Antimycin A-resistant oxygen consumption in rabbit reticulocytes]

In rabbit reticulocytes there exists an Antimycin A-resistent oxygen consumption. It amounts to about 20% of the total oxygen consumption, independently of the degree of maturation of the cells and of the presence of external substrates. The main substrate of the Antimycin A-resistent oxygen consumption is glucose, which is metabolized by the pentose phosphate pathway. NADP-dependent substrates provide more CO2 in the presence of Antimycin A. The 14CO2-formation from metabolites of the citric acid cycle and of metabolites directly connected with this cycle is decreased in the presence of Antimycin A, whereas no 14CO2 is formed from long-chain fatty acids. A H2O2-formation by a NADPH-oxygenase is postulated. The mitochondria contribute reducing equivalents to the cytosolic oxygen consumption. The postulated interactions include hydrogen transfer and the malate-shuttle.     

The specific radioactivity of the tissue free amino acid pool as a basis for measuring the rate of protein synthesis in the rat in vivo

1. Rats were infused in vivo with [U-(14)C]glycine for periods of 2-6h, during which time the specific radioactivity of the free glycine in plasma and tissue approached a constant value. 2. Free serine also became labelled. The ratio of specific radioactivity of serine to that of glycine in the protein of liver, kidney, brain, jejunum, heart, diaphragm and gastrocnemius muscle was closer to the ratio in the free amino acid pool of the tissue than that of the plasma. 3. The kinetics of incorporation of [(14)C]glycine and [(14)C]serine into the protein of gastrocnemius muscle further suggested that the plasma free amino acids were not the immediate precursors of protein. 4. Infusion of rats with [U-(14)C]serine resulted in labelling of free glycine. The ratio of specific radioactivity of glycine to serine in the protein of liver, kidney, brain, jejunum and heart again suggested incorporation from a pool similar to the free amino acid pool of the tissue. 5. Rates of tissue protein synthesis calculated from the incorporation into protein of both radioactive glycine and serine, either infused or derived, were very similar when the precursor specific radioactivity was taken to be that in the total free amino acids of the tissue. Except for gastrocnemius muscle and diaphragm during the infusion of radioactive serine, the rates of tissue protein synthesis calculated from the specific radioactivity of the free glycine and serine in plasma differed markedly.     

Gluconeogenesis from glycine and serine in fasted normal and diabetic rats.

1. Non-anaesthetized normal and diabetic rats were fasted for 1 day, and [U-14C]glycine, or [U-14C]serine, or [U-14C]- plus [3-3H]-glucose was injected intra-arterially. The rates of synthesis de novo/irreversible disposal for glycine, serine and glucose, as well as the contribution of carbon atoms by the amino acids to plasma glucose, were calculated from the integrals of the specific-radioactivity-versus-time curves in plasma. 2. The concentrations of both glycine and serine in blood plasma were lower in diabetic than in fasted normal animals. 3. The rates of synthesis de novo/irreversible disposal of both amino acids tended to be lower in diabetic animals, but the decrease was statistically significant only for serine (14.3 compared with 10.5 mumol/min per kg). 4. Of the carbon atoms of plasma glucose, 2.9% arose from glycine in both fasted normal and diabetic rats, whereas 4.46% of glucose carbon originated from serine in fasted normal and 6.77% in diabetic rats. 5. As judged by their specific radioactivities, plasma serine and glycine exchange carbon atoms rapidly and extensively. 6. It was concluded that the turnover of glycine remains essentially unchanged, whereas that of serine is decreased in diabetic as compared with fasted normal rats. The plasma concentration of both amino acids was lower in diabetic rats. Both glycine and serine are glucogenic. In diabetic rats the contribution of carbon atoms from glycine to glucose increases in direct proportion to the increased glucose turnover, whereas the contribution by serine becomes also proportionally higher.     

Pools and protein synthesis in mammalian cells.

