Alanine Aminotransferase and Aspartate Aminotransferase in Leishmania tarentolae1

SYNOPSIS. Culture stages (promastigotes) of Leishmania tarentolae were tested for alanine aminotransferase (E.C.2.6.1.2) and aspartate aminotransferase (E.C.2.6.1.1.). Neither enzyme was detected in crude cell extracts. After starch block electrophoresis, however, both transaminase activities were found in proteins migrating toward the anode. Only one species of each enzyme was found. Using coupled enzyme assay systems, the following physical and kinetic properties were seen: 1) aspartate aminotransferase was inhibited by α-ketoglutarate concentrations above 1.68 × 10^?2 M and alanine aminotransferase was inhibited by concentrations higher than 1.34 × 10^?2 M; 2) the Michaelis constant (Km[α-ketoglutarate]) was 5.4 × 10^?4 M for aspartate aminotransferase and 3.0 × 10^?4 M for alanine aminotransferase; 3) maximum activity was found at ?pH 8.5 (broad range between pH 7.75–9.0) for aspartate aminotransferase whereas maximum activity for alanine aminotransferase was ?pH 7.2 (range between pH 7.0–7.5); 4) both enzymes lost half of their activity after 4 days at 8 C; 5) aspartate aminotransferase was most active at 35 C and completely inactivated at 59.5 C, alanine aminotransferase exhibited maximum activity at 29.5 C and was completely inactivated at 61 C; and 6) neither enzyme showed enhanced activity with added pyridoxal phosphate.     

The effect of cosubstrates on tricarboxylic acid cycle dynamics during pyruvate oxidation: the formation of alpha-ketoglutarate and utilization of glutamate by mitochondria from rabbit brain.

—Data comparing tricarboxylic acid cycle dynamics in mitochondria from rabbit brain using [2- or 3-¹⁴C]pyruvate with and without cosubstrates (malate, α-ketoglutarate, glutamate) are reported. With a physiological concentration of an unlabelled cosubstrate, from 90-99% of the isotope remained in cycle intermediates. However, the liberation of ¹⁴CO₂ and the presence of ¹⁴C in the C-1 position of α-ketoglutarate indicated that multiple turns of the cycle occurred. Entry of pyruvate into the cycle was greater with malate than with either α-ketoglutarate or glutamate as cosubstrate. With malate as cosubstrate for [¹⁴C]pyruvate the amount of [¹⁴C]citrate which accumulated averaged 30nmol/ml or 23% of the pyruvate utilized while α-ketoglutarate averaged 45 nmol/ml or 35% of the pyruvate utilized. With α-ketoglutarate as cosubstrate for [¹⁴C]pyruvate, the average amount of [¹⁴C]citrate which accumulated decreased to 8 nmol/ml or 10% of the pyruvate utilized while [¹⁴C]α-ketoglutarate increased slightly to 52 nmol/ml or an increase to 62%, largely due to a decrease in pyruvate utilization. The percentage of ¹⁴C found in α-ketoglutarate was always greater than that found in malate, irrespective of whether α-ketoglutarate or malate was the cosubstrate for either [2- or 3-¹⁴C]pyruvate. The fraction of ¹⁴CO₂ produced was slightly greater with α-ketoglutarate as cosubstrate than with malate. This observation and the fact that malate had a higher specific activity than did α-ketoglutarate when α-ketoglutarate was the cosubstrate, indicated a preferential utilization of α-ketoglutarate formed within the mitochondria. When l -glutamate was a cosubstrate for [¹⁴C]pyruvate the principal radioactive product was glutamate, formed by isotopic exchange of glutamate with [¹⁴C] α-ketoglutarate. If malate was also added, [¹⁴C]citrate accumulated although pyruvate entry did not increase. Due to retention of isotope in glutamate, little [¹⁴C]succinate, malate or aspartate accumulated. When [U-¹⁴C]l -glutamate was used in conjunction with unlabelled pyruvate more ¹⁴C entered the cycle than when unlabelled glutamate was used with [¹⁴C]pyruvate and led to α-ketoglutarate, succinate and aspartate as the major isotopic products. When in addition, unlabelled malate was added, total and isotopic α-ketoglutarate increased while [¹⁴C]aspartate decreased. The increase in [¹⁴C]succinate when [¹⁴C] glutamate was used indicated an increase in the flux through α-ketoglutarate dehydrogenase and was accompanied by a decrease of pyruvate utilization as compared to experiments when either α-ketoglutarate or glutamate were present at low concentration. It is concluded that the tricarboxylic acid cycle in brain mitochondria operates in at least three open segments, (1) pyruvate plus malate (oxaloacetate) to citrate; (2) citrate to α-ketoglutarate and; (3) α-ketoglutarate to malate, and that at any given time, the relative rates of these segments depend upon the substrate composition of the environment of the mitochondria. These data suggest an approach to a steady state consistent with the kinetic properties of the tricarboxylic acid cycle within the mitochondria.     

