Effect of phenylpyruvate on pyruvate dehydrogenase activity in rat brain mitochondria

1. The effects of phenylpyruvate, a metabolite produced in phenylketonuria, on the pyruvate dehydrogenase-complex activity were investigated in rat brain mitochondria. 2. Pyruvate dehydrogenase activity was measured by two methods, one measuring the release of (14)CO(2) from [1-(14)C]pyruvate and the other measuring the acetyl-CoA formed by means of the coupling enzyme, pigeon liver arylamine acetyltransferase (EC 2.3.1.5). In neither case was there significant inhibition of the pyruvate dehydrogenase complex by phenylpyruvate at concentrations below 2mm. 3. However, phenylpyruvate acted as a classical competitive inhibitor of the coupling enzyme arylamine acetyltransferase, with a K(i) of 100mum. 4. It was concluded that the inhibition of pyruvate dehydrogenase by phenylpyruvate is unlikely to be a primary enzyme defect in phenylketonuria.     

The effect of phenylpyruvate on oxidative-phosphorylation in brain mitochondria

Abstract— A mitochondrial fraction isolated from the cerebral cortex of immature rats was shown to possess the characteristics of intact, functional mitochondria. Phenylpyruvate, in low concentrations, had an inhibitory effect upon oxygen utilization by this system without uncoupling phosphorylation. The inhibition was specific for the keto acid and to pyruvate as substrate. It was concluded that phenylpyruvate inhibits the oxidation of pyruvate and the implications of this are discussed in relation to phenylpyruvic oligophrenia.     

Regulation of pyruvate metabolism via pyruvate carboxylase in rat brain mitochondria

1. The fixation of CO(2) by pyruvate carboxylase in isolated rat brain mitochondria was investigated. 2. In the presence of pyruvate, ATP, inorganic phosphate and magnesium, rat brain mitochondria fixed H(14)CO(3) (-) into tricarboxylic acid-cycle intermediates at a rate of about 250nmol/30min per mg of protein. 3. Citrate and malate were the main radioactive products with citrate containing most of the radioactivity fixed. The observed rates of H(14)CO(3) (-) fixation and citrate formation correlated with the measured activities of pyruvate carboxylase and citrate synthase in the mitochondria. 4. The carboxylation of pyruvate by the mitochondria had an apparent K(m) for pyruvate of about 0.5mm. 5. Pyruvate carboxylation was inhibited by ADP and dinitrophenol. 6. Malate, succinate, fumarate and oxaloacetate inhibited the carboxylation of pyruvate whereas glutamate stimulated it. 7. The results suggest that the metabolism of pyruvate via pyruvate carboxylase in brain mitochondria is regulated, in part, by the intramitochondrial concentrations of pyruvate, oxaloacetate and the ATP:ADP ratio.     

Inhibitory effect of high oxygen pressure on potassiuminduced activation of pyruvate dehydrogenase and glucose metabolism in rat brain slices

The effects of high oxygen pressure on pyruvate dehydrogenase (pyruvate: lipoate oxidoreductase (decarboxylating and acceptor-acylating), EC 1.2.4.1) activity, tissue concentration of ATP, and CO₂ production from glucose were studied in rat brain cortical slices. The increase in pyruvate dehydrogenase activity and the lowering of cellular ATP, occurring during potassium-induced depolarization at 1 atm of oxygen, were reversed by increasing the oxygen pressure to 5 atm. When brain slices were incubated at 1 atm oxygen with [U-¹⁴C]glucose, a high potassium medium approximately doubled the production of ¹⁴CO₂. Oxygen at 5 atm abolished this potassium-dependent increase in ¹⁴CO₂ production with no significant effect on glucose oxidation in normal Krebs-Ringer phosphate medium. Adding 4 atm helium to 1 atm oxygen did not interfere with the ability of potassium ions to activate pyruvate dehydrogenase, lower ATP, or increase glucose oxidation. The results show that toxic effects of hyperbaric oxygen, not manifest in “resting” tissue, may be revealed during stress such as potassium depolarization. The site of the toxic effects of oxygen is probably the cell membrane where excess oxygen appears to interfere with the action of the sodium pump, calcium transport or other processes stimulated by increased concentrations of extracellular potassium.     

Effect of phenylpyruvate on enzymes involved in fatty acid synthesis in rat brain

1. The effects of phenylpyruvate, a metabolite produced in phenylketonuria, on the pyruvate dehydrogenase-complex activity were investigated in rat brain mitochondria. 2. Pyruvate dehydrogenase activity was measured by two methods, one measuring the release of ¹⁴CO₂ from [1-¹⁴C]pyruvate and the other measuring the acetyl-CoA formed by means of the coupling enzyme, pigeon liver arylamine acetyltransferase (EC 2.3.1.5). In neither case was there significant inhibition of the pyruvate dehydrogenase complex by phenylpyruvate at concentrations below 2mm. 3. However, phenylpyruvate acted as a classical competitive inhibitor of the coupling enzyme arylamine acetyltransferase, with a K_i of 100μm. 4. It was concluded that the inhibition of pyruvate dehydrogenase by phenylpyruvate is unlikely to be a primary enzyme defect in phenylketonuria.     

The effect of propionate on the metabolism of pyruvate and lactate in the perfused rat liver

1. Rates of gluconeogenesis in the perfused rat liver from propionate, l-lactate, pyruvate and the combination of propionate with either lactate or pyruvate were measured. Less than additive rates were obtained with either propionate plus lactate or propionate plus pyruvate. 2. The uptake of pyruvate plus lactate from the perfusion medium was decreased more seriously when propionate was present with lactate than with pyruvate. 3. The use of [2-(14)C]pyruvate in the presence of propionate showed that the decreased disappearance of pyruvate plus lactate did not result in their formation from propionate. 4. The addition of sodium butyrate to the perfusion medium caused an inhibition of gluconeogenesis from propionate and stimulated gluconeogenesis and uptake of pyruvate and lactate. 5. The observations are consistent with there being a sparing effect of propionate on lactate and pyruvate metabolism.     

