Ammonia assimilation in Bacillus polymyxa. 15N NMR and enzymatic studies

Pathways of ammonia assimilation into glutamic acid and alanine in Bacillus polymyxa were investigated by 15N NMR spectroscopy in combination with measurements of the specific activities of glutamate dehydrogenase, glutamine synthetase, glutamate synthetase, alanine dehydrogenase, and glutamic-alanine transaminase. Ammonia was found to be assimilated into glutamic acid predominantly by NADPH-dependent glutamate dehydrogenase with a Km of 2.9 mM for NH4+ not only in ammonia-grown cells but also in nitrate-grown and nitrogen-fixing cells in which the intracellular NH4+ concentrations were 11.2, 1.04, and 1.5 mM, respectively. In ammonia-grown cells, the specific activity of alanine dehydrogenase was higher than that of glutamic-alanine transaminase, but the glutamate dehydrogenase/glutamic-alanine transaminase pathway was found to be the major pathway of 15NH4+ assimilation into [15N]alanine. The in vitro specific activities of glutamate dehydrogenase and glutamine synthetase, which represent the rates of synthesis of glutamic acid and glutamine, respectively, in the presence of enzyme-saturating concentrations of substrates and coenzymes are compared with the in vivo rates of biosynthesis of [15N]glutamic acid and [alpha,gamma-15N]glutamine observed by NMR, and implications of the results for factors limiting the rates of their biosynthesis in ammonia- and nitrate-grown cells are discussed

Glutamate biosynthesis in Bacillus azotofixans. 15N NMR and enzymatic studies

Pathways of ammonia assimilation into glutamic acid in Bacillus azotofixans, a recently characterized nitrogen-fixing species of Bacillus, were investigated through observation by NMR spectroscopy of in vivo incorporation of 15N into glutamine and glutamic acid in the absence and presence of inhibitors of ammonia-assimilating enzymes, in combination with measurements of the specific activities of glutamate dehydrogenase, glutamine synthetase, glutamate synthase, and alanine dehydrogenase. In ammonia-grown cells, both the glutamine synthetase/glutamate synthase and the glutamate dehydrogenase pathways contribute to the assimilation of ammonia into glutamic acid. In nitrate-grown and nitrogen-fixing cells, the glutamine synthetase/glutamate synthase pathway was found to be predominant. NADPH-dependent glutamate dehydrogenase activity was detectable at low levels only in ammonia-grown and glutamate-grown cells. Thus, B. azotofixans differs from Bacillus polymyxa and Bacillus macerans, but resembles other N2-fixing prokaryotes studied previously, as to the pathway of ammonia assimilation during ammonia limitation. Implications of the results for an emerging pattern of ammonia assimilation by alternative pathways among nitrogen-fixing prokaryotes are discussed, as well as the utility of 15N NMR for measuring in vivo glutamate synthase activity in the cell

Effect of the nitrogen source on glutamine and alanine biosynthesis in Neurospora crassa. An in vivo 15N nuclear magnetic resonance study

The influences of different nitrogen sources on the relative rates of biosynthesis of glutamine and alanine have been studied by 15N nuclear magnetic resonance spectroscopy of intact Neurospora crassa mycelia suspensions. The rate of glutamine synthesis was fastest after growth in media deficient in free ammonium ion, whereas it was slowest following growth in media containing both glutamic acid and glutamine. The reverse trend was observed for the biosynthesis of alanine. A competition between the two biosynthetic pathways for the same substrate, glutamic acid, was found to limit the rate of alanine synthesis when glutamine synthesis was rapid. The observed in vivo rates of these reactions are compared to the reported specific activities of the enzymes catalyzing the reactions, and implications of these results for nitrogen regulation of these pathways under various physiological conditions are discussed

β-Catenin asymmetry is regulated by PLA1 and retrograde traffic in C. elegans stem cell divisions

Asymmetric division is an important property of stem cells. In Caenorhabditis elegans, the Wnt/β-catenin asymmetry pathway determines the polarity of most asymmetric divisions. The Wnt signalling components such as β-catenin localize asymmetrically to the cortex of mother cells to produce two distinct daughter cells. However, the molecular mechanism to polarize them remains to be elucidated. Here, we demonstrate that intracellular phospholipase A1 (PLA1), a poorly characterized lipid-metabolizing enzyme, controls the subcellular localizations of β-catenin in the terminal asymmetric divisions of epithelial stem cells (seam cells). In mutants of ipla-1, a single C. elegans PLA1 gene, cortical β-catenin is delocalized and the asymmetry of cell-fate specification is disrupted in the asymmetric divisions. ipla-1 mutant phenotypes are rescued by expression of ipla-1 in seam cells in a catalytic activity-dependent manner. Furthermore, our genetic screen utilizing ipla-1 mutants reveals that reduction of endosome-to-Golgi retrograde transport in seam cells restores normal subcellular localization of β-catenin to ipla-1 mutants. We propose that membrane trafficking regulated by ipla-1 provides a mechanism to control the cortical asymmetry of β-catenin