THE EFFECT OF HYPERTHERMIA UPON OXYGEN CONSUMPTION AND UPON ORGANIC PHOSPHATES, GLYCOLYTIC METABOLITES, CITRIC ACID CYCLE INTERMEDIATES AND ASSOCIATED AMINO ACIDS IN RAT CEREBRAL CORTEX

The influence of hyperthermia on cerebral blood flow, cerebral metabolic rate for oxygen and cerebral metabolite levels was studied by increasing body temperature from 37° to 40°C and 42°C in rats under nitrous oxide anaesthesia maintained at constant arterial CO₂ tension. The metabolic rate for oxygen increased by 5-6% per degree centigrade. At 42°C the increase in cerebral blood Row was comparable to that in the metabolic rate. The increased temperatures were not accompanied by changes in organic phosphates (phosphocreatine, ATP, ADP or AMP) or in lactate/pyruvate ratio. There was an increase in the tissue to blood glucose concentration ratio. At steady state, there was an increase in glucose-6-phosphate but no other changes in glycolytic metabolites or citric acid cycle intermediates, and the only change in amino acids studied (glutamate, glutamine, aspartate, alanine and GABA) was an increase in glutamate concentration.     

CEREBRAL METABOLIC CHANGES IN PROFOUND, INSULIN-INDUCED HYPOGLYCEMIA, AND IN THE RECOVERY PERIOD FOLLOWING GLUCOSE ADMINISTRATION

Severe hypoglycemia was induced by insulin in lightly anaesthetized (70°_o N₂O) and artificially ventilated rats. Brain tissue was frozen in situ after spontaneous EEG potentials had disappeared for 5. 10. 15 or 30 min and cerebral cortex concentrations of labile organic phosphates, glycolytic metabolites, ammonia and amino acids were determined. In other experiments, recovery was induced by glucose injection at the end of the period of EEG silence. All animals with an isoelectric EEG showed extensive deterioration of the cerebral energy state. and gross perturbation of amino acid concentrations. The latter included a 4-fold rise in aspartate concentration and reductions in glutamate and glutamine concentrations to 20 and 5^o_o of control levels respectively. There was an associated rise in ammonia concentration to about 3μmol-g^-1. Administration of glucose brought about extensive recovery of cerebral energy metabolism. For example, after an isoelectric period of 30 min tissue concentrations of phosphocreatine returned to or above normal, the accumulation of ADP and AMP was reversed, there was extensive resynthesis of glycogen and glutamine and full normalisation of tissue concentrations of pyruvate. α-ketoglutarate. GABA and ammonia. However, even after 3 h of recovery there was a reduction in the ATP concentration and thereby in adenine nucleotide pool, moderate elevations of lactate content and the lactate pyruvate ratio, and less than complete restoration of the amino acid pool. It is concluded that some cells may have been irreversibly damaged by the hypoglycemia.     