The effect of hyperphenylalaninaemia on glycine metabolism in developing rat brain.

The brains of 3--16-day-old rats that were rendered hyperphenylalaninaemic by daily injections of alpha-methylphenylalanine plus phenylalanine were subjected to biochemical analysis. Fluctuations throughout the treatment period in the concentrations of branched-chain amino acids, methionine and serotonin were in agreement with the known interference of excess plasma phenylalanine with transport. The glycine content, however, became abnormal only by day 5, remained so through the treatment, and the elevation was equally apparent at 4, 8 or 24 h after the last daily injections. On the last day of treatment there were small increases in the taurine, glutamate, aspartate and 4-aminobutyrate concentrations, attributable mainly to the diencephalon or brain stem. After day 3 of treatment there were persistent elevations in the specific activity of phosphoserine phosphatase and glycine synthase (but not serine hydroxymethyltransferase) of the brain in each of the regions analysed. The observations indicate that chronic hyperphenylalaninaemia interferes with the normal regulation of intracerebral glycine metabolism during a critical period of early postnatal development, and suggest that the resulting excess in this amino acid (particularly marked in the cortex) contributes to the behavioural abnormalities that these animals exhibit in later life.     

The effects of adenine nucleotides on pyruvate metabolism in rat liver

1. The effects of adenine nucleotides on pyruvate metabolism by isolated liver cells and isolated mitochondria have been investigated. The amount of pyruvate carboxylated has been estimated by determining the tricarboxylic acid-cycle intermediates, glutamate and aspartate accumulating in the incubation medium. The extent of pyruvate oxidation has been assessed by measuring oxygen uptake and the yield of ¹⁴CO₂ from [1-¹⁴C]pyruvate and [2-¹⁴C]pyruvate. 2. When catalytic amounts of adenine nucleotides (1–2mm) were added to suspensions of isolated liver cells incubated with pyruvate an ATP:ADP ratio greater than 6:1 was maintained. Both pyruvate oxidation to acetyl-CoA and the oxidation of acetyl-CoA through the tricarboxylic acid cycle were stimulated but pyruvate carboxylation was not affected. The production of acetyl-CoA exceeded the capacity of the cells for the oxidation of acetyl-CoA and the excess was converted into ketone bodies. 3. If a low ATP:ADP ratio was maintained in isolated cells or mitochondria by incubating them with dinitrophenol or hexokinase, pyruvate carboxylation was grossly inhibited, oxygen uptake depressed and ketone-body formation stimulated. Measurement of oxaloacetate concentrations confirmed that under these conditions oxaloacetate was rate-limiting for the oxidation of acetyl-CoA via the tricarboxylic acid cycle. The inclusion in the incubation medium of fumarate (1·25mm) completely prevented the ketogenic action of dinitrophenol or hexokinase. 4. When ADP (5mm) was added to a suspension of isolated liver cells incubated with pyruvate an actual ADP concentration of about 1mm was attained. This brought about effects on pyruvate metabolism similar to those obtained with dinitrophenol or hexokinase. 5. These results support the concept that the relative concentrations of adenine nucleotides within the liver cell may play a role in governing the rates of pyruvate oxidation and carboxylation. In addition, they provide further evidence that the availability of oxaloacetate in the liver cell can play a key role in determining whether acetyl-CoA arising from pyruvate is oxidized through the tricarboxylic acid cycle or converted into ketone bodies.     

Inhibitory effect of high oxygen pressure on potassium- induced activation of pyruvate dehydrogenase and glucose metabolism in rat brain slices

The effects of high oxygen pressure on pyruvate dehydrogenase (pyruvate: lipoate oxidoreductase (decarboxylating and acceptor-acylating), EC 1.2.4.1) activity, tissue concentration of ATP, and CO2 production from glucose were studied in rat brain cortical slices. The increase in pyruvate dehydrogenase activity and the lowering of cellular ATP, occurring during potassium-induced depolarization at 1 atm of oxygen, were reversed by increasing the oxygen pressure to 5 atm. When brain slices were incubated at 1 atm oxygen with [U-14C]glucose, a high potassium medium approximately doubled the production of 14CO2. Oxygen at 5 atm abolished this potassium-dependent increase in 14CO2 production with no significant effect on glucose oxidation in normal Krebs-Ringer phosphate medium. Adding 4 atm helium to 1 atm oxygen did not interfere with the ability of potassium ions to activate pyruvate dehydrogenase, lower ATP, or increase glucose oxidation. The results show that toxic effects of hyperbaric oxygen, not manifest in "resting" tissue, may be revealed during stress such as potassium depolarization. The site of the toxic effects of oxygen is probably the cell membrane where excess oxygen appears to interfere with the action of the sodium pump, calcium transport or other processes stimulated by increased concentrations of extracellular potassium.     

The effect of irradiation on dopamine level and metabolism in the rat brain

In experiments on rats it was shown that after 20 Gy irradiation dopamine and homovanilic acid content increases in the caudate nucleus and limbic structures of the forebrain: dopamine disappears more readily when its biosynthesis is blocked. The rate of the mediator degradation in the brain increases by 1.5-2 times, and the rate of the synthesis, by 3-5 times at early times after irradiation.