13N isotope studies of glutamine assimilation pathways in Neurospora crassa

Abstract

L-[amide-'3N]glutamine in Neurospora crassa is metabolized to [13N]glutamate by glutamate synthase and to [13N]ammonium by the glutamine transamina-w-amidase pathway. The [13Njammonium relased is assimilated by glutamate dehydrogenase and glutamine synthetase, confirming the operation of a glutamine cycle. Most of the nitrogen is retained during cycling between glutamate and glutamine.In Neurospora crassa, glutamine, in addition to participating in transamidation reactions, is converted to 2-oxoglutarate and ammonium by the enzymes of the glutamine transaminase-w-amidase pathway, in which a transaminase catalyzes transfer of amino nitrogen of glutamine to various oxo acids. 2-Oxoglutaramate thus produced is hydrolyzed by w-amidase to 2-oxoglutarate and ammonium ion (2).The amine nitrogen of glutamine is also assimilated by the action of glutamate synthase (glutamine oxoglutarate amidotransferase [GOGAT]), which synthesizes two molecules of glutamate from glutamine and 2-oxoglutarate (1). From the results obtained with several inhibitors and mutants, Calder6n and Mora concluded that the ammonium ion released by the w-amidase pathway is assimilated by glutamate dehydrogenase (GDH) and by glutamine synthetase (1, 2). These enzymes, when combined with the glutamine transaminase and w-amidase, yield a glutamine cycle in which glutamine is continually degraded and resynthesized (1). We report here the data obtained by using L-[amide-"3N]glutamine as a tracer to study the metabolic pathways of glutamine assimilation in N. crassa. 13N, the longest-lived radioisotope of nitrogen (t112 = 9.96 min), was used to label ammonia (7). L-[amide-'3N]glutamine was prepared from [13N]ammonia by using glutamine synthetase immobilized onto CNBr-activated Sepharose as previously described (16).The N. crassa cultures were grown for 8 h at 30°C on Vogel minimal medium (17) containing 1.5% sucrose and 25 mM NH4NO3 as the nitrogen source. Six milliliters of this medium was centrifuged, and after decantation, 50 ,ul of L-[amide-13N]glutamine (1 to 5 mCi [50 to 100 ,uM]) was added to the cell suspension. Uptake of label was stopped by adding 2 ml of water, and after centrifugation and a further wash, the metabolites were extracted with 0.2 ml of ice-cold 1% (wt/vol) picric acid. The '3N-labeled metabolites were analyzed by using a high-pressure liquid chromatography system (Series 4; The Perkin-Elmer Corp., Norwalk, Conn.) in which the effluent was continuously monitored with a Ramona D flowthrough y detector (5).A Partisil 10 SCX 10 ,uM (4.6 by 250 mm) (Phenomenex, Rancho Palo Verdes, Calif.) analytical cation-exchange column was used for separation; the elution conditions were as reported previously (5). In some cases glutamate was mea-* Corresponding author. sured by high-pressure liquid chromatography of the ophthaldialdehyde derivates (10), and ammonia content was determined enzymatically with glutamate dehydrogenase (4).The relative radioactivity incorporated into the a-amino and amide groups of glutamine was determined as follows. A...

Isolation and characterization of a Neurospora crassa mutant altered in the alpha polypeptide of glutamine synthetase

“…This metabolic adjustment in reducing the cycling of glutamine results in similar growth of the parental and the a-me-MSO-resistant mutant strains in low ammonia. These data point out that an excess of GS activity ensures a higher glutamine cycling, something that allows for a wider range adjustment in the glutamate/glutamine ratio, which is also in accordance with the proposal that the function of GOGAT in N. crassa is to recycle glutamine to glutamate (1,3,15).…”

“…It has been found that GS is involved in glutamine cycling, in which this amino acid is continually assimilated by GOGAT, degraded by the glutamine transaminase-wamidase pathway, and resynthesized (1)(2)(3)(4). It has been proposed that this cycle is necessary for carbon utilization (11).…”

Glutamine and glutamate metabolism in normal and heat shock conditions in Drosophila Kc cells: Conditions supporting glutamine synthesis maximize heat shock polypeptide expression

“…1) is consistent with, but does not prove, the possibility that glutamine is the source of this NH,. Glutaminase activity has been shown to be present (Chase and Kankel, 19871, but other routes of glutamine breakdown or cycling which do not release free NH, exist in other systems (see e.g., Calderon et al, 1989;Haussinger et al, 1985) and remain uninvestigated possibilities.…”

Glutamine metabolism and cycling in Neurospora crassa.

“…glutamine have demonstrated that the glutamine cycle operates during growth of wild-type N. crassa and have given information about the rate and amount of nitrogen cycling (6). After the addition of L-[amide-13N]glutamine to the wild-type strain, glutamate was more highly labeled than other nitrogen-containing metabolites.…”