Characterization of d-amino acid aminotransferase from Lactobacillus salivarius

We searched a UniProt database of lactic acid bacteria in an effort to identify d-amino acid metabolizing enzymes other than alanine racemase. We found a d-amino acid aminotransferase (d-AAT) homologous gene (UniProt ID: Q1WRM6) in the genome of Lactobacillus salivarius. The gene was then expressed in Escherichia coli, and its product exhibited transaminase activity between d-alanine and α-ketoglutarate. This is the first characterization of a d-AAT from a lactic acid bacterium. L. salivarius d-AAT is a homodimer that uses pyridoxal-5′-phosphate (PLP) as a cofactor; it contains 0.91 molecules of PLP per subunit. Maximum activity was seen at a temperature of 60 °C and a pH of 6.0. However, the enzyme lost no activity when incubated for 30 min at 30 °C and pH 5.5 to 9.5, and retained half its activity when incubated at pH 4.5 or 11.0 under the same conditions. Double reciprocal plots of the initial velocity and d-alanine concentrations in the presence of several fixed concentrations of α-ketoglutarate gave a series of parallel lines, which is consistent with a Ping-Pong mechanism. The K_m values for d-alanine and α-ketoglutarate were 1.05 and 3.78 mM, respectively. With this enzyme, d-allo-isoleucine exhibited greater relative activity than d-alanine as the amino donor, while α-ketobutylate, glyoxylate and indole-3-pyruvate were all more preferable amino acceptors than α-ketoglutarate. The substrate specificity of L. salivarius d-AAT thus differs greatly from those of the other d-AATs so far reported.     

Effect of quinolinate on aminotransferases

Quinolinate inhibits several aminotransferases (ornithine, alanine, and aspartate). However, it is considerably more potent as an inhibitor of liver and heart cytoplasmic aspartate aminotransferase. It is a much less potent inhibitor of mitochondrial aspartate aminotransferases. Quinolinate is bound to the active site of cytoplasmic aspartate aminotransferase. It has a much greater affinity for the pyridoximine-P than the pyridoxal-P form of the enzyme. According to kinetic results, the inhibition or dissociation constant of quinolinate is 0.2 and 20 mm, respectively, for the pyridoxamine-P and the pyridoxal-P forms of the enzyme. Since quinolinate is mainly bound to the pyridoxamine-P form: (a) it is a potent competitive inhibitor of α-ketoglutarate but has little effect when α-ketoglutarate is saturating even if the level of aspartate is low; (b) it decreases the effect of α-ketoglutarate on the absorption spectrum of the pyridoxamine-P form; and (c) it enhances the effect of glutamate on the absorption spectrum of the pyridoxal-P form. Quinolinate is also apparently bound to the apoenzyme since it inhibits reconstitution by either pyridoxamine-P or pyridoxal-P. Since quinolinate is a competitive inhibitor of α-ketoglutarate, it is possible that part of the inhibitory effect of quinolinate on hepatic gluconeogenesis could result from quinolinate inhibiting the conversion of aspartate to oxalacetate by the cytoplasmic aspartate aminotransferase. Quinolinate has no effect on either rat or bovine liver glutamate dehydrogenase or on kidney glutamate dehydrogenase.     

THE INFLUENCE OF HYPOCAPNIA UPON INTRACELLULAR pH AND UPON SOME CARBOHYDRATE SUBSTRATES, AMINO ACIDS AND ORGANIC PHOSPHATES IN THE BRAIN

Abstract— In order to evaluate the influence of hypocapnia upon the energy metabolism of the brain, lightly anaesthetized rats were hyperventilated to arterial CO₂ tensions of 26, 15 and 10 mm Hg respectively, with subsequent measurements of intracellular pH and of tissue concentrations of carbohydrate substrates, amino acids and organic phosphates. At P_co1= 26 there was a moderate increase in the intracellular pH but when the P_co2 was reduced further to 10 mm Hg the intracellular pH returned to normal, or slightly subnormal, values. The reduction in P_Co2 was accompanied by increased cerebral cortical concentrations of lactate, pyruvate, citrate, α-ketoglutarate, malate and glutamate and by decreased aspartate concentrations. It is concluded that the accumulation of metabolic acids explains the normal value for intracellular pH at very low CO₂ tensions. Previous results obtained in man indicate that there is an increased anaerobic production of lactic acid in the brain in extreme hypocapnia. At comparable CO₂ tensions the present results showed a small fall in phosphocreatine and a small rise in ADP. However, since the ammonia concentrations were normal or decreased and since there was an increase in citrate, the results give no direct support to the hypothesis of an activation of phosphofructokinase. Since the cerebral venous P_o2 was reduced to 20 mm Hg at an arterial CO₂ tension of 10 mm Hg the accumulation of acids was probably secondary to tissue hypoxia. However, since there was no, or only a very small, increase in the calculated cytoplasmic NADH/NAD⁺ ratio, it appears less likely that acids accumulated due to lack of NAD⁺.     