Faster flux of neurotransmitter glutamate during seizure — Evidence from ¹³C-enrichment of extracellular glutamate in kainate rat model

<div><p>The objective is to examine how the flux of neurotransmitter glutamate from neurons to the extracellular fluid, as measured by the rate of ¹³C enrichment of extracellular glutamate (GLU_ECF), changes in response to seizures in the kainate-induced rat model of temporal-lobe epilepsy. Following unilateral intrahippocampal injection of kainate, GLU_ECF was collected by microdialysis from the CA1/CA3 region of awake rats, in combination with EEG recording of chronic-phase recurrent seizures and intravenous infusion of [2,5-¹³C]glucose. The ¹³C enrichment of GLU_ECF C5 at ~ 10 picomol level was measured by gas-chromatography mass-spectrometry. The rate of ¹³C enrichment, expressed as the increase of the fractional enrichment/min, was 0.0029 ± 0.0001/min in frequently seizing rats (<i>n</i> = 4); this was significantly higher (<i>p</i> < 0.01) than in the control (0.00167 ± 0.0001/min; <i>n</i> = 6) or in rats with infrequent seizures (0.00172 ± 0.0001/min; <i>n</i> = 6). This result strongly suggests that the <i>flux</i> of the excitatory neurotransmitter from neurons to the extracellular fluid is significantly increased by frequent seizures. The extracellular [¹²C + ¹³C]glutamate <i>concentration</i> increased progressively in frequently seizing rats. Taken together, these results strongly suggest that the observed seizure-induced high flux of glutamate overstimulated glutamate receptors, which triggered a chain reaction of excitation in the CA3 recurrent glutamatergic networks. The rate of ¹³C enrichment of extracellular glutamine (GLN_ECF) at C5 was 0.00299 ± 0.00027/min in frequently seizing rats, which was higher (<i>p</i> < 0.05) than in controls (0.00227 ± 0.00008/min). For the first time in vivo, this study examined the effects of epileptic seizures on <i>fluxes</i> of the neurotransmitter glutamate and its precursor glutamine in the <i>extracellular fluid</i> of the hippocampus. The advantages, limitations and the potential for improvement of this approach for pre-clinical and clinical studies of temporal-lobe epilepsy are discussed.</p></div

EEG recording of a chronic-phase seizure in a freely behaving KA rat.

<p><b>(A)</b><i>Top</i>: The kainate-injected ipsilateral hippocampus shows a quiescent period with a single inter-ictal spike (IIS) followed by a seizure (enclosed in box). <i>Bottom</i>: The contralateral hippocampus was seizure-free. <b>(B)</b><i>Top</i>: Expanded plot of the seizure with a time-scale of 10 s. The inset shows an expanded plot of the peak in the box with a time scale of 10 ms; the wave pattern is characteristic of a hypersynchronous population burst from glutamatergic neurons. <i>Bottom</i>: Corresponding recording from the seizure-free contralateral hippocampus.</p

The time course of total [¹²C + ¹³C]GLU_ECF.

<p>Total GLU_ECF showed little changes in the control (open square) or in infrequently seizing rats (black diamond), but in frequently seizing rats (black square) showed gradual elevation between <i>t</i> = 80–120 min and a significant elevation between <i>t</i> = 125–167 min (Fig 4). See text for the explanation of the significant differences at <i>t</i> = 97, 117, 144 and 167 min. Likely explanations for the observed multi-phase increases in the frequently seizing KA rats are described in Discussion.</p

Progressive ¹³C enrichment of GLU_ECF during i.v. infusion of [2,5-¹³C]glucose, as observed by GCMS.

<p>The top left panel shows a GC chromatogram of 10 picomol of <i>t</i>BDMS-GLU. On the right are shown the structures of <i>t</i>BDMS-GLU and of the fragment ions m/z 432 and m/z 330. The loss of another <i>t</i>-butyl group from the latter gives rise to the m/z 272 ion. The bottom panel shows representative mass spectra of the ion pair m/z 330/331 of the <i>t</i>BDMS derivative of GLU_ECF (which contain C2-C5 of GLU_ECF) collected from the ipsilateral hippocampus of an epileptic rat (R1125 from group III) at the indicated time points during the i.v. infusion of [2,5-¹³C]glucose. A progressive increase in ¹³C enrichment was observed from the increase in the peak area of m/z 331 ion relative to that of the m/z 330 ion, shown by the arrows.</p

Rates of ¹³C enrichments of GLU_ECF C5 and GLN_ECF C5 during [2,5-¹³C]glucose infusion in control and KA rats undergoing infrequent or frequent seizures.

<p>Rates of ¹³C enrichments of GLU_ECF C5 and GLN_ECF C5 during [2,5-¹³C]glucose infusion in control and KA rats undergoing infrequent or frequent seizures.</p