Effects of ammonium, L-glutamate, and L-glutamine on nitrogen catabolism in Aspergillus nidulans

During growth of Aspergillus nidulans in medium containing ammonium the specific activities of most enzymes involved in catabolism of nitrogen sources are low (ammonium repression). The gdhA10 lesion, which results in loss of nicotinamide adenine dinucleotide phosphate-linked glutamate dehydrogenase activity, has been shown to lead to partial relief of ammonium repression of three amidase enzymes as well as histidase. The areA102 lesion led to altered levels of these enzymes but did not greatly affect ammonium repression. The double mutant areA102,gdhA10 was almost completely insensitive to ammonium repression of two of the amidase enzymes and histidase. This suggests that an interaction between the areA and gdhA genes in determining responses to ammonium occurs. Growth of mycelium in medium containing l-glutamate has been found to result in lowered levels of all four enzymes, and this occurs in strains insensitive to ammonium repression. Very strong repression in all strains occurred during growth in medium containing l-glutamine. Relief of these repressive effects of glutamate and glutamine was blocked by cycloheximide. Glutamate and glutamine had similar effects on the production of extracellular protease activity, and growth on glutamine led to low levels of urate oxidase. In contrast to the above enzymes, nitrate reductase was insensitive to the effects of glutamine and glutamate, even though this enzyme is very sensitive to ammonium repression. Although other possibilities exist, it is suggested that there may be mechanisms of general control of nitrogen-catabolic enzymes other than ammonium repression.     

Rapid induction of the intrinsic apoptotic pathway by L-glutamine starvation

While the amino acid L-glutamine is known to play a role in the survival of several cell types, the underlying molecular mechanisms are still poorly defined. We show in this report that L-glutamine starvation rapidly triggered apoptosis in Sp2/0-Ag14 hybridoma cells. This process involved the activation of both caspases-9 and -3, suggesting that L-glutamine deprivation initiated an intrinsic apoptotic pathway in Sp2/0-Ag14 cells. Supporting this idea, the cytosolic release of the mitochondrial proteins SMAC/DIABLO and cytochrome c (Cyt c) was observed, with an initial limited leakage occurring during the first 30 min of L-glutamine deprivation, followed by a greater release after 60 min. The latter occurred simultaneously with the translocation of the pro-apoptotic protein Bax to the mitochondria. Finally, a decline in XIAP levels and the activation of caspases-3 and -9 were observed. Thus, L-glutamine deprivation of Sp2/0-Ag14 cells rapidly triggers intracellular events, which target the mitochondria, leading to the cytosolic release of apoptogenic factors, the activation of caspases-9 and -3, and the commitment to the death program. This work introduces the Sp2/0Ag14 hybridoma as a unique model for the study of the molecular events underlying the pro-survival function of L-glutamine.     

Transamination pathways influencing L-glutamine and L-glutamate oxidation by rat enterocyte mitochondria and the subcellular localization of L-alanine aminotransferase and L-aspartate aminotransferase

Using analytical subcellular fractionation techniques, 12% of the total L-alanine aminotransferase activity and 26% of the total L-aspartate aminotransferase activity was localized in enterocyte mitochondria. Alanine and aspartate were products from the oxidation of glutamine and glutamate by enterocyte mitochondria. At low concentrations, malate stimulated aspartate synthesis but was inhibitory at higher concentrations. The malate inhibition of aspartate synthesis, which increased in the presence of pyruvate, was accompanied by an increase in alanine synthesis. With glutamine as substrate in the presence of pyruvate and malate, alanine synthesis was increased by 127% on addition of purified L-alanine aminotransferase, in spite of large amounts of glutamate generated. It was concluded that when pyruvate is available the important route for glutamine or glutamate oxidation by transamination was via L-alanine:2-oxoglutarate aminotransferase and not via L-aspartate:2-oxoglutarate aminotransferase. Results suggested that mitochondria may account for 50% of alanine production from glutamine in the enterocyte despite the relatively low activity of L-alanine aminotransferase therein.     

Physiological and morphological changes during short term starvation of marine bacterial islates

Three marine bacteria were examined for physiological and morphological changes in the initial phase of starvation. It was found that the starvation process was induced in a similar way irrespective of whether the cells were suspended in nutrient and energy free artificial seawater (NSS) or NSS supplemented with nitrogen and phosphorus. An initial phase of increased activity was consistent with a decreased response to added nutrients. Recovery from starvation exhibited the same response in both these starvation regimes, measured throughout the starvation period. Cells in nitrogen or phosphorus deprived starvation regimes, showed a high and rapid increased activity, followed by a delayed and more pronounced decline in respiratory activity. The initial phase of starvation also included a loss of poly--hydroybutyrate as observed by transmission electron microscopy (TEM). Two bacterial strains showed formation of small vesicles on the outer cell layer when examined by TEM. This formation and release of vesicles was related to the continuous size reduction during starvation survival. The results are discussed in terms of defining the mechanisms of initial cellular responses to nutrient deprivation.Abbreviation NSS nine salt solution     

Changes in erythrocyte 2,3 diphosphoglycerate in women following short term maximal exercise.