THE EFFECT OF ELEVATED AMINO ACIDS ON AMINOTRANSFERASE LEVELS IN BRAIN AND LIVER OF THE MOUSE

Abstract— Adult mice were fed standard diets that were enriched with selected amino acids, i.e. 3% methionine, 6% valine, or 8% lysine. These diets caused the following changes in the amino acid pool of the brain measured at 7 and 21 days. The high methionine diet resulted in 50-fold higher levels of methionine and cysteine and somewhat lower levels of serine and glutamine. The valine and lysine-enriched diets also caused 2- to 4-fold increases in valine and lysine contents of brain, respectively. In spite of the large changes in amino acid levels, however, there were essentially no changes in aspartate: α-ketoglutarate, alanine: α-ketoglutarate, ornithine: α-ketoglutarate, methionine: α-ketoglutarate, and the branched chain aminotransferase activities of brain 3, 10, and 21 days after the onset of the dietary regimen. In contrast, these diets produced significant changes in some of these enzyme activities in liver. Changes in liver included a 2-fold increase in ornithine and alanine aminotransferase activities with the methionine-enriched diet. Liver ornithine aminotransferase activity also increased slightly in animals fed the valine-enriched or lysine-enriched diet.     

TRANSAMINATION OF AMINO ACIDS IN HOMOGENATES OF RAT BRAIN

Abstract— The aminotransferase activity of homogenates of brains from adult and neonatal rats has been investigated. Aminotransferase activity was demonstrated wtih 15 of 22 amino acids incubated with seven keto acids. The basic amino acids exhibited little or no activity.

• 1 The greatest activity was obtained when glutamate or aspartate was incubated with α-ketoglutarate or oxaloacetate. Significant activity was also observed when the neutral aliphatic and aromatic amino acids were incubated with these two keto acids.
• 2 Activity with pyruvate was obtained principally upon incubation with glutamate and alanine. Most of the other amino acids that underwent transamination with α-ketoglutarate also did so with pyruvate, although at a lower rate.
• 3 When phenylpyruvate was added to the medium, glutamate, phenylalanine and tyrosine transaminated most actively.
• 4 Incubations with 11 amino acids and glyoxylic acid demonstrated aminotransferase activity, with glutamate and ornithine being the most active substrates.
• 5 α-Ketoisocaproate and α-ketoisovalerate accepted amino groups primarily from the branched-chain amino acids. Except for glutamate, activity with other amino acids was low or not detectable.
• 6 A comparison of aminotransferase activity in the newborn brain with that in the adult brain showed that the greatest change in activity occurred for glutamate with pyruvate or for alanine with α-ketoglutarate, these activities increasing about 10-fold from birth to adulthood; during this time activities with most other amino acids increased two- to threefold. Amino transfers from the branched-chain amino acids showed no increase with maturation, and some reactions, such as that with methionine and a number of keto acids, decreased from birth to adulthood.
• 7 Our results correspond in general to previous studies of aminotransferase activity in brain and in liver. However, our study also indicates a possible second aminotransferase acting on the branched-chain amino acids, the presence of aminotransferase activity for methionine and asparagine, and relatively high aminotransferase activity for glutamine or ornithine when incubated with glyoxylic acid rather than other keto acids. Moreover, phenylpyruvate and glyoxylate are active in amino transfers and may serve as substrates for a number of aminotransferases.

