Synthesis and degradation of nitrate reductase in Escherichia coli

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Abstract
The biosynthesis, insertion and in vivo stability of nitrate reductase were examined by following the amount of labeled enzyme present in both membranes and cytoplasm at varying times after a short pulse of radioactive sulfate. Nitrate reductase levels were measured by autoradiography of immunoprecipitated material after fractionation on sodium dodecyl sulfate-polyacrylamide gels. Subunits A and B were synthesized in the cytoplasm and subsequently inserted into membranes. The insertion of subunits was dependent on the synthesis of another protein, and the rate of synthesis of this protein determined the rate of insertion of subunits A and B. The nitrate reductase produced by the chlA mutant was inserted into membranes in the normal fashion; the nitrate reductase produced by the chlC and chlE mutants was poorly incorporated. The nitrate reductase in the wild type was completely stable in vivo under inducing or noninducing conditions; in the chlC and chlE mutants nitrate reductase was degraded extensively in both the cytoplasm and membranes, even under inducing conditions. Under similar conditions, nitrate reductase was stable in the chlA mutant.