Abstract
Abstract In the presence of NADH and cyanide, NADH-nitrate oxidoreductase (EC 1.6.6.1) from Chlorella vulgaris is converted to an inactive form which is readily reactivated by ferricyanide. Experiments with H14CN indicated that the inactivation process is associated with the firm binding to the protein of 0.066 nmole of cyanide per unit of enzyme inactivated. This cyanide binding was linearly proportional to the amount of inactivation. No firm cyanide binding and no inactivation occurred in the absence of NADH or an equivalent reductant. The bound 14C was released when the enzyme was reactivated by ferricyanide. After the cells had been treated with ammonia for several hours prior to disruption, the crude cell extracts contained the nitrate reductase primarily in the inactive form. Critical examination of the methods of cell disruption suggested that the inactivation of the enzyme had truly occurred in vivo. After 300-fold purification, activation of this in vivo inactivated enzyme resulted in the release of 0.066 nmole of HCN per unit of enzyme activated. We conclude that the inactivation of nitrate reductase in vivo involves the formation of a firmly bound complex of reduced enzyme and cyanide. The reaction between reduced enzyme and HCN may be written: Er + HCN (ka)/⇄/(kd) Er-HCN The measured value for ka was 1.25 x 106 m-1 min-1, for kd, 4.5 x 10-4 min-1, giving a dissociation constant, Kd = 3.6 x 10-10 m.
Highlights
The present study addresses itself to these questions and establishes that the in &JO inactivated nitrate reductase contains bound cyanide
That when nitrate is added to the cell suspension immediately prior to sonication, the extracts contain a considerable proportion of the enzyme in the active form (Table inactivate the enzyme (II), Experiment 1)
Presence of Bound Cyanide in in Vivo-inactivated EnzymeThe procedure developed for the purification of activated nitrate reductase can be applied well to the purification of the Release of HCN associated with activation nitrate reductase of naturally inactivated
Summary
Growth of Chlorella-The cells were grown in continuous white light on a mineral salts medium, with nitrate as the only source of nitrogen, in a stream of 57, (v/v) CO, in air, at 2(f22”, as previously described [1, 12]. 500.ml flasks (10 to 12 ml of cells) were harvested by centrifugation, washed with 2 liters of nitrate-free culture medium (20 mM MgSO4, 20 mM KHtP04, and 35 mM NaCl), and suspended in 3 liters of the same nitrate-free salt solution containing, in addition, 20 mM NH&l. The slow activation caused by nitrate is insignificant on the time scale under consideration It was, reasoned that if nitrate was added to the cell suspension immediately prior to cell disruption, its presence should prevent any inactivation which might occur during or after the disruption process. In earlier studies from this laboratory, water-washed cells were disrupted by sonication and consistently yielded extracts in which most of the enzyme was inactive. That when nitrate is added to the cell suspension immediately prior to sonication, the extracts contain a considerable proportion of the enzyme in the active form (Table II, Experiment 1)
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