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At least 811 records · Page 45Linked to original sources
PubMed1987

A simple spectrophotometric assay for arogenate dehydratase.

A simple spectrophotometric assay for arogenate dehydratase, the enzyme that catalyzes the formation of L-phenylalanine from L-arogenate, is presented. The method couples the arogenate dehydratase reaction with that of an aromatic aminotransferase partially purified from Acinetobacter calcoaceticus. In the presence of 2-ketoglutarate, phenylpyruvate formation is measured at 320 nm at basic pH. The method was compared with two other methods already in use in our laboratory for arogenate dehydratase. The new method is simple, quick, fairly sensitive, and especially suitable for the screening of a large number of samples.

Acinetobacter↗
PubMed1979

Enzymology of l-Tyrosine Biosynthesis in Mung Bean (Vigna radiata [L.] Wilczek).

The enzymes of the 4-hydroxyphenylpyruvate (prephenate dehydrogenase and 4-hydroxyphenylpyruvate aminotransferase) and pretyrosine (prephenate aminotransferase and pretyrosine dehydrogenase) pathways of l-tyrosine biosynthesis were partially purified from mung bean (Vigna radiata [L.] Wilczek) seedlings. NADP-dependent prephenate dehydrogenase and pretyrosine dehydrogenase activities coeluted from ion exchange, adsorption, and gel-filtration columns, suggesting that a single protein (52,000 daltons) catalyzes both reactions. The ratio of the activities of partially purified prephenate to pretyrosine dehydrogenase was constant during all purification steps as well as after partial inactivation caused by p-hydroxymercuribenzoic acid or heat. The activity of prephenate dehydrogenase, but not of pretyrosine dehydrogenase, was inhibited by l-tyrosine at nonsaturating levels of substrate. The K(m) values for prephenate and pretyrosine were similar, but the specific activity with prephenate was 2.9 times greater than with pretyrosine.Two peaks of aromatic aminotransferase activity utilizing l-glutamate or l-aspartate as amino donors and 4-hydroxyphenylpyruvate, phenylpyruvate, and/or prephenate as keto acid substrates were eluted from DEAE-cellulose. Of the three keto acid substrates, 4-hydroxyphenylpyruvate was preferentially utilized by 4-hydroxyphenylpyruvate aminotransferase whereas prephenate was best utilized by prephenate aminotransferase. The identity of a product of prephenate aminotransferase as pretyrosine following reaction with prephenate was established by thin layer chromatography of the dansyl-derivative.

Journal Article↗
PubMed1991

Hexose metabolism in pancreatic islets. Activation of the Krebs cycle by nutrient secretagogues.

In rat pancreatic islets, a rise in extracellular D-glucose concentration is known to cause a greater increase in the oxidation of D-[6-14C]glucose than utilization of D-[5-3H]glucose. In the present study, such a preferential stimulation of acetyl residue oxidation relative to glycolytic flux was mimicked by nutrient secretagogues such as 2-aminobicyclo[2,2,1]heptane-2-carboxylate, 3-phenylpyruvate, L-leucine, 2-ketoisocaproate, D-fructose and ketone bodies. The preferential stimulation of D-[6-14C]glucose oxidation by these nutrients was observed at all hexose concentrations (0.5, 6.0 and 16.7 mM), coincided with an unaltered rate of D-[3,4-14C]glucose oxidation, was impaired in the absence of extracellular Ca2+, and failed to be affected by NH4+. Although the ratio between D-[6-14C]glucose oxidation and D-[5-3H]glucose utilization in islets exposed to other nutrient secretagogues could be affected by factors such as isotopic dilution and mitochondrial redox state, the present data afford strong support to the view that the preferential stimulation of oxidative events in the Krebs cycle of nutrient-stimulated islets is linked to the activation of key mitochondrial dehydrogenases, e.g. 2-ketoglutarate dehydrogenase. The latter activation might result from the mitochondrial accumulation of Ca2+, as attributable not solely to stimulation of Ca2+ inflow into the islet cells but also to an increase in ATP availability.

Amino Acids↗
PubMed1993

Phenylalanine determined in plasma with use of phenylalanine dehydrogenase and a centrifugal analyzer.