From the kinetics of incorporation into protein shown by four amino acids and one amino acid analogue in suspension cultured HeLa S-3 cells, two distinctly different patterns were observed under the same experimental conditions. An initial slow exponential incorporation followed by linear kinetics was characteristic of the two non-essential amino acids, glycine and proline, whereas the two essential amino acids studied, phenylalanine and leucine, showed linear kinetics of incorporation with no detectable delay. The analogue amino acid, p-fluorophenylalanine also showed immediate linear kinetics of incorporation. There was a poor correlation between the rate of formation of acid-soluble pools and incorporation kinetics. However, the rate of formation of the freely diffusible pool of amino acids correlated more closely with incorporation kinetics. The lack of direct involvement of the acid-soluble pool in protein synthesis was also demonstrated by pre-loading of pools before treatment of cells with labelled amino acids. The results partially support the hypothesis that precursor amino acids for protein synthesis come from the external medium rather than the acid-soluble pool, but suggest that the amino acid which freely diffuses into the cell from the external medium could also be the source.     

The 1986 Borden award lecture. The role of the kidney in amino acid metabolism and nutrition

Measurement of the arteriovenous differences for free amino acids across rat kidney reveals that glycine and citrulline are removed and serine and arginine are added to the circulation. In addition, glutamine is taken up in large quantities by kidneys of animals that need to excrete large quantities of acid (e.g., diabetic animals, NH4Cl-fed animals, and animals fed a high protein diet). Glutamine is the major precursor of urinary ammonia and thus renal glutamine metabolism plays a key role in acid-base homeostasis. This process occurs primarily in the cells of the convoluted proximal tubule. Glutamine carbon is converted to glucose in acidotic rats and is totally oxidized in dogs. Regulation of glutamine metabolism occurs at two levels: acute regulation and chronic regulation. Acute regulation is, in part, mediated through a fall in intracellular [H+]. This activates alpha-ketoglutarate dehydrogenase and, ultimately, glutaminase. Chronic regulation involves induction of key enzymes, including, in the rat, glutaminase, glutamate dehydrogenase, and phosphoenolpyruvate carboxykinase. During the acidosis of prolonged starvation, the kidneys' requirement for glutamine must be met from muscle proteolysis and thus becomes a drain on lean body mass. Serine synthesis occurs by two separate pathways: from glycine by the combined actions of the glycine cleavage enzyme and serine hydroxymethyltransferase and from gluconeogenic precursors using the phosphorylated-intermediate pathway. Both pathways are located in the cells of the proximal tubule. Conversion of glycine to serine is ammoniagenic and the activity of the glycine cleavage enzyme is increased in acidosis. The function of serine synthesis by the phosphorylated-intermediate pathway is not apparent.(ABSTRACT TRUNCATED AT 250 WORDS)     

Serine metabolism in human pregnancy

Serine plays an important role in intermediary metabolism as a source of one carbon pool for nucleotide biosynthesis, as a precursor for glycine and glucose, and as a contributor to cysteine biosynthesis. A unique serine-glycine cycling between the liver and the placenta has been demonstrated in the sheep fetus. We hypothesized that, because of serine's role in growth and development, significant changes in serine metabolism will occur in pregnancy with advancing gestation. The rate of appearance (R(a)) of serine and its metabolism were quantified in healthy women longitudinally through pregnancy with a [2-(15)N(13)C]serine tracer. The contribution of serine N to urea and the rate of oxidation of serine were measured using the precursor-product relation. Plasma serine concentrations and serine R(a) were lower in pregnant (P) women, in both early and late gestation, compared with nonpregnant (NP) women [plasma serine: NP, 113 +/- 24.5; P early, 71.9 +/- 6.2; P late, 68.5 +/- 9.6 micromol/l; serine R(a): NP (n = 7), 152.9 +/- 42.8; P early (n = 12), 123.7 +/- 21.5; P late (n = 8), 102.8 +/- 18.2 micromol x kg(-1) x h(-1)]. Serine contributed approximately 6% to urea N and 15-20% to the plasma glycine pool, and oxidation of serine represented approximately 8% of R(a). There was no significant difference between P and NP subjects. Glucose infusion, at 3 mg x kg(-1) x min(-1) in P subjects, resulted in a decrease in serine R(a) and an increase in oxidation. The decrease in serine turnover in pregnancy may represent a decrease in alpha-amino nitrogen turnover related to a decreased rate of branched-chain amino acid transamination and caused by pregnancy-related hormones aimed at nitrogen conservation and accretion.     

