Glutamine synthetase assays

Figure 9.41 Chromatograms of enzyme assay media. (A) Elution profile of the assay medium of glutamine synthetase. Alder root nodule enzyme plus assay mixture was incubated for IS minutes. (B) Elution profile of the assay medium of glutamate synthetase. Rice leaves enzyme plus assay mixture was incubated for 15 minutes. (From Martin et al., 1982.)...

Figure 9.42 Chromatograms corresponding to a glutamine synthetase in situ assay. (a) Sample taken at zero time, (b) Sample taken after completion of the assay. (From Marques et al., 1989.)...

Bressler, S. L., and Ahmed, S. I. (1984). Detection of glutamine synthetase activity in marine phytoplankton Optimization of the biosynthetic assay. Mar. Ecol. Prog. Ser. 14, 207—217. 

Rees, T. A. V., Larson, T. R., Heldens, J. W. G., and Huning, E. G. J. (1995). In situ glutamine synthetase activity in a marine unicellular alga (Development of a sensitive colorimetric assay and the effects of nitrogen status on enzyme activity). Plant Physiol. 109, 1405—1410. 

Pahuja, S. L., and Reid, T. W. (1982). Radioisotope assay for glutamine synthetase using thin-layer chromatography. J. Chromatogr. 235, 249—255. 

Prusiner, S., and Milner, L. (1970). A rapid radioactive assay for glutamine synthetase, glutaminase, asparagines synthetase, and asparaginase. Anal. Biochem. 37, 429—438. 

Slawyk, G., and Rodier, M. (1988). Biosynthetically active glutamine synthetase in the marine diatom Phaeodactylum tricornutum Optimization of the forward-reaction assay. Mar. Biol. 97, 269-274. 

This reaction requires catalytic amounts of nucleotide. Activity in the transferase assay generally gives rates several times higher than those obtained in the synthetase assays [Eqs. (1) and (2)] and for this reason is often used for measurement of glutamine synthetase, especially in relatively crude preparations. However caution should be exercised in the use of the transferase assay as a similar reactitm can also be catalyzed by some glutaminases and certain other amidases (Meister et al., 1955), although in these cases the reaction is not dependent on ADP, phosphate, or divalent metal cation. Levintow et al. (1955) demonstrated that the synthetase activity and the ADP phosphate divalent cation dependent transferase activity are functions of the same enzyme. 

Initial attempts to assay the enzyme utilized an assay principle employed for glutamine synthetase involving the formation of /8-aspartyl hydroxamate from hydroxylamine. 

Care should be taken when determining the kinetics of the ammonia-dependent reaction that glutamine synthetase is not present in the extracts. Glutamine could easily be formed from glutamate and ammonia in the presence of ATP and Mg and act as the substrate for the AS reaction being assayed. 

Figure 9.44 Chromatogram of assay of asparagine synthetase. Peaks 1, o-phthaldialdehyde aspartate 2, glutamate 3, asparagine 4, glutamine. A mixture of 0.025 mAf of each amino acid was made and 20 yu.L injected. (From Unnithan et al., 1984.)...

In the enzyme catalysis of the first committed step in the de novo synthesis of purines, an amino group from L-glutamine is transferred to 5-phosphoribosyl-l-pyrophosphate to form glutamate and 5-phosphoribosyl-1-amine. The assay includes glycinamide ribonucleotide synthetase, which converts 5-phosphoribosyl-l-amine to glycinamide ribonucleotide, which is the reaction product quantitated. 

Aspartate kinase is usually subject to very strong inhibition by lysine and/or threonine (Miflin et ai, 1979). Even if the worker can be certain that the activity that is measured is due to the action of asparagine synthetase, there is still no information on whether glutamine or ammonia is acting as the amino donor. It is for these reasons that the hydroxamate assay is not recommended. 

Route c involves the conversion of aspartate to asparagine by glutamine-dependent asparagine synthetase (EC 6.3.5.4). The enzyme has had a checkered career and it has proved extremely difficult to prepare extracts with high activity. Joy and Ireland (1990) have described in detail suitable assay methods and discussed possible factors that may prevent the determination of maximum rates of activity. A heat-stable, dialyzable inhibitor was found in pea leaf homogenates that inhibited the lupin cotyledon enzyme (Joy et al. 1983). The presence of this inhibitor may well account for the current lack of detection of asparagine synthetase in green leaf tissue. 

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