Neuronal Glutamine Utilization: Glutamine/Glutamate Homeostasis in Synaptosomes

The synaptosomal metabolism of glutamine was studied under in vitro conditions that simulate depolarization in vivo. With [2-15N]glutamine as precursor, the [glutamine]i was diminished in the presence of veratridine or 50 mM KCl, but the total amounts of [15N]glutamate and [15N]aspartate formed were either equal to those of control incubations (veratridine) or higher (50 mM [KCl]). This suggests that depolarization decreases glutamine uptake and independently augments glutaminase activity. Omission of sodium from the medium was associated with low internal levels of glutamine which indicates that influx occurs as a charged Na(+)-amino acid complex. It is postulated that a reduction in membrane potential and a collapse of the Na+ gradient decrease the driving forces for glutamine accumulation and thus inhibit its uptake and enhance its release under depolarizing conditions. Inorganic phosphate stimulated glutaminase activity, particularly in the presence of calcium. At 2 mM or lower [phosphate] in the medium, calcium inhibited glutamine utilization and the production of glutamate, aspartate, and ammonia from glutamine. At a high (10 mM) medium [phosphate], calcium stimulated glutamine catabolism. It is suggested that a veratridine-induced increase in intrasynaptosomal inorganic phosphate is responsible for the enhancement of flux through glutaminase; calcium affects glutaminase indirectly by modulating the level of free intramitochondrial [phosphate]. Because phosphate also lowers the Km of glutaminase for glutamine, augmentation of the amino acid breakdown may occur even when depolarization lowers [glutamine]i. Reducing the intrasynaptosomal glutamate to 26 nmol/mg of protein had little effect on glutamine catabolism, but raising the pH to 7.9 markedly increased formation of glutamate and aspartate. It is concluded that phosphate and H+ are the major physiologic regulators of glutaminase activity.     

Studies of Sulfate Utilization by Algae. 7. In vivo Metabolism of Thiosulfate by Chlorella

Chlorella pyrenoidosa Chick (Emerson strain 3) utilizes thiosulfate for growth as effectively as sulfate, and more effectively than a variety of organic sulfur compounds containing sulfur in various oxidation states. Thiosulfates, differentially labeled with ³⁵S in either the SH— or SO₃ — sulfur moieties, were used to follow the incorporation of thiosulfate-sulfur into constituents of the insoluble fraction and of the soluble pools. Labeled sulfate was also used for purposes of comparison. Label from both sulfur atoms of thiosulfate and from sulfate is incorporated into the cysteine, homocysteine, and glutathione of the soluble pools, and into the methionine and cystine of protein in the insoluble fraction. Label from SO₃-sulfur of thiosulfate is incorporated more slowly into protein methionine and cystine than label from the SH-sulfur. Moreover, the SO₃-sulfur of thiosulfate is recovered largely as sulfate in both the soluble pools and the insoluble fraction, while only a trace of SH-sulfur is recovered as sulfate in either case. Consistent with this, the metabolism of the SO₃-sulfur of thiosulfate more closely resembles the metabolism of sulfate. Thus it would appear that exogenous thiosulfate undergoes early dismutation in which the SO₃-sulfur is preferentially oxidized, and the SH-sulfur is preferentially incorporated in a reduced state. These results are discussed in relation to the conversion of sulfate to thiosulfate by cell-free extracts of Chlorella previously described.     

Neuronal Glutamine Utilization: Pathways of Nitrogen Transfer tudied with [15N]Glutamine

Gas chromatography-mass spectrometry was used to evaluate the metabolism of [15N]glutamine in isolated rat brain synaptosomes. In the presence of 0.5 mM glutamine, synaptosomes accumulated this amino acid to a level of 25-35 nmol/mg protein at an initial rate greater than 9 nmol/min/mg of protein. The metabolism of [15N]glutamine generated 15N-labelled glutamate, aspartate, and gamma-aminobutyric acid (GABA). An efflux of both [15N]glutamate and [15N]aspartate from synaptosomes to the medium was observed. Enrichment of 15N in alanine could not be detected because of a limited pool size. Elimination of glucose from the incubation medium substantially increased the rate and amount of [15N]aspartate formed. It is concluded that: (1) With 0.5 mM external glutamine, the glutaminase reaction, and not glutamine transport, determines the rate of metabolism of this amino acid. (2) The primary route of glutamine catabolism involves aspartate aminotransferase which generates 2-oxoglutarate, a substrate for the tricarboxylic acid cycle. This reaction is greatly accelerated by the omission of glucose. (3) Glutamine has preferred access to a population of synaptosomes or to a synaptosomal compartment that generates GABA. (4) Synaptosomes maintain a constant internal level of glutamate plus aspartate of about 70-80 nmol/mg protein. As these amino acids are produced from glutamine in excess of this value, they are released into the medium. Hence synaptosomal glutamine and glutamate metabolism are tightly regulated in an interrelated manner.     

