Depending on the organism, assimilation of ammonia takes place via the activities of glutamine synthetase, glutamate synthetase, and/or glutamate dehydrogenase. The assays described, which measure glutamate and/or glutamine formation and disappearance, were developed for use in situ and in vitro.
In the HPLC assay method described by Martin et al. (1982), glutamic acid and glutamine are separated by reversed-phase HPLC after derivatization with o-phthaldialdehyde/2-mercaptoethanol (OPA). The separation was carried out on a C18 column (/xBondapak) using a mobile phase of 20 mM sodium phosphate buffer (pH 6.8) and methanol (64:36 v/v). The column was eluted isocratically and monitored at 340 nm. The separation, carried out at room temperature, is shown in Figure 9.40. The reaction mixtures were prepared according to published procedures. The reactions were terminated using either chloride or TCA. Precipitates were removed by centrifugation, and the supernatant solution was used for the assay. Figure 9.41, which shows chromatographic profiles obtained after incubation for 15 minutes, also indicates the formation of glutamine (gin) and glutamate (glu).
The enzymes were prepared by published procedures either from root nodules or from rice leaves.
In the assay described by Marques et al. (1989), the o-phthaldialdehyde derivatives of glutamine and glutamate were separated at 45°C on a ¿iBonda-pak Qg or a Novapak C18 column (3.9 mm x 150 mm). The mobile phase was 20 mM sodium phosphate buffer (pH 6.5) containing 22% (v/v) methanol and 2% (v/v) tetrahydrofuran. Fluorescence detection was used, with excitation and emission at 338 and 425 nm, respectively.
Glutamine synthetase activity was determined by following glutamine formation. The reaction contained 50 mM Hepes-NaOH (50 mM). The reaction was started by addition of ATP and stopped after 15 minutes of incubation at 30°C by addition of 0.6 mL of 1 N HC1.
Glutamate synthetase activity was determined by following glutamate formation. The reaction mixture contained in a final volume of 0.9 mL: 45 jumol potassium phosphate buffer (pH 7.0), 5 ¿imol L-glutamine, 1 /¿mol 2-oxoglutarate, 5 ¿imol aminooxyacetate, 10 nmol Synechococcus ferredoxin, and enzyme. The reaction was started by adding 0.8 mg of sodium dithionite freshly dissolved in 0.1 mL of 0.12 M NaHC03, and stopped after 15 minutes at 30°C by the addition of 0.6 mL of 1 N HC1.
Glutamate dehydrogenase aminating activity was determined as glutamate formation in a reaction mixture that contained in 1 mL: 85 ftmol Tris-HCl buffer (pH 8.0), 10 /¿mol 2-oxoglutarate, 50 ¿¿mol NH4C1, 0.2 /¿mol NADPH, and enzyme. The reaction was started by adding NH4C1, and stopped after 15 minutes of incubation at 30°C by the addition of 0.6 mL of 1 N HC1.
With each assay described, samples were processed by taking 0.4 mL from the reaction mixture, centrifuging, and diluting an aliquot of the supernate
I r min
I r min
Figure 9.40 Chromatograms of o-phthaldialdehyde, glutamic acid, and glutamine standards. Sample contained 5 nmol of glutamic acid and 10 nmol of glutamine. (From Martin et al„ 1982.)
25-fold with 50 mM potassium phosphate buffer (pH 7.5). For each assay, 50 /aL of diluted sample was mixed with 150 /xL of a derivatizing solution prepared as follows: 27 mg o-phthaldialdehyde were dissolved in a mixture containing 0.5 mL of methanol, 4.5 mL of 0.4 M sodium borate buffer (pH 10.0), and 20 /xL of 2-mercaptoethanol. After a 90-second incubation at 22°C, 40 fiL of sample was injected into the HPLC system.
The sources of enzyme assayed was Synechococcus PCC 6301 and PCC 6803. For in situ assays, cells were permeabilized by mixed alkyltrimethylam-monium bromide or toluene.
Figures 9.42 and 9.43 show chromatograms of enzyme 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 15 minutes, (fl) 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.)
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