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Abstract

FNR, the activator of anaerobic respiratory genes of Escherichia coli, has previously only been isolated as a protein of M_r 29,000, which lacks nine N-terminal amino acid residues. The underlying proteolytic events have been studied with the aim of isolating intact FNR and determining whether cleavage is the result of a physiologically significant intracellular processing mechanism or proteolytic degradation during isolation. The FNR protein was present in aerobically and anaerobically grown bacteria as the intact protein (M_r 30,000). Proteolysis only occurred during and shortly after disruption of the bacteria. The production of FNR (M_r 29,000) must therefore be regarded as an isolation artefact. The proteolysis was caused by a protease which is located outside the cytoplasmic membrane or activated upon disruption of the membrane. Protease inhibitors directed against serine, cysteine or metalloproteases failed to prevent cleavage of FNR. In E. coli strain CAG627, proteolysis was greatly reduced making it possible to isolate FNR of M_r 30,000. The N-terminal sequence of FNR (M_r 30,000) was identical to that predicted from the fnr gene starting with the initiating methionine residue and including a four-cysteine cluster (16)Cys-X₃-Cys-X₂-Cys-X₅-Cys(29).