Etude des mutants chlorate-resistants d'escherichia coli K12. IV. Isolement, purification et etude de la nitrate-reductase reconstituee In vitro par complementation

Study on chlorate-resistants mutants of Escherichia coli K12. IV. Isolation, purification and study of nitrate-reductase restored in vitro by complementation

By mixing the cell-free extracts of the two mutants chl A^? and chl B^? of Escherichia coli K12, previously freed from particle membranes, we achieved restoration of nitrate reductase activity. The activity is restored first in a soluble form, then in a particulate form. This mechanism is called “complementation”. In the soluble state, the purified enzyme reduces NO₃^? and ClO₃^?, using reduced benzyl viologen or FMNH₂ as electron donors. It is sensitive to KCN, NaN₃, p-hydroxymercuribenzoate (1 mM) and N-ethylmaleimide (0.1 mM)

The soluble form is sensitive neither to phospholipase C, nor to 2-n-heptyl-4-hydroxyquinoline-N-oxide; it associates with phospholipids and cytochrome b₁ to form particles in which nitrate reductase activity is no longer sensitive to ethyl N-maleimide and p-hydroxymercuribenzoate, but, conversely, becomes sensitive to 2-n-heptyl-4-hydroxyquinoline-N-oxide.

These results clearly demonstrate that it is possible to study the mechanism of integration of the enzyme leading to active membranes particles without any previous solubilisation of the original material.