Measuring asparagine synthetase activity in crude plant extracts

Asparagine synthetase B (AS) is the primary enzyme responsible for asparagi ne synthesis in plants. Routine biochemical studies of this enzyme's activi ty have been hindered by several problems including enzyme instability and rapid physiological turnover, endogenous inhibitors, competing pathways, an d asparaginase activity. We describe an extraction procedure and assay cond itions that protide a reliable, direct assay for the determination of AS ac tivity in crude plant extracts. This assay performed well with several legu minous species and the enzyme preparation retained activity for up to 3 wee ks when stored at -80 degrees C. Radio-HPLC detection enabled quantitative measurement of de novo aspargine synthesis in the extracts. Optimal activit y was obtained with 1 nM glutamine and 10 mM ATP in the reaction assay. Ami nooxyacetic acid (AOA, 1 mM) which prevents the assimilation of aspartate i nto the TCA cycle, was necessary to measure AS activity in peas, but not in lupine or soybean.