     

Rat brain aspartate β-decarboxylase. A comparative study with the liver enzyme

Aspartate β-decarboxylase (AspD), which catalyses the β-decarboxylation of aspartate (Asp) to alanine (Ala), was found in significant quantities only in the brain, kidney and liver. This enzyme has an optimum pH at 7.4. Addition of exogenous pyridoxal 5′-phosphate did not increase enzyme activity presumably because of firmly bound cofactor. However, aminooxyacetic acid is a potent inhibitor.There is an apparent 8-fold variation in AspD in the seven brain regions studied, with the highest activities in the cortex and the lowest in the striatum and hippocampus. In the presence of α-ketoglutarate, the production of ¹⁴CO₂ from [¹⁴C]Asp may no longer represent AspD activity due to active transamination of Asp, presumably by aspartate aminotransferase, to oxaloacetate. Under such conditions, comparable AspD activities were observed in all seven brain regions.Kinetic analysis showed that the liver and kidney enzymes have identical affinity for Asp (K_m = 3.5 mM) while the brain enzyme has a higher affinit (K_m = 1.3 mM). The V_max values obtained indicated that the enzyme populations in liver, kidney and brain are in the ratio 18:4:1. Various amino acids were found to inhibit both brain and liver AspD. Serine, however, activated the liver enzyme but inhibited competitively the kidney and brain enzymes. These results indicate that AspD may exist as two or more isozymes.     

Studies on utilization of 2-ketoglutarate,glutamate and other amino acids by the unicellular alga Cyanidium caldarium

Two strains of Cyanidium caldarium which possess different biochemical and nutritional characteristics were examined with respect to their ability to utilize amino acids or 2-ketoglutarate as substrates.One strain utilizes alanine, glutamate or aspartate as nitrogen sources, and glutamate, alanine or 2-ketoglutarate as carbon and energy sources for growth in the dark. The growth rate in the dark on 2-ketoglutarate is almost twice as high or higher than that on glutamate or alanine. During growth or incubation of this alga on amino acids, large amounts of ammonia are formed; however, ammonia formation is strongly inhibited by 2-ketoglutarate. The capacity of the alga to form ammonia from amino acids is inducible and develops fully only when the cells are grown or incubated in the presence of glutamate.By contrast, the other strain of Cyanidium caldarium cannot utilize alanine or aspartate as nitrogen sources. It utilizes glutamate only very poorly and does not excrete ammonia into the external medium. This strain is unable to utilize amino acids or 2-ketoglutarate as carbon and energy sources for heterotrophic growth.Cell-free extracts were tested for the occurrence of enzymes which could account for amino acid metabolism and ammonia formation.     

CEREBRAL METABOLIC AND CIRCULATORY CHANGES INDUCED BY HYPOXIA IN STARVED RATS

In order to study cerebral metabolic and circulatory effects of hypoxia under conditions of restricted glucose supply, the arterial P_o2, was reduced to 25–30mm Hg in artificially ventilated and lightly anaesthetized rats that were starved for 24 or 48 h prior to experiments. Arterial glucose concentrations, that were initially around 6μmol g^-1, were significantly reduced after 15min of hypoxia, and decreased to 50^o of control after 30min. In animals studied after 30min of hypoxia (24 h of starvation), cerebral blood flow had increased 4-fold and there was a moderate (25%) rise in cerebral oxygen consumption. During the course of hypoxia, cerebral cortical concentrations of glucose fell to low values. In spite of this, concentrations of pyruvate and lactate rose with time, and the sum of citric acid cycle intermediates (citrate, α-ketoglutarate, fumarate. malate and oxaloacetate) increased. Changes in amino acids were dominated by a fall in aspartate and a rise in alanine concentration. There was a moderate reduction in phosphocreatine and a slight rise in ADP concentration, but concentrations at ATP and AMP were unchanged. The changes observed are similar to those previously obtained in fed animals. It is concluded that even if blood glucose concentrations fall to 3μmol g^-1, and cerebral energy flux is maintained, substrate supply is sufficient to cover the energy requirements of the tissue. Hypoxia was accompanied by increases in the lactate/pyruvate and β-hydroxybutyrate acetoacetate ratios of blood. In the tissue, NADH/NAD⁺ ratios derived from the lactate, malate and β-hydroxybutyrate dehydrogenase systems rose, while that derived from the glutamate dehydrogenase reaction fell. It is concluded that the latter system is not well suited for estimating mitochondrial redox changes in brain tissue.     