15 untrained women were subjected to a walking treadmill test to determine the influence of maximal exercise upon synthesis of erythrocyte 2,3 DPG. Although there was a 9.8% increase in the 2,3 DPG content following exercise, there was a concomitant 9.4% increase in the hemoglobin level; therefore, when 2,3 DPG is expressed as a ratio to hemoglobin (See Article), there was no significant change as a result of exercise stress. It was suggested that three additive factors produced during strenuous exercise; decreased pH; increased hemoglobin concentration; and increased CO2 production result in by-product inhibition of 2,3 DPG synthesis. It is concluded that 2,3 DPG does not provide a physiologic benefit in the adaptation of the oxygen transport system to exercise.     

Metabolic response to short term starvation in non-pregnant and late pregnant cafeteria-obese rats

We have determined the blood metabolite responses to a 24-h starvation period of cafeteria obese rats, in both non-pregnant and late pregnant states. In the fed condition the concentrations of glucose, lactate, pyruvate, glycerol and urea do not differ when compared in control and obese rats, but acetoacetate and 3-hydroxybutyrate levels are higher in the obese group. The overall response of the cafeteria-obese rats to starving seems characterized by decreased rates of glucose and amino-acids utilization, substituted by a more intense utilization of lipid fuels, with excess ketone bodies production and increased utilization of the mobilized glycerol. What we observed in the obese pregnant response to starvation can be summarized as the additional or superimposed effects of excess fat reserves. In the obese pregnant starved rats a less severe hypoglycaemia, lower levels of glycerol (as a consequence of increased utilization), reduced urea levels, and increased acetoacetate and 3-hydroxybutyrate levels were observed. It can be assumed that the pregnant obese rat response to starvation is related to the size of the fat deposits: the more obese, the more hyperketonaemia and less hypoglycaemia, and even diminished rates of amino-acid utilization, as indicated by a lower levels, when compared to the lean pregnant.     

Study on the effect of hypergravity stress on L-glutamate uptake by rat brain nerve endings

Using rat brain synaptosomes, we have investigated the effect of hypergravity on the kinetic parameters of Na(+)-dependent, high-affinity L-glutamate transport activity. The time-course of L-[14C]-glutamate uptake and dependence of L-[14C]-glutamate uptake velocity on glutamate concentrations were analyzed. K(m) and Vmax of this process have been determined. The hypergravity stress was created by centrifugation of rats for 1 hour at 10 g. We observed no differences in K(m) values between the control rats (10.7 +/- 2.5 microM) and animals exposed to hypergravity (6.7 +/- 1.5 microM). The similarity of this parameter for the two studied groups of animals showed that affinity of glutamate transporter to substrate was not sensitive to hypergravity stress. In contrast, the maximal velocity of glutamate uptake changed in hypergravity conditions. Vmax reduced from 12.5 +/- +/- 3.2 nmol/min per 1 mg of protein (control group) to 5.6 +/- 0.9 nmol/min per 1 mg of protein (animals, exposed to hypergravity stress). The possible mechanisms of attenuation of the glutamate transporter activity without modifying K(m) of glutamate uptake were discussed.     

The uptake of L-glutamate by the central nervous system of the cockroach, Periplaneta americana.

A concentrative uptake mechanism for L-glutamate with the following characteristics has been identified in the abdominal nerve cord: 1. The uptake can be divided into Na+-sensitive and Na-plus-insensitive components. 2. The Na-plus-sensitive component showed the typical saturation kinetics of a carrier mediate process. It had a V of 15.9 x 10(6) times 10(6) muM/mg wet weight/min and a Km of 0-33 mm. Its magnitude was proportional to the first power of the Na-plus concentration of the medium. The uptake was specific for L-dicarboxylic amino acids and was sensitive to the presence of metabolic inhibitors. 3. The Na-plus-insensitive component was linearly related to the glutamate concentration of the medium. An isosmotic saline is described for use with the isolated intact abdominal nerve cord of P. americana.     