Extracellular Alkaline Phosphatase in Multicellular Marine Algae and Their Utilization of Glycerophosphate

The production of extracellular alkaline phosphatase by multicellular marine algae in axenic culture has been investigated. The algae studied were five species of Rhodophyta: Asterocytis ramosa, Goniotrichum elegans, Nemalion helminthoides, Polysiphonia urceolata and Rhodosorus marinus; and one species of Phaeophyta: Ecrocarpus confervoides. The extent of enzyme activity varies from one species to another. It also varies with the phosphorus conditions under which the alga is grown. The pattern of glycerophosphate utilization suggests that this type of compound is not taken up directly by the alga but split by the external enzyme before uptake of the phosphate-ion only. The enzyme performs its action outside the organism and appears both associated with the cells and free in the surrounding water. Assays with culture filtrate of Asterocytis and Ectocarpus show that the enzyme is an unspecific phosphomonoesterase with optimum activity far to the alkaline side. It is activated by Zn²⁺.     

Volatilization of Mercury by Algae

The presence of Chlorella cells in a medium containing HgCl₂ causes a rapid decrease of the mercury content in the algal suspension. The rate of decrease depends on the inoculum concentration. The presence of Hg in the medium induces a lag in the growth, whose length depends on the initial concentration of Hg and of the inoculum. Binding and/or uptake of mercury by the cells is not dependent on temperature. The mercury content per cell declines somewhat at the time at which the culture resumes growth at a rate similar to that of the controls without mercury.     

Glutamine utilization by Rhizobium etli

We undertook the study of the use of glutamine (Gln) as the source of carbon and energy by Rhizobium etli. Tn5-induced mutagenesis allowed us to identify several genes required for Gln utilization, including those coding for two broad-range amino acid transporters and a glutamate dehydrogenase. The isolated mutants were characterized by the analysis of their capacity i) to grow on different media, ii) to transport Gln (uptake assays), and iii) to utilize Gln as the C energy source (CO2 production from Gln). We show that Gln is degraded through the citric acid cycle and that its utilization as the sole C source is related to a change in the bacterial cell shape (from bacillary to coccoid form) and a high susceptibility to a thiol oxidative insult. Both these data and the analysis of ntr-dependent promoters suggested that Gln-grown bacteria are under a condition of C starvation and N sufficiency, and as expected, the addition of glucose counteracted the morphological change and increased both the bacterial growth rate and their resistance to oxidative stress. Finally, a nodulation analysis indicates that the genes involved in Gln transport and degradation are dispensable for the bacterial ability to induce and invade developing nodules, whereas those involved in gluconeogenesis and nucleotide biosynthesis are strictly required.     

The Adaptation of Plankton Algae

The variation in Skeletonema cells grown at 3 klux continuous illumination and 20°C is reported. Four different types of lamps gave no difference in the photosynthetic characteristics. The average diameter of the cells decreased from 8–3.5 μ during their six months vegetative period. The ratio between the pigment content in the largest and the smallest cells was about 2:1. A good correlation between cell volume and chlorophyll a content was found for this species. The content of chlorophyll c generally varied between 4 and 17 per cent of the chlorophyll a content. — A distinct correlation between the chlorophyll a content and the rate of photosynthesis per unit of cells at low light intensity was found. The rate of photosynthesis, in mg C per mg chlorophyll a and hour at 1 klux, varied between 0.40 and 0.70 for all 60 experiments with an average value of 0.56. The corresponding value for cells deficient in phosophorus was 0.19 and for cells deficient in nitrogen 0.09. — The material also showed a good correlation between the rate of photosynthesis per cell at 1 klux and the light-saturated rate of photosynthesis. I_k varied between 7 and 13 klux.     

The Adaptation of Plankton Algae

The various aspects of the adaptation of plankton algae lo light and temperature are discussed. The shape of a light intensity-photosynthesis curve is shown to be an important means of describing the physiological adjustment of an algal population. If the algae are not exposed to adverse influences such as poisons, pronounced nutrient deficiency or light shocks, the rate of real photosynthesis per mg chlorophyll a at 1 Klux (incandescent light) should be about 0.4–0.6 mg C/hour. Hence this rate presents an excellent means of judging the quality of experiments. Experiments are presented where Chlorella pyrenoidosa was adapted to light intensities between 0.32 klux and 21 Klux. This alga adapts to different light intensities by varying the amount of pigments per cell. Algae grown at 1 Klux have about 10 times more chlorophyll per cell than those grown at 21 klux. Other species of algae—but by no means all—are shown to behave in the same way. The problem of algal resistance to photo-oxidation at high light intensities is discussed. Adaplation is shown to he one of the mechanisms which make the algae resistent. “Chlorophyll inactivation” is another. Experiments with the diatom Skeletonema costatum concerning adaptation to different temperatures have been performed. The fact that the alga has essentially the same rate of photosynthesis per cell at all light intensities at 20°C and 7°C, may be attributed to an increase of all the enzymes at the low temperature. The amount of protein per cell was twice as high at 7°C as at 20°C.     

The Adaptation of Plankton Algae

A survey of the adaptation of plankton algae in Nature to different light intensities and temperatures is given. The adaptation is always important for the plankton populations at all depths. However, the shade adaptation of the algae in the lower part of the photic zone is often of relatively little importance for the integral primary production per unit of surface, except on the by no means rare occasions when the bulk of the algae is found in this lower part. It is shown that the I_k of surface plankton during summer in the Arctic is high despite the low temperatures present. This is due to an increase of the enzyme quantities per cell. Daily fluctuations in I_k are due partly to photooxidation, partly to the fact that the periods for the production of chlorophyll and photosynthetic enzymes are mutually displaced. Despite the daily fluctuations in I_k the difference between “sun” pbytoplankton and “shade” pbytoplankton is distinct.