The synthesis of nitrate reductase by a parental Escherichia coli K12 strain and its isogenic chlA and chlB mutants has been analyzed by protein double labelling with L- [4,5-3H]leucine and sulphur-35 and by immunoprecipitation using specific antiserum. The chlA and chlB mutants although defective in nitrate reductase activity retain the ability to synthesise the different polypeptides that are normally required for functional enzyme activity. In addition the data shows the following.1. These polypeptides are present in unequal quantities in the membrane and in the cytoplasm of the cells. The chlB mutant synthesizes three times more nitrate reductase than the chlA mutant.2. The subunit composition of the membrane-bound nitrate reductase present in the two mutants is different.3. Membrane preparations from the chZB mutant contain the three subunits c~, P, y in a ratio which is similar to the 'wild type.4. In the chlA mutant the two subunits p and y are missing and the level of CI subunit is very low. In the same membrane a 48 000-M, subunit (polypeptide p') precipitable by nitrate reductase antiserum has been found. The chZA and chlB mutants accumulate the three subunits c~, /s and y in different proportion and concentrations in the cytoplasm unlike the parental strain. 5.The cytoplasm from the chlA mutant also contains the P' polypeptide found in the membrane fraction of this mutant and in addition contain another polypeptide designated c~' of molecular weight 105 000 which is precipitated by the nitrate reductase antiserum.The formation of particulate active nitrate reductase can be achieved by mixing the supernatant fractions of the chlA and chlB mutants (complementation) and procedes by two distinct but mutually dependent stages. Following reconstitution of activity the two peptides c~' and p' present in the supernatant fraction of the chlA mutant, disappear. Analysis of the immunoprecipitate polypeptides present in both the soluble and particulate nitrate reductase protein after reconstitution suggests that these polypeptides are precursors of the CI and / 3 subunits following a process that remains to be elucidated.

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