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The intrinsic fluorescence of the enzyme rhodanese is quenched by as much as 30% when sulfur is transferred to the free enzyme form, E, giving the sulfur-substituted enzyme, ES. This fluorescence change (lambda ex = 295 nm and lambda em = 335 nm) has been used to quantitate the E and ES forms which are isolatable, obligatory intermediates in rhodanese catalysis. Fluorescence titration was performed using cyanide to irreversibly remove sulfur from ES. The results show a stoichiometry corresponding to 1 bound sulfur/molecule of the ES form of rhodanese (Mr = 33,000). The fluorescence changes were used to measure the concentrations of E and ES when these were in reversible equilibria induced by interactions with the substrates S2O3(2-) and SO3(2-). These results were compared with an equilibrium constant derived from published kinetic studies for the reaction (formula; see text) The very close agreement between the physical and kinetic methods indicate that there are no significant concentrations of intermediates other than E and ES. Overall, the results are compatible with the formation of a persulfide intermediate in rhodanese catalysis and are consistent with conclusions from x-ray crystallography and absorption spectroscopy. In addition, these procedures offer a facile method to measure equilibria between catalytic intermediates in the rhodanese reaction using functionally relevant concentrations. 相似文献
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Horowitz BZ 《The Western journal of medicine》1989,151(1):68-69
In Los Angeles, a substantial increase in the number of infections due to Neisseria meningitidis has been noted. More alarming has been an associated mortality rate of 21%. The prompt recognition of meningococcal disease based on a high index of suspicion and the early institution of effective antibiotic treatment are essential for preventing complications and reducing mortality. 相似文献
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Gerald A. Merrill David Miller John Chirgwin Paul M. Horowitz 《Journal of Protein Chemistry》1992,11(2):193-199
Rhodanese has been utilized as a model enzyme for the study of protein structure-function relationships. The enzyme has recently been cloned and the recombinant enzyme is now available for investigation. However, prior to use in structure-function studies, the recombinant enzyme must be shown to have the same structure and activity as the bovine liver enzyme used in the previous studies. An immunological study of the conformations of these enzyme conformers is described. Three antibodies (two monoclonal and one polyclonal, site-directed antibody) were shown to detect distinct and nonoverlapping epitopes. The epitopes of the monoclonal antirhodanese antibodies (R207 and MAB11) were mapped to the same CNBr digest fragment of the amino terminal domain of rhodanese, and the epitope of the site-directed antibody prepared against the interdomain tether sequence of rhodanese (PAT-T1) was mapped to that region of rhodanese (residues 142–156). The rhodanese conformers were studied by monitoring the accessibility of the epitopes recognized by each antibody in each conformer using an indirect ELISA. None of the antibodies could detect its epitope on the purified liver enzyme. Two of the antibodies (R207 and PAT-T1) could also not detect their epitopes on the recombinant enzyme. However, MAB11 did detect a conformational difference between the natural and recombinant rhodanese conformers, indicating the conformational difference is localized in the first 73 amino acids of rhodanese. This difference presumably reflects the difference in the histories of the two enzymes and may be due to differences in enzyme folding, differences in the purification procedures, and differences in storage conditions—all of which could influence the final conformation of the enzyme. 相似文献
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Response of microbial populations to environmental disturbance 总被引:18,自引:0,他引:18
Taxonomic and genetic diversities of microbial communities disturbed by chemical pollutants were lower than in undisturbed
reference communities. The dominant populations within the disturbed communities had enhanced physiological tolerances and
substrate utilization capabilities, indicating that generalized physiological versatility is an adaptive characteristic of
populations that successfully compete within disturbed communities. 相似文献
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Nurit Eyal Nurit Firon Sylvia Wilder Edwin H. Kolodny Mia Horowitz 《Human genetics》1991,87(3):328-332
Summary Single-stranded cDNA was prepared from RNA obtained from a patient with type 1 Gaucher disease. The cDNA was amplified in vitro and analyzed by sequencing. Three base-pair changes were identified which included a G to C transversion at nucleotide 3119 of the active gene (Asp140His), an A to C transversion at nucleotide 3170 (Lys157Gln) and a G to A change at nucleotide 5309 (Glu326Lys). To study the mode of inheritance of the three different base-pair changes, genomic DNA was prepared from blood or skin fibroblasts of several family members. Genomic glucocerebrosidase DNA sequences were amplified and subjected to hybridization with allele-specific oligonucleotides (ASOs). The hybridization profiles demonstrated that two of the basepair changes originated from the mother and were transmitted to her two affected sons and to a grandchild, while the third base-pair change, originating from the father, was transmitted to his two affected sons, a carrier daughter and a second grandchild. Tests of other patients with Gaucher disease failed to disclose the presence of the three base-changes. This is a unique family with three base-pair changes tightly linked to Gaucher disease. 相似文献