Regulation of microautophagy and basal protein turnover in rat liver. Effects of short-term starvation

Basal rates of long-lived (resident) protein degradation in rat liver, measured during perfusion after amino acid suppression of macroautophagy, were shown to be strongly regulated by caloric deprivation, decreasing 70% over 48 h in animals fed a high protein diet and 50% in normal controls. Intralysosomal pools of degradable protein correlated directly with basal turnover over this range, yielding a slope (0.09 min-1) that was virtually identical with previous estimates of macroautophagic turnover. The specific radioactivity of valine released from lysosomes in previously labeled livers was the same as that in plasma in both basal and deprivation-induced states. Quantitative electron microscopy revealed a significant decrease with starvation in the absolute volume of a class of secondary lysosome (type A) previously associated with basal or microautophagy. By contrast, the volumes of other microautophagic forms, which comprised roughly 10% of the total, did not change. Taking 0.087 min-1 as the turnover constant of degradable intralysosomal protein and assuming that the concentration of sequestered protein was the same in all vacuoles as that in cytoplasm, we obtained close agreement between predicted and observed rates of basal protein turnover over the range of regulation. The results support the view that the lysosomal system is the final step in the basal degradation of long-lived proteins in the hepatocyte and that a specific class of secondary lysosome (type A) plays a direct role in its regulation during caloric starvation.     

Energy requirement for L-glutamate uptake and utilization by Hansenula subpelliculosa cells

Shieh, K. Z. (Illinois Institute of Technology, Chicago), and L. R. Hedrick. Energy requirement for l-glutamate uptake and utilization by Hansenula subpelliculosa cells. J. Bacteriol. 92:1638-1644. 1966.-Cells of the yeast Hansenula subpelliculosa require an energy source for the uptake of glutamate. A lag period of 20 to 40 min was required after the addition of glucose to the cells before glutamate uptake was initiated. When cells were preincubated in glucose, and washed with distilled water prior to the addition of glutamate, there was no lag period. Preincubation in glucose and glutamate lowered both the rate and the total uptake of glutamate as compared with cells preincubated in glucose alone. This is attributed to the partial utilization of the glucose-metabolite by glutamate or to the partial saturation of binding sites by glutamate during the preincubation period. Transport of glutamate by these yeast cells appears to be via a carrier, where energy is required for the binding of the amino acid to nonspecific binding sites. In addition to total uptake, some aspects of the C(14)-glutamate utilization were measured. Of the total uptake, 58% was metabolized and converted to CO(2), 25.2% remained in the soluble pool, and 16.8% was incorporated into trichloroacetic acid-insoluble products. When the available energy source was depleted, the processes of uptake, metabolism, and incorporation ceased, even though there was an ample supply of glutamate present within the cells. Removal of cells from glutamate and addition of glucose reinitiated the incorporation of glutamate into proteins and other trichloroacetic acid-insoluble compounds. Therefore, an additional energy source is required with this species of yeast for glutamate uptake, for the priming of mechanisms required for its metabolism, and for its incorporation.     

Ferrous-iron-dependent uptake of L-glutamate by a mesophilic,mixotrophic iron-oxidizing bacterium strain OKM-9

Strain OKM-9 is a mesophilic, mixotrophic iron-oxidizing bacterium that absolutely requires ferrous iron as its energy source and L-amino acids (including L-glutamate) as carbon sources for growth. The properties of the L-glutamate transport system were studied with OKM-9 resting cells, plasma membranes, and actively reconstituted proteoliposomes. L-Glutamate uptake into resting cells was totally dependent on ferrous iron that was added to the reaction mixture. Potassium cyanide, an iron oxidase inhibitor, completely inhibited the activity at 1 mM. The optimum pH for Fe2+-dependent uptake activity of L-glutamate was 3.5-4.0. Uptake activity was dependent on the concentration of the L-glutamate. The Km and Vmax for L-glutamate were 0.4 mM and 11.3 nmol x min(-1) x mg(-1), respectively. L-Aspartate, D-aspartate, D-glutamate, and L-cysteine strongly inhibited L-glutamate uptake. L-Aspartate competitively inhibited the activity, and the apparent Ki for this amino acid was 75.9 microM. 2,4-Dinitrophenol, carbonyl cyanide m-chlorophenylhydrazone, gramicidin D, valinomycin, and monensin did not inhibit Fe2+-dependent L-glutamate uptake. The OKM-9 plasma membranes had approximately 40% of the iron-oxidizing activity of the resting cells and approximately 85% of the Fe2+-dependent uptake activity. The glutamate transport system was solubilized from the membranes with 1% n-octyl-beta-D-glucopyranoside and reconstituted into a lecithin liposome. The L-glutamate transport activity of the reconstituted proteoliposomes was 8-fold than that of the resting cells. The Fe2+-dependent L-glutamate uptake observed here seems to explain the mixotrophic nature of this strain, which absolutely requires Fe2+ oxidation when using amino acids as carbon sources.