Effect of Methionine Sulfoximine on Asparaginase Activity and Ammonium Levels in Pea Leaves

Abstract

In developing leaves of Pisum sativum the levels of ammonium did not change during the light-dark photoperiod even though asparaginase (EC 3.5.1.1) did; asparaginase activity in detached leaves doubled during the first 2.5 hours in the light. When these leaves were supplied with I millimolar methionine sulfoximine (MSX, an inhibitor of glutamine synthetase, GS, activity) at the beginning of the photoperiod, levels of ammonium increased 8-to 10-fold, GS activity was inhibited 95%, and the light-stimulated increase in asparaginase activity was completely prevented, and declined to less than initial levels. When high concentrations of ammonium were supplied to leaves, the light-stimulated increase of asparaginase was partially prevented. However, it was also possible to prevent asparaginase increase, in the absence of ammonium accumulation, by the addition of MSX together with aminooxyacetate (AOA, which inhibits transamination and some other reactions of photorespiratory nitrogen cycling). AOA alone did not prevent light-stimulated asparaginase increase; neither MSX, AOA, or elevated ammonium levels inhibited the activity of asparaginase in vitro. These resuits suggest that the effect of MSX on asparaginase increase is not due solely to interference with photorespiratory cycling (since AOA also prevents cycling, but has no effect alone), nor to the production of high ammonium concentration or its subsequent effect on photosynthetic mechanisms. MSX must have further inhibitory effects on metabolism.It is concluded that accumulation of ammonium in the presence of MSX may underestimate rates of ammonium tumover, since liberation of ammonium from systems such as asparaginase is reduced by the effects of MSX.The degradation of asparagine is important in supplying nitrogen for amino acid and protein synthesis in developing leaves, and '5N-labeling studies have indicated that up to 75% of the nitrogen required for growth in half-expanded pea leaves was derived from the amide-nitrogen of asparagine (25). The amide-nitrogen of asparagine can be liberated directly by asparaginase (EC 3.5.1.1.) (8) port (21), and, asparaginase activity is also regulated by other factors (22). Increased asparaginase activity should result in increased production of aspartate and ammonium, which are quickly metabolized (25). The oxidation of glycine during photorespiration produces appreciable amounts ofammonium in the light, and estimates based on treatments with MSX3 (an irreversible inhibitor of GS [24]), have suggested that release of photorespiratory ammonium exceeds by 10-fold or more the rate ofproduction from nitrate reduction (10). To demonstrate that nitrogen enters the photorespiratory pathway through the reaction catalyzed by serine-glyoxylate transaminase, AOA (an inhibitor of transamination reactions and glycine decarboxylation [5]) has been used in conjunction with MSX, since when supplied together, ammonium production resulting from photorespiration was assumed to be abolished (13,29). Similar results were obtained in t...

Internal ammonium excess induces ROS-mediated reactions and causes carbon scarcity in rice

“…Therefore, L-methionine-D,L-sulfoximine (MSX), a potent inhibitor of the primary NH 4 + assimilation pathway mediated by the activity of glutamine synthetases [35] was applied (1 mM) for 4 h in the presence of high NH 4…”

Solute content and energy status of roots of barley plants cultivated at different pH on nitrate‐ or ammonium‐nitrogen

“…A possible source for ammonium liberation in roots might be glutamine dehydrogenase (Robinson et al, 1991), or asparaginase (EC 3.5.1.1). The activity of the latter enzyme undergoes diurnal fluctuations, with increasing activity in the light (Sieciechowicz, Ireland & Joy, 1985;Sieciechowicz, Joy & Ireland, 1989).…”

Internal ammonium excess induces ROS-mediated reactions and causes carbon scarcity in rice

“…Meanwhile, 7 and 5 folds higher concentrations of free NH 4 + were measured in roots and shoots, respectively (Fig. 1c) [35] was applied (1 mM) for 4h in the presence of high NH 4 + (20 mM).…”