RESUMEN
The electron-transferring proteins, trimethylamine dehydrogenase (TMAD) and electron-transferring flavoprotein (ETF) from the bacterium Methylophilius methylotrophus, were studied in vitro by fluorescence spectroscopy. Flavin adenine dinucleotide (FAD) was found to be capable of a slow and spontaneous release from ETF, which is accompanied by an increase in flavin fluorescence. At a rather high ionic strength (0.1 M NaCl or 50 mM phosphate), the FAD release is sharply activated by TMAD preparations that induce a local conformational transition in ETF. The values of tryptophan fluorescence polarization and lifetime and the use of the Levshin-Perrin equation helped show that the size of protein particles remain unchanged upon the TMAD and ETF mixing; i.e., these proteins themselves do not form a stable complex with each other. The protein mixture did not release flavin from ETF in the presence of trimethylamine and formaldehyde. In this case, a stable complex between the proteins appeared to be formed under the action of formaldehyde. Upon a short-term incubation of ETF with ferricyanide, FAD was hydrolyzed to flavin mononucleotide (FMN) and AMP. This fact explains the previous detection of AMP in ETF preparations by some researches. A fluorescence method was proposed for distinguishing FAD from FMN in solution using ethylene glycol. The English version of the paper: Russian Journal of Bioorganic Chemistry, 2004, vol. 30, no. 3; see also http://www.maik.ru.
Asunto(s)
Flavoproteínas Transportadoras de Electrones/química , Flavina-Adenina Dinucleótido/química , Methylophilus methylotrophus/enzimología , Oxidorreductasas N-Desmetilantes/química , Glicol de Etileno/química , Mononucleótido de Flavina/análisis , Mononucleótido de Flavina/química , Flavina-Adenina Dinucleótido/análisis , Methylophilus methylotrophus/metabolismo , Conformación Proteica , Espectrometría de FluorescenciaRESUMEN
Cell DNAs of various species of Enterobacteriaceae were hybridized with the probes based on IS285 and IS100, mobile genetic elements of Yersinia pestis. These IS elements are found only in the genomes of all tested Y. pestis strains and a number of strains of a related bacterium Y. pseudotuberculosis. Phylogenetic relations between the tested strains and correlation of fingerprints with the geographical origin of the strains were revealed by analysis of the hybridization profiles of Y. pestis and Y. pseudotuberculosis chromosomal DNAs with IS probes. Comparison of the chromosomal IS100 profiles of Y. pestis wild strain and its nonpigmented mutant helped us determine the minimal extension of the genetic rearrangement and detect at least three copies of the IS element in the mutant region.