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. 1968 Jun;43(6):930–940. doi: 10.1104/pp.43.6.930

Some Characteristics of Nitrate Reductase From Higher Plants 1

L E Schrader 1, G L Ritenour 1,2, G L Eilrich 1, R H Hageman 1
PMCID: PMC1086950  PMID: 16656864

Abstract

With respect to cofactor requirements, NADH, and FMNH2 were equally effective as electron donors for nitrate reductase obtained from leaves of maize, marrow, and spinach, when the cofactors were supplied in optimal concentrations. The concentration of FMNH2 required to obtain half-maximal activity was from 40- to 100-fold higher than for NADH. For maximal activity with the corn enzyme, 0.8 millimolar FMNH2 was required. In contrast, NADPH was functional only when supplied with NADP:reductase and exogenous FMN (enzymatic generation of FMNH2).

All attempts to separate the NADH2- and FMNH2-dependent nitrate reductase activities were unsuccessful and regardless of cofactor used equal activities were obtained, if cofactor concentration was optimal. Unity of NADH to FMNH2 activities were obtained during: A) purification procedures (4 step, 30-fold); B) induction of nitrate reductase in corn seedlings with nitrate; and C) inactivation of nitrate reductase in intact or excised corn seedlings. The NADH- and FMNH2-dependent activities were not additive.

A half-life for nitrate reductase of approximately 4 hours was estimated from the inactivation studies with excised corn seedlings. Similar half-life values were obtained when seedlings were incubated at 35° in a medium containing nitrate and cycloheximide (to inhibit protein synthesis), or when both nitrate and cycloheximide were omitted.

In those instances where NADH activity but not FMNH2 activity was lost due to treatment (temperature, removal of sulfhydryl agents, addition of p-chloromercuribenzoate), the loss could be explained by inactivation of the sulfhydryl group (s) required for NADH activity. This was verified by reactivation with exogenous cysteine.

Based on these current findings, and previous work, it is concluded that nitrate reductase is a single moiety with the ability to utilize either NADH or FMNH2 as cofactor. However the high concentration of FMNH2 required for optimal activity suggests that in vivo NADH is the electron donor and that nitrate reductase in higher plants should be designated NADH:nitrate reductase (E.C. 1.6.6.1).

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Selected References

These references are in PubMed. This may not be the complete list of references from this article.

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