Institution: | Laboratoire de Chimie Bactérience, C.N.R.S., 31, chemin J. Aiguier, Marseille, France |
Abstract: | 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. |