Identification and purification of a protein involved in the activation of nitrate reductase in the soluble fraction of a chlA mutant of Escherichia coli K12.
The isolation and purification of a protein which is the presumed product of the chlA gene has been achieved. This protein, which we have named Protein PA, has been isolated from the soluble fraction of a chlB mutant. The protein was identified by its ability to activate nitrate reductase (EC.1.7.99.4) when mixed with a soluble fraction derived from a chlA mutant. The protein has a molecular weight of about 72 000 and is composed of a single polypeptide chain. Antiserum specific for Protein PA has been produced. Removal of Protein PA from the soluble fraction of chlB mutant by immunoprecipitation with this antiserum leads to the loss of the ability of the preparation to activate nitrate reductase when mixed with a soluble fraction from a chlA mutant. Protein PA, therefore, performs an essential but as yet undefined role in the activation process. Employing this antiserum Protein PA could be quantified by rocket immunoelectrophoretic analysis. The activity of the isolated Protein PA is low, since comparatively large amounts of Protein PA are required to activate the nitrate reductase present in the soluble fraction of the chlA mutant. The mixing of Protein PA with the chlA mutant soluble fraction leads to activation of nitrate reductase in both a soluble and a membranous form, as is the case when the complete soluble fraction of the chlB mutant is used in place of Protein PA. After activation, however, only a small proportion (15%) of the Protein PA is associated with the newly formed membranous material.
D001426:Bacterial Proteins / Q000302:isolation & purification* / Q000494:pharmacology; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D004789:Enzyme Activation; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D007122:Immunoelectrophoresis; D009154:Mutation; D009565:Nitrate Reductases / Q000378:metabolism*
D001426:Bacterial Proteins; D009565:Nitrate Reductases