Archenteron formation induced by ascorbate and alpha-ketoglutarate in sea urchin embryos kept in SO2- 4 -free artificial seawater

In permanent blastulae of the sea urchin, which were obtained by culture in SO^2?₄-free artificial seawater from the time of fertilization, ascorbate and α-ketoglutarate, activators of protocollagen proline hydroxylase, induced the formation of archenteron. By adding either ascorbate or α-ketoglutarate to the SO^2?₄-free culture at 12 hr of fertilization, spherical embryos with archenteron were obtained by successive 12 hr cultures at 20°C. The embryos thus obtained did not develop to plutei. Archenteron formation induced by these compounds in SO^2?₄-free-cultured embryos, as well as in the normal embryos, was inhibited by α,α′-dipyridyl, an inhibitor of protocollagen proline hydroxylase. Glutamate, malate, citrate, and fumarate did not stimulate archenteron formation in SO^2?₄-free cultured embryos. In the SO^2?₄-free-cultured embryos exposed to [¹⁴C]proline, considerable radioactivity was found in hot trichloroacetic acid-extractable proteins but the radioactivity of [¹⁴C]hydroxyproline residue, produced by hydroxylation of proline residue of protocollagen, was markedly lower than that in normal embryos. In the presence of ascorbate and α-ketoglutarate, the radioactivity of [¹⁴C]hydroxyproline residue became high and was lowered by α,α′-dipyridyl. Archenteron formation induced by ascorbate and α-ketoglutarate in the embryos kept in SO^2?₄-free artificial seawater probably results from the stimulated protocollagen hydroxylation.     

Regulation of the α-ketoglutarate dehydrogenasecomplex during hibernation in a small mammal,the Richardson's ground squirrel (Urocitellus richardsonii)

The citric acid cycle (CAC) is a central metabolic pathway that links carbohydrate, lipid, and amino acid metabolism in the mitochondria and, hence, is a crucial target for metabolic regulation. The α-ketoglutarate dehydrogenase complex (KGDC) is the rate-limiting step of the CAC, the three enzymes of the complex catalyzing the transformation of α-ketoglutarate to succinyl-CoA with the release of CO₂ and reduction of NAD to NADH. During hibernation, the metabolic rate of small mammals is suppressed, in part due to reduced body temperature but also active controls that suppress aerobic metabolism. The present study examined KGDC regulation during hibernation in skeletal muscle of the Richardson's ground squirrel (Urocitellus richardsonii). The KGDC was partially purified from skeletal muscle of euthermic and hibernating ground squirrels and kinetic properties were evaluated at 5°, 22°, and 37 °C. KGDC from hibernator muscle at all temperatures compared with euthermic controls exhibited a decreased affinity for CoA as well as reduced activation by Ca²⁺ ions at 5 °C from both euthermic and hibernating conditions. Co-immunoprecipitation was employed to isolate the E1, E2 and E3 enzymes of the complex (OGDH, DLST, DLD) to allow immunoblot analysis of post-translational modifications (PTMs) of each enzyme. The results showed elevated phospho-tyrosine content on all three enzymes during hibernation as well as increased ADP-ribosylation and succinylation of hibernator OGDH. Taken together these results show that the KGDC is regulated by posttranslational modifications and temperature effects to reorganize enzyme activity and mitochondrial function to aid suppression of mitochondrial activity during hibernation.     

