In Vitro Stability of Nitrate Reductase from Wheat Leaves

Abstract

Wben a crude extract from 8-day-old wheat (Trincum aestivum L. cv. Olympic) leaves was fractionated by a combination of ammonium sulfate precipitation and Sephadex G-100 chromatography the presence of three factors which have a marked effect on the stability of highly purified nitrate reductase was revealed. Two of these factors (I and Ill) have a positive effect and the other factor (II) has a negative effect on stability. Factors I and III can each overcome the instability-promoting effect of II; however, this was apparently not due to a direct effect on factor II.Both factors I and III have been subjected to further purification. Factor I can be separated into at least four fractions, each with stability-promoting activity. Factor III appears to be a single factor.The in vitro activity and stability of nitrate reductase in crude extracts were found to vary diurnally. Stability and activity were highest 4 hours after the start of the light period and both were minimal 1 to 3 hours after the end of the lght period. When crude extract was fractionated as described above and an assessment made of the relative amounts of I, II, and III, there appeared to be a distinct diurnal variation in their levels. Factors I and III were highest when in vitro nitrate reductase activity and stability were highest. Factor 11 was apparently out of phase in that maximum activity coincided with the time of minimum in vitro nitrate reductase activity and stability.

Rhythmicity in Nitrate Reductase Activity in Wheat Embryos During Germination

“…Therefore, the endogenous rhythmicity of activity may result from oscillations in the degradation of NR enzyme protein (Cohen and Gumming, 1974). The observed rhythmic fluctuations in in vitro activit>^ and stability of NR agrees with the report of Sherrard, Kennedy and Dalling (1979).…”

Differential Light Induction of Nitrate Reductases in Greening and Photobleached Soybean Seedlings

“…In soybean plants the regulation of NR activity may be particularly complicated because two isozymes with quite different properties may be present (1,19). One manifestation of this complexity is the two diumal peaks in extractable NR activity in soybean leaves (8) as compared to the one diurnal peak in NR activity of maize (8), and wheat (25).…”

“…The large amplitude of daily rhythms for NR3 activity extracted from higher plants (8,25) and the toxic effects of NO2, the product of NR activity, in higher plants (4) suggest that in many species NR activity is highly regulated and that the close regulation of NR is necessary for healthy plant function. Phytochrome (11,12,22), blue light photoreceptors (16,22) synthetic pigments (6), , reduced nucleotides (21), ATP:ADP ratios (21), polyribosome levels (28), and specific proteinaceous inhibitors and activators of NR (25) have been shown to affect levels of NR activity which may be extracted from higher plants and have been implicated as regulators of NR activity.…”

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The large amplitude of daily rhythms for NR3 activity extracted from higher plants (8,25) and the toxic effects of NO2, the product of NR activity, in higher plants (4) suggest that in many species NR activity is highly regulated and that the close regulation of NR is necessary for healthy plant function.

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“…Phytochrome (11,12,22), blue light photoreceptors (16,22) synthetic pigments (6), , reduced nucleotides (21), ATP:ADP ratios (21), polyribosome levels (28), and specific proteinaceous inhibitors and activators of NR (25) have been shown to affect levels of NR activity which may be extracted from higher plants and have been implicated as regulators of NR activity. In soybean plants the regulation of NR activity may be particularly complicated because two isozymes with quite different properties may be present (1,19).…”