Serine incorporation into the selenocysteine moiety of glutathione peroxidase

The selenium in mammalian glutathione peroxidase is present as a selenocysteine ([Se]Cys) moiety incorporated into the peptide backbone 41-47 residues from the N-terminal end. To study the origin of the skeleton of the [Se]Cys moiety, we perfused isolated rat liver with 14C- or 3H-labeled amino acids for 4 h, purified the GSH peroxidase, derivatized the [Se]Cys in GSH peroxidase to carboxymethylselenocysteine ([Se]Cys(Cm)), and determined the amino acid specific activity. Perfusion with [14C]cystine resulted in [14C]cystine incorporation into GSH peroxidase without labeling [Se]Cys(Cm), indicating that cysteine is not a direct precursor for [Se]Cys. [14C]Serine perfusion labeled serine, glycine (the serine hydroxymethyltransferase product), and [Se]Cys(Cm) in purified GSH peroxidase, whereas [3-3H]serine perfusion only labeled serine and [Se]Cys(Cm), thus demonstrating that the [Se]Cys in GSH peroxidase is derived from serine. The similar specific activities of serine and [Se]Cys(Cm) strongly suggest that the precursor pool of serine used for [Se] Cys synthesis is the same or similar to the serine pool used for acylation of seryl-tRNAs.     

Effect of Type of Carbohydrate on Amino Acid Accumulation and Utilization by Tetrahymena

Both concentration and pattern of free amino acids in Tetrahymena pyriformis, as well as utilization, synthesis, and excretion of amino acids, were affected by type of carbohydrate in growth media. With glucose, cell pool concentrations were higher than with dextrin, and alanine and glycine together accounted for an average of 45% of the total pool. These two amino acids accumulated in similar proportions when their synthesis from essential amino acids was a prerequisite, as when they were provided by growth media, but they constituted only 15 to 18% of the free amino acids in cells from media with dextrin. Alanine, glycine, and glutamic acid were excreted into the medium regardless of whether they were provided by starting media; hence, they appear to be end products of the required metabolism of some of the essential amino acids. Both accumulation and excretion of the above non-essential amino acids were nearly twice as extensive in media providing glucose as the only carbohydrate as when the carbohydrate supplied was dextrin or dextrin plus glucose. Thus, the observed differences are not attributable to differences in osmolarity of monosaccharide and polysaccharide media. Free amino acid differences do not appear to be the immediate cause of the different growth-stimulating properties of such carbohydrates.     

Synthesis of proteins and nucleic acids by the pea aphid Acyrthosiphon pisum (aphididae) in the absence of a full complement of dietary amino acids

Protein, nucleic acids, and nucleotide syntheses were studied in pea aphids, Acyrthosiphon pisum (Harris), by feeding them labeled ¹⁴C-amino acids and [5-³H]-orotic acid in sucrose. It was demonstrated that in the absence of dietary essential amino acids, aphids were capable of synthesizing nucleic acids, nucleotides, and proteins when provided with a single dietary amino acid in sucrose. It is suggested that other required amino acids were possibly supplied by the symbionts present in the pea aphid and/or were obtained from the amino acid pool in the hemolymph or glucose, one of the end products of sucrose digestion. Of the various amino acids tested, synthesis of measurable amounts of protein or other compounds occurred when alanine, aspartic acid, glutamic acid, glycine, proline, or serine were provided, but no synthesis occurred with cysteine.     