Hypothermia, Metabolic Stress, and NMDA-Mediated Excitotoxicity

Abstract: Isolated embryonic retinas were metabolically stressed by inhibition of glycolysis either with iodoacetate (IOA) or by glucose withdrawal plus 10 mM 2-deoxy-D-glucose, and the effects of hypothermia were examined. Incubation at 30 versus 37°C during 30 min of hypoglycemia with IOA completely reduced the rapid swelling-related GABA release [6 ± 2 vs. 68 ± 10 nmol/100 mg of protein (mean ± SEM) for 30 and 37°C, respectively]. Histology of the retina immediately following 30 min of metabolic stress at 30°C appeared normal, whereas that at 37°C showed a pattern of acute edema, characteristic of NMDA-mediated acute excitotoxicity. Coincubation with a competitive or noncompetitive NMDA antagonist, respectively, CGS-19755 (10 μM) or MK-801 (1 μM), during 30 min of hypoglycemia at 37°C completely prevented tissue swelling, whereas extracellular GABA content remained at basal levels, indicating that the cytotoxic effects of IOA treatment for 30 min at 37°C were NMDA receptor mediated. Longer periods of hypoglycemia at 37° C produced acute toxicity that was only partially NMDA receptor mediated. Hypothermia delayed the onset of NMDA-mediated toxicity by 30–60 min. At 30°C, the rate of loss of ATP was slowed during the first several minutes of hypoglycemia (82 and 58% of maximal tissue levels at 30 and 37° C, respectively, at 5 min), but by 10 min, ATP levels were comparably reduced. After a transient exposure of retina to 50 μM NMDA in Mg²⁺-free medium, hypothermia significantly attenuated acute GABA release by 30%. At 24 h of recovery, lactate dehydrogenase release was decreased by 37%. Hypothermia had no effect when the exposure was done in medium containing physiological concentrations of Mg²⁺. The above results suggest that the protective effect of hypothermia during the metabolic insult is predominately directed at the cellular events that lead up to NMDA receptor involvement. Reduction in the rate of loss of ATP, however, does not fully account for the delay in involvement of NMDA receptors during metabolic stress at 30°C. The attenuation of direct NMDA-mediated toxicity in Mg²⁺-free medium further suggests that decreased temperature may result in altered channel properties during situations when the Mg²⁺ block is lifted.     

Acute effects of ammonia on the enzymes of citric acid cycle in rat brain

Activities of the enzymes of citric acid cycle were determined along with aspartate and alanine aminotransferases and NADP⁺-isocitrate dehydrogenase in the brains of rats treated with an acute dose of ammonium acetate and compared with those of normal animals. Elevation in the activities of pyruvate, α-ketoglutarate and succinate dehydrogenases and citrate synthase was observed in hyperammonemic animals. The activities of malate, NADP⁺-isocitrate dehydrogenases and aminotransferases decreased under these conditions. The results suggest that ammonia toxicity might not be due to the depletion of α-ketoglutarate from citric acid cycle.     

Regulation of oxaloacetate, aspartate, and malate formation in mesophyll protoplast extracts of three types of c(4) plants

The use of mesophyll protoplast extracts from various C₄ species has provided an effective method for studying light-and substrate-dependent formation of oxaloacetate, malate, and asparate at rates equivalent to whole leaf C₄ photosynthesis. Conditions regulating the formation of the C₄ acids were studied with protoplast extracts from Digitaria sanguinalis, an NADP-malic enzyme C₄ species, Eleusineindica, an NAD-malic enzyme C₄ species, and Urochloa panicoides, a phosphoenolpyruvate (PEP) carboxykinase C₄ species. Light-dependent induction of CO₂ fixation by the mesophyll extracts of all three species was relatively low without addition of exogenous substrates. Pyruvate, alanine and α-ketoglutarate, or 3-phosphoglycerate induced high rates of CO₂ fixation in the mesophyll extracts with oxaloacetate, malate, and aspartate being the primary products. In all three species, it appears that pyruvate, alanine, or 3-phosphoglycerate may serve as effective precursors to the formation of PEP for carboxylation through PEP-carboxylase in C₄ mesophyll cells. Induction by pyruvate or alanine and α-ketoglutarate was light-dependent, whereas 3-phosphoglycerate-induced CO₂ fixation was not.     

Effects of α-ketoglutarate on neutrophil intracellular amino and α-keto acid profiles and ROS production

The aim of this study was to determine the effects of α-ketoglutarate on neutrophil (PMN), free α-keto and amino-acid profiles as well as important reactive oxygen species (ROS) produced [superoxide anion (O₂ ^?), hydrogen peroxide (H₂O₂)] and released myeloperoxidase (MPO) acitivity. Exogenous α-ketoglutarate significantly increased PMN α-ketoglutarate, pyruvate, asparagine, glutamine, asparatate, glutamate, arginine, citrulline, alanine, glycine and serine in a dose as well as duration of exposure dependent manner. Additionally, in parallel with intracellular α-ketoglutarate changes, increases in O₂ ^– formation, H₂O₂-generation and MPO acitivity have also been observed. We therefore believe that α-ketoglutarate is important for affecting PMN “susceptible free amino- and α-keto acid pools” although important mechanisms and backgrounds are not yet completely explored. Moreover, our results also show very clearly that changes in intragranulocytic α-ketoglutarate levels are relevant metabolic determinants in PMN nutrition considerably influencing and modulating the magnitude and quality of the granulocytic host defense capability as well as production of ROS.     

