Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:3.5.1.1 (asparaginase)
 2,695 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Asparagine synthetase B (AS) is the primary enzyme responsible for asparagine synthesis in plants. Routine biochemical studies of this enzyme's activity have been hindered by several problems including enzyme instability and rapid physiological turnover, endogenous inhibitors, competing pathways, and asparaginase activity. We describe an extraction procedure and assay conditions that provide a reliable, direct assay for the determination of AS activity in crude plant extracts. This assay performed well with several leguminous species and the enzyme preparation retained activity for up to 3 weeks when stored at -80 degrees C. Radio-HPLC detection enabled quantitative measurement of de novo aspargine synthesis in the extracts. Optimal activity was obtained with 1 mM glutamine and 10 mM ATP in the reaction assay. Aminooxyacetic acid (AOA, 1 mM) which prevents the assimilation of aspartate into the TCA cycle, was necessary to measure AS activity in peas, but not in lupine or soybean.
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PMID:Measuring asparagine synthetase activity in crude plant extracts. 1082 80

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 1 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 results 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 turnover, since liberation of ammonium from systems such as asparaginase is reduced by the effects of MSX.
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PMID:Effect of methionine sulfoximine on asparaginase activity and ammonium levels in pea leaves. 1666 14