Glycine and serine catabolism in non-photosynthetic higher plant cells: their role in C1 metabolism

Glycine and serine are two interconvertible amino acids that play an important role in C1 metabolism. Using 13C NMR and various 13C-labelled substrates, we studied the catabolism of each of these amino acids in non-photosynthetic sycamore cambial cells. On one hand, we observed a rapid glycine catabolism that involved glycine oxidation by the mitochondrial glycine decarboxylase (GDC) system. The methylenetetra- hydrofolate (CH2-THF) produced during this reaction did not equilibrate with the overall CH2-THF pool, but was almost totally recycled by the mitochondrial serine hydroxymethyltransferase (SHMT) for the synthesis of one serine from a second molecule of glycine. Glycine, in contrast to serine, was a poor source of C1 units for the synthesis of methionine. On the other hand, catabolism of serine was about three times lower than catabolism of glycine. Part of this catabolism presumably involved the glycolytic pathway. However, the largest part (about two-thirds) involved serine-to-glycine conversion by cytosolic SHMT, then glycine oxidation by GDC. The availability of cytosolic THF for the initial SHMT reaction is possibly the limiting factor of this catabolic pathway. These data support the view that serine catabolism in plants is essentially connected to C1 metabolism. The glycine formed during this process is rapidly oxidized by the mitochondrial GDC-SHMT enzymatic system, which is therefore required in all plant tissues.     

Disruption of thymidylate synthesis and glycine-serine interconversion by L-methionine and L-homocystine in Raji cells

Excessive concentrations of L-methionine inhibited the folate-dependent de novo synthesis of thymidylic acid (TMP) in Raji cells, demonstrating the usefulness of this cell line for the study of methionine-folate antagonism. The effect was also produced by L-homocystine but not by other amino acids including D-methionine and L-ethionine, suggesting that this effect is exerted by a common intermediate of methionine and homocystine metabolism. L-Methionine, L-homocysteine, S-adenosylmethionine (SAM), and S-adenosylhomocysteine (SAH) are not inhibitors of thymidylate synthase activity. On the other hand the capacity of the cells to incorporate serine 3-carbon and glycine 2-carbon into DNA is impaired by the presence of L-methionine or L-homocystine. Studies with cell-free extracts demonstrated that the glycine cleavage enzyme is inhibited by 45% by L-methionine, L-homocysteine, SAM or SAH. Serine hydroxymethylase on the other hand was slightly stimulated by these sulfur-containing compounds and this stimulation was shown to occur in the intact cell as well. These findings suggest that when levels of L-methionine metabolites are elevated, there is an increase in the use of glycine to maintain the intracellular concentration of serine, which is required for homocysteine detoxification by conversion to cystathionine. The reduction in TMP synthesis caused by excess L-methionine or L-homocystine may result from increased utilization of one-carbon units for serine synthesis.     

Urate synthesis in the perfused chick liver

Urate synthesis was studied in a perfused chicken liver preparation. The perfused liver had an ATP/ADP ratio of 0.29+/-0.05(6) compared with 0.34+/-0.07(10) in liver obtained from chicks under ether anaesthesia. Lactate/pyruvate ratios were 9.4+/-1.7(5) in the perfused liver and 14.8+/-1.8(5) in the rapidly sampled liver. Urate synthesis was only marginally stimulated by glycine, glutamine, aspartic acid or NH(4)Cl, but significant increases were observed with phosphoribosyl pyrophosphate, aminoimidazolecarboxylic acid riboside, inosine, inosinic acid and xanthine. Urate synthesis from glycine, glutamine, NH(4)Cl, asparagine, alanine, histidine and a mixture of 21 amino acids was obtained on inclusion of insulin in the perfusion medium. Evidence for the inclusion of the carbon of histidine into uric acid was obtained. Aspects of the energy consumption associated with the conversion of excess of amino acid into uric acid are considered.     

The role of energy,serine, glycine,and 1-carbon units in the cost of nitrogen excretion in mammals and birds

