the enzyme is maintained in spite of the mutation, with the catalytic cysteine (C166), lysine (K134) and glutamate (E59) in similar positions to those of the wild-type enzyme. In the case of the E142L mutant a chloride ion is located in the position occupied by E142 Oε1 in the wild-type enzyme and interacts with the active site lysine. In the case of the E142D mutant this site is occupied by D142 Oδ1 . In neither case is an atom located at the position of E142 Oε2 in the wild-type enzyme. The active site cysteine of the E142L mutant was found to form a Michael adduct with acrylamide, which is a substrate of the wild-type enzyme. This is due to an interaction that places the double bond of the acrylamide rather than the amide carbonyl carbon adjacent to the active site cysteine. Our results demonstrate that in the wild-type active site, a crucial role is played by the hydrogen bond between E142 Oε2 and the substrate amino group in positioning the substrate with the correct stereoelectronic alignment to enable the nucleophilic attack on the carbonyl carbon by the catalytic cysteine.Journal of Biological Chemistry 08/2013; · 4.65 Impact Factor
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ABSTRACT: The role of chromosomal toxin-antitoxin (TA) modules in bacterial physiology remains enigmatic despite their abundance in the genomes of many bacteria. Mycobacterium smegmatis contains three putative TA systems, VapBC, MazEF, and Phd/Doc, and previous work from our group has shown VapBC to be a bona fide TA system. In this study, we show that MazEF and Phd/Doc are also TA systems that are constitutively expressed, transcribed as leaderless transcripts, and subject to autoregulation, and expression of the toxin component leads to growth inhibition that can be rescued by the cognate antitoxin. No phenotype was identified for deletions of the individual TA systems, but a triple deletion strain (ΔvapBC, mazEF, phd/doc), designated ΔTA(triple), exhibited a survival defect in complex growth medium demonstrating an essential role for these TA modules in mycobacterial survival. Transcriptomic analysis revealed no significant differences in gene expression between wild type and the ΔTA(triple) mutant under these conditions suggesting that the growth defect was not at a transcriptional level. Metabolomic analysis demonstrated that in response to starvation in complex medium, both the wild type and ΔTA(triple) mutant consumed a wide range of amino acids from the external milieu. Analysis of intracellular metabolites revealed a significant difference in the levels of branched-chain amino acids between the wild type and ΔTA(triple) mutant, which are proposed to play essential roles in monitoring the nutritional supply and physiological state of the cell and linking catabolic with anabolic reactions. Disruption of this balance in the ΔTA(triple) mutant may explain the survival defect in complex growth medium.Journal of Biological Chemistry 12/2011; 287(8):5340-56. · 4.65 Impact Factor