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Fig. 6. Changes in the accumulation of glycogen, glutamate and ammonia. (A) Natural-abundance proton-decoupled ¹³C-NMR spectra (125.13 MHz, 25°C, pH 7.2) of perchloric acid extracts from wild-type (WT) and mutant (KO) cells grown to late exponential phase. Resonances from carbons C1 (100.6 ppm) and C4 (77.8 ppm) of glycogen (Gly 1:4) and carbons C3 (27.7 ppm), C4 (34.2 ppm) and C5 (181.9 ppm) of glutamate (Glu) are shown in representative spectra of wild-type (left) and mutant cells (right). Arrows highlight the disappearance of glycogen and glutamate resonances. (B) Similar amount of wild-type and mutant cells were washed and deposited on nitrocellulose filters over cellulose pads. After 5 hours of starvation at 22°C, cells were taken off the filter for protein quantification, and the ammonia released to the underneath cellulose pad was determined by a colorimetric assay; ammonia concentration (mM) normalized to the number of cells (left panel) and total protein content (right panel). (C) Wild-type and mutant cells were deposited on filters for development with one or two PDF-soaked cellulose pads. Photographs were taken at the indicated times. When one cellulose pad was used under the filter, the wild-type cells culminated normally, whereas the mutant remained at the slug stage. This delay in culmination was rescued in midA^- when two cellulose pads were used underneath the filter to dilute the secreted ammonia. Bar, 0.5 mm.

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