The efficiency with which dietary protein is used affects the nitrogen excretion by the animal and the environmental impact of animal production. Urea and uric acid are the main nitrogen excretion products resulting from amino acid catabolism in mammals and birds, respectively. Nitrogen excretion can be reduced by using low-protein diets supplemented with free amino acids to ensure that essential amino acids are not limiting performance. However, there are questions whether the capacity to synthesize certain nonessential amino acids is sufficient when low-protein diets are used. This includes glycine, which is used for uric acid synthesis. Nitrogen excretion not only implies a nitrogen and energy loss in the urine, but energy is also required to synthesize the excretion products. The objective of this study was to quantify the energy and metabolic requirements for nitrogen excretion products in the urine. The stoichiometry of reactions to synthesize urea, uric acid, allantoin, and creatinine was established using information from a publicly available database. The energy cost was at least 40.3, 60.7, 64.7, and 65.4 kJ/g excreted N for urea, uric acid, allantoin, and creatinine, respectively, of which 56, 56, 47, and 85% were retained in the excretion product. Data from a broiler study were used to carry out a flux balance analysis for nitrogen, serine, glycine, and so-called 1-carbon units. The flux balance indicated that the glycine intake was insufficient to cover the requirements for growth and uric acid excretion. The serine intake was also insufficient to cover the glycine deficiency, underlining the importance of the de novo synthesis of serine and glycine. One-carbon units are also a component of uric acid and can be synthesized from serine and glycine. There are indications that the de novo synthesis of 1-carbon units may be a “weak link” in metabolism, because of the stoichiometric dependency between the synthesized 1-carbon units and glycine. The capacity to catabolize excess 1-carbon units may be limited, especially in birds fed low-protein diets. Therefore, there may be an upper limit to the 1-carbon-to-glycine requirement ratio in relation to nutrients that supply 1-carbon units and glycine. The ratio can be reduced by increasing uric acid excretion (i.e., reducing protein deposition) or by dietary supplementation with glycine. The hypothesis that the 1-carbon-to-glycine requirement ratio should be lower than the supply ratio provides a plausible explanation for the growth reduction in low-protein diets and the positive response to the dietary glycine supply.     

Studies on the cholesterol synthesis in the human adipose tissue. II. Mechanism of metabolic shifts and regulation of cholesterol through ascorbic acid.

Ratios of yields of cholesterol in different media with different metabolites (200 estimations) show that if the initial normal quantity in a system is X, glucose makes it 4X which is then reduced by the amino acids and ascorbic acid. Alanine, Serine, Threonine, Cysteine, Cystine and Lysine reduce it to 2X. Glycine, Valine, Leucine, Aspartic, Phenylalanine, Tyrosine, Tryptophan roughly increase it to 6X. Other amino acids are intermediate between the above sets. Balanced amino acids and fatty acids keep up the glucose value 4X but the individual amino acids behave as above. Ascorbic acid reduces the quantity of cholesterol everywhere from one third to one half bringing it back to normal X. Since it is not directly involved in the cholesterol synthesis, it must be acting through the pyridine nucleotides. The interesting feature is that the C/H ratio in the amino acids corresponds to the yield of cholesterol with respect to glucose saline yield = 1. Mechanism of metabolic shifts has been discussed on the empirical basis as also on the basis of the change in ratios of NAD+ (NADP+) to NADH2 (NADPH2) through ascorbic acid.     

Chemotaxis by Pseudomonas aeruginosa.

Chemotaxis by Pseudomonas aeruginosa RM46 has been studied, and conditions required for chemotaxis have been defined, by using the Adler capillary assay technique. Several amino acids, organic acids, and glucose were shown to be attractants of varying effectiveness for this organism. Ethylenediaminetetraacetic acid was absolutely required for chemotaxis, and magnesium was also necessary for a maximum response. Serine taxis was greatest when the chemotaxis medium contained 1.5 X 10(-5) M ethylenediaminetetraacetic acid and 0.005 M magnesium chloride. It was not necessary to include methionine in the chemotaxis medium. The strength of the chemotactic responses to glucose and to citrate was dependent on prior growth of the bacteria on glucose and citrate, respectively. Accumulation in response to serine was inhibited by the addition of succinate, citrate, malate, glucose, pyruvate, or methionine to the chemotaxis medium. Inhibition by succinate was not dependent on the concentration of attractant in the capillary. However, the degree to which glucose and citrate inhibited serine taxis was dependent on the carbon source utilized for growth. Further investigation of this inhibition may provide information about the mechanisms of chemotaxis in P. aeruginosa.     

