NADH:nitrate reductase (EC 1.6.6.1) and NAD(P)H:nitrate reductase (EC 1.6.6.2) were purified from wild-type soybean (Glycine max IL.i Meff., cv Williams) and nr1-mutant soybean plants. Purification Apparent Michaelis constants for nitrate were 5 millimolar (c,NR) and 0.19 millimolar (c2NR). The iNR from the mutant had a pH optimum of 7.5, sn,, of 7.6, and was less mobile on polyacrylamide gels than c,NR and c2NR. The iNR preferred NADH over NADPH and had an apparent Michaelis constant of 0.13 millimolar for nitrate.Thus, wild-type soybean contains two forms of constitutive nitrate reductase, both differing in their physical properties from nitrate reductases common in higher plants. The inducible nitrate reductase form present in soybeans, however, appears to be similar to most substrateinduced nitrate reductases found in higher plants.The NR3 enzyme of most higher plants uses NADH as electron donor to reduce nitrate to nitrite (1, 9). This ubiquitous enzyme has a pH optimum around 7.4, and Michaelis constants for nitrate and NADH of approximately 200 and 2 ,M, respectively (1,9).

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Nelson, R. S., Streit, L. and Harper, J. E. 1984. Biochemical characterization of nitrate and nitrite reduction in the wild-type and a nitrate reductase mutant of soybean. -Physiol. Plant. 61: 384-390.Enzyme activities involved in nitrate assimilation were analyzed from crude leaf extracts of wild-type (cv. Williams) and mutant {nr,) soybean [Glycitie max (L.) Mcrr.| plants lacking constitutive nitrate reductase (NR) activity. The nr, soybean mulant (formerly LNR-2), had decreased NADH-NR, FMNH,-NR and cytochrome c reductase activities, all of which were associated with the loss of constitutive NR activity. Measurement of FMNHj-NR activity, by nitrite determination, was accurate since nitrite reductase could not use FMNH, as a rcductant source. Nitrite reductase activity was normal in the nr, plant type in the presence of reduced methyl viologen. Assuming that constitutive NR is similar in structure to nitrate rcductases from other plants, presence of xanthiiic dehydrogenaso activity and loss of cytochrome c reductase activity indicated that the apoprotcin and not the molybdenum cofactor had been affected in the constitutive enzyme of the mutant. Constitutive NR from urea-grown wild-type plants had 1) greater ability to use FMNHj as an electron donor, 2) a lower pH optimum, and 3) decreased ability to distinguish between NOj and HCO5, compared with inducible NR from NOj-grown ;;/-, plants. The presence in soybean leaves of a nitrate reductase with a pH optimum of 7.5 is contrary to previous reports and indicates that soybean is not an exception among higher plants for this activity.Additional key words -Gtycine max. isozymes, molybdenum cofactor, nitrite reductase, xanthine dehydrogenase. R. S. Nelson and L. Streit. Dept o] Agronomy, and J. E. Harper (reprint reqttcsts).

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