RESUMO
Methionine sulphoxide reductases (Msr) reduce methionine sulphoxide to methionine and protect bacteria against reactive oxygen intermediates (ROI) and reactive nitrogen intermediates (RNI). Many organisms express both MsrA, active against methionine-(S)-sulphoxide, and MsrB, active against methionine-(R)-sulphoxide. Mycobacterium tuberculosis (Mtb) expresses MsrA, which protects DeltamsrA-Escherichia coli from ROI and RNI. However, the function of MsrA in Mtb has not been defined, and it is unknown whether Mtb expresses MsrB. We identified MsrB as the protein encoded by Rv2674 in Mtb and confirmed the distinct stereospecificities of recombinant Mtb MsrA and MsrB. We generated strains of Mtb deficient in MsrA, MsrB or both and complemented the mutants. Lysates of singly deficient strains displayed half as much Msr activity as wild type against N-acetyl methionine sulphoxide. However, in contrast to other bacteria, single mutants were no more vulnerable than wild type to killing by ROI/RNI. Only Mtb lacking both MsrA and MsrB was more readily killed by nitrite or hypochlorite. Thus, MsrA and MsrB contribute to the enzymatic defences of Mtb against ROI and RNI.
Assuntos
Proteínas de Bactérias/metabolismo , Ácido Hipocloroso/metabolismo , Mycobacterium tuberculosis/enzimologia , Nitritos/metabolismo , Oxirredutases/metabolismo , Sequência de Aminoácidos , Animais , Proteínas de Bactérias/genética , Regulação Bacteriana da Expressão Gênica , Genes Bacterianos , Teste de Complementação Genética , Metionina Sulfóxido Redutases , Camundongos , Camundongos Endogâmicos C57BL , Dados de Sequência Molecular , Mutagênese Insercional , Mycobacterium tuberculosis/genética , Oxirredutases/genética , Espécies Reativas de Nitrogênio/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Alinhamento de Sequência , Especificidade por SubstratoRESUMO
This work investigates the introduction of methanol and a salt modifier to molecular weight cut-off membrane-based centrifugal filters (MWCO) to enrich sub-microgram peptide quantities. Using a neuropeptide standard, bradykinin, sample loss was reduced over two orders of magnitude with and without undigested protein present. Additionally, a bovine serum albumin tryptic digestion was investigated. Twenty-seven tryptic peptides were identified from MALDI mass spectra after enriching with methanol while only two tryptic peptides were identified after MWCO separation using H(2)O. The strategy presented here enhances recovery from MWCO separation for sub-µg peptide samples.