Water Stress Induced Changes in the Amounts of Some Photosynthetic Assimilation Products and Respiratory Metabolites of Sunflower Leaves

Sunflower leaves, water-stressed under controlled conditions,contained greater amounts of amino acids as their water potentialdecreased, with glycine, serine, and glutamate increasing morethan alanine and aspartate. Proline accumulated only at severestress. Low O₂ concentration altered the amounts of amino acids,principally decreasing the amount of glycine and increasingserine. The changes in total pool size are related to previousresults on the accumulation of 14C and the specific activityof products. Photorespiration was large under water stress,where leaves accumulated carbon in glycine of low specific activity,and in 21% O₂, where both total amount and specific activityof glycine was greater than in 1.5% O₂. This suggests that thereare two pools of glycine, one controlled by O₂ and closely relatedto photosynthesis, the other non-photosynthetic and affectedby water stress. The organic acids suocinate, citrate, and fumarate increasedat small leaf-water potentials. Sucrose decreased in amountwith stress and was absent at the most severe stress; therewas less glucose and fructose. The amount of carbon lost fromsugars was similar to the amount accumulated in amino acidstogether with the carbon lost in respiration. It is concluded that stress decreased the flux of carbon fromphotosynthesis for the synthesis of amino acids and sugars butmore carbon from stored materials, principally sucrose, wasused in the production of organic acids and amino acids.     

Acute cold exposure and the metabolism of glucose and some of its precursors in the liver of the fed and fasted sheep.

Measurements of total body oxygen consumption, visceral and hepatic blood flow, oxygen consumption, exchanges of amino acids, lactate, pyruvate and glucose were made on sheep fed 3--6 h or 21 h before the experiment and exposed for 3 h to a neutral environment (15 degrees C) or a cold environment (0.5 to 4 degrees C with clipped coat and wind speed 2 m.s-1). Recent feeding significantly increasedd the total oxygen consumption and the oxygen consumption of the viscera and liver. No general release of amino acids from the viscera or uptake by the liver after feeding was detected although the arterial plasma concentration of essential amino acids did increase significantly after feeding. The plasma concentration of most non-essential amino acids also increased except that of glycine, which decreased significantly. Cold exposure increased the total oxygen consumption and reduced the respiratory quotient significantly. Release of amino acids from the viscera was stimulated by cold exposure. There was a variable increase in the hepatic uptake of lactate and alanine when the sheep were fasted and cold-exposed. The liver's glucose output doubled and the blood (arterial) glucose concentration significantly increased in the cold.     

Correlation between the consumption of amino acids and the production of the antibiotic gallidermin by Staphylococcus gallinarum

The correlation between the consumption of amino acids and the production of the polypeptide antibiotic gallidermin by Staphylococcus gallinarum Tü 3928 was investigated by on-line determination of amino acids and pulse experiments. A prolonged production phase together with an increase in gallidermin formation of about 25% was obtained during pulse and fed-batch experiments with the amino acids glutamic acid, glycine, serine and threonine.     

Uptake of the neutral amino acids glutamine, leucine, and serine by Pneumocystis carinii.

Experiments to elucidate the mechanism by which Pneumocystis carinii transports glutamine, leucine, and serine were performed in this study. Uptake of all three radiolabeled amino acids exhibited first-order, saturation kinetics as extracellular substrate concentrations were increased, thus ruling out simple diffusion and indicating carrier-mediated transport. Kinetic analyses of amino acid uptake and the results of competitive inhibition experiments suggested that leucine, serine, and glutamine were taken up via a common transporter system. The uptake of serine was examined in greater detail to characterize the nature of the carrier. Serine uptake was not affected by N, N'-dicyclohexylcarbodiimide, carbonyl cyanide m-chlorophenyl hydrazone, ouabain, gramicidin, valinomycin, sodium azide, salicylhydroxamine acid (SHAM), iodoacetate, iodoacetate plus SHAM, KCN, and azide. Thus serine uptake did not require sodium or energy from ATP, an electrochemical proton gradient or a membrane potential across the cell surface (i.e., proton-motive force). Serine uptake was dependent on glucose in the extracellular compartment. In the presence of glucose, serine uptake was inhibited by chloramphenicol but not cycloheximide. The results from these experiments are most consistent with facilitated diffusion as the mechanism. After 30 min of incubation, most of the radioactivity was in the cellular soluble fraction. In most cases, incorporation into the extractable total lipids and the remaining particulate cellular components were detectable after this incubation period.