Studies on the apparent instability of bovine kidney alpha-ketoglutarate dehydrogenase complex

The α-ketoglutarate dehydrogenase complex in extracts of bovine kidney and liver mitochondria is inactivated rapidly at 25 °C. This inactivation is not accompanied by loss of activity of the three component enzymes of the complex. This inactivation can be prevented by extensive washing of the mitochondria with dilute phosphate buffer prior to rupturing the mitochondria by freezing and thawing. Evidence is presented that the washings contain a protease which cleaves a peptide bond or bonds in the dihydrolipoyl transsuccinylase component of the α-ketoglutarate dehydrogenase complex, and this limited proteolysis results in dissociation of α-ketoglutarate dehydrogenase and dihydrolipoyl dehydrogenase from the transsuccinylase.The protease appears to be specific for the transsuccinylase component of the mammalian α-ketoglutarate dehydrogenase complex. It does not affect the activity of the mammalian pyruvate dehydrogenase complex or the Escherichia coli α-ketoglutarate dehydrogenase complex. The protease has been purified about 100-fold from extracts of unwashed mitochondria from bovine kidney. It requires a thiol for activity and it is not affected by treatment with diisopropyl phosphorofluoridate or phenylmethyl sulfonylfluoride.A component has been detected in highly purified preparations of the bovine kidney α-ketoglutarate dehydrogenase complex by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, which is present in trace amounts, if at all, in purified preparations of the bovine heart α-ketoglutarate dehydrogenase complex. This component is tightly bound to the transsuccinylase.     

Effects of Dietary Supplementation with Vitamin C and Vitamin E and Their Combination on Growth Performance,Some Biochemical Parameters,and Oxidative Stress Induced by Copper Toxicity in Broilers

This study investigated effects of dietary supplementation with vitamin C, vitamin E on performance, biochemical parameters, and oxidative stress induced by copper toxicity in broilers. A total of 240, 1-day-old, broilers were assigned to eight groups with three replicates of 10 chicks each. The groups were fed on the following diets: control (basal diet), vitamin C (250 mg/kg diet), vitamin E (250 mg/kg diet), vitamin C + vitamin E (250 mg/kg?+?250 mg/kg diet), and copper (300 mg/kg diet) alone or in combination with the corresponding vitamins. At the 6th week, the body weights of broilers were decreased in copper, copper + vitamin E, and copper + vitamin C + vitamin E groups compared to control. The feed conversion ratio was poor in copper group. Plasma aspartate aminotransferase, alanine aminotransferase, alkaline phosphatase activities, iron, copper concentrations, and erythrocyte malondialdehyde were increased; plasma vitamin A and C concentrations and erythrocyte superoxide dismutase were decreased in copper group compared to control. Glutathione peroxidase, vitamin C, and iron levels were increased; aspartate aminotransferase, alanine aminotransferase, alkaline phosphatase, and copper levels were decreased in copper + vitamin C group, while superoxide dismutase, glutathione peroxidase, and vitamin E concentrations were increased; aspartate aminotransferase, alanine aminotransferase, and alkaline phosphatase were decreased in copper with vitamin E group compared to copper group. The vitamin C concentrations were increased; copper, uric acid, aspartate aminotransferase, alanine aminotransferase, alkaline phosphatase, and malondialdehyde were decreased in copper + vitamin C + vitamin E group compared to copper group. To conclude, copper caused oxidative stress in broilers. The combination of vitamin C and vitamin E addition might alleviate the harmful effects of copper as demonstrated by decreased lipid peroxidation and hepatic enzymes.