Quantitative determination of plasma phenylalanine (Phe) is essential for the diagnosis of phenylketonuria (PKU) and the control of dietary therapy of PKU patients. We have adapted a spectrophotometric method (Clin Chim Acta 1991;201:95-8) based on phenylalanine dehydrogenase (EC 1.4.1.-) for rapid and accurate determinations of Phe with the Cobas Fara II centrifugal analyzer. The method is based on the NAD(H)-dependent reductive deamination of Phe with large amounts of phenylalanine dehydrogenase, which catalyzes the formation of phenylpyruvate at pH 10.8. Combining the specificity of the phenylalanine dehydrogenase with the precision, accuracy, and considerable time-saving of an automated system is useful for monitoring PKU patients in a children's hospital.

Amino Acid Oxidoreductases↗
PubMed1989

Purification and characterization of a dimeric phenylalanine dehydrogenase from Rhodococcus maris K-18.

NAD+-dependent phenylalanine dehydrogenase (EC 1.4.1.) was purified to homogeneity from a crude extract of Rhodococcus maris K-18 isolated from soil. The enzyme had a molecular mass of about 70,000 daltons and consisted of two identical subunits. The enzyme catalyzed the oxidative deamination of L-phenylalanine and several other L-amino acids and the reductive amination of phenylpyruvate and p-hydroxyphenylpyruvate. The enzyme required NAD+ as a natural coenzyme. The NAD+ analog 3-acetylpyridine-NAD+ showed much greater coenzyme activity than did NAD+. D-Phenylalanine, D-tyrosine, and phenylethylamine inhibited the oxidative deamination of L-phenylalanine. The enzyme reaction was inhibited by p-chloromercuribenzoate and HgCl2. Initial-velocity and product inhibition studies showed that the reductive amination proceeded through a sequential ordered ternary-binary mechanism. NADH bound first to the enzyme, followed by phenylpyruvate and then ammonia, and the products were released in the order L-phenylalanine and NAD+. The Michaelis constants were as follows: L-phenylalanine, 3.8 mM; NAD+, 0.25 mM; NADH, 43 microM; phenylpyruvate, 0.50 mM; and ammonia, 70 mM.

Amino Acid Oxidoreductases↗
PubMed1992

Parallel control of hepatic proteolysis by phenylalanine and phenylpyruvate through independent inhibitory sites at the plasma membrane.

Intracellular protein degradation in the rat hepatocyte is regulated by 7 amino acids of which Leu, Gln, and Tyr play major roles. Although Phe is known to inhibit proteolysis as effectively as Tyr at high concentrations, it has not been regarded as an active regulator because of its rapid hydroxylation to Tyr. We now show that proteolytic responses to Phe during liver perfusion differ strikingly from effects of the multiphasic regulators Leu, Gln, and Tyr in eliciting mirror image responses at half-normal and normal plasma concentrations. Since response curves to phenylpyruvate and Phe were identical, we considered the possibility that phenylpyruvate mediated its anomalous inhibition intracellularly. However, when phenylpyruvate was produced from phenyllactate intracellularly at a rate providing the same rate of transamination (and intracellular concentration) as that derived from the uptake of phenylpyruvate, no response was obtained. Hence, the effect of phenylpyruvate was not initiated within the cell but more likely from the plasma membrane. Comparable evidence for Phe is less direct. Recent findings indicate that recognition sites for Leu and Gln are located at the plasma membrane. Since Phe augments the concerted inhibition by Leu and Gln at 4-fold normal levels, Phe is probably recognized in close proximity to them. However, the failure of phenylpyruvate to substitute for Phe in this interaction suggests that proteolytic inhibition by phenylpyruvate and Phe is mediated through similar, although independent, plasma membrane sites.

Animals↗
PubMed1983

Mechanism of 3-phenylpyruvate-induced insulin release. Metabolic aspects.

1. The metabolism and metabolic effects of 3-phenylpyruvate were examined in rat pancreatic islets. 2. Islet homogenates catalysed transamination reactions between 3-phenylpyruvate and L-glutamate, L-leucine, L-norleucine or L-valine. 3-Phenylpyruvate failed to activate glutamate dehydrogenase. 3. 3-Phenylpyruvate rapidly entered into islet cells, was extensively converted into phenylalanine but slowly oxidized. 4. The conversion of phenylpyruvate into phenylalanine coincided with a fall in the content of several amino acids (especially glutamate and aspartate) in the islets and incubation medium, the accumulation of 2-oxoglutarate and a modest fall in the NH4+ production rate. 5. 3-Phenylpyruvate failed to affect 14CO2 output from islets prelabelled with [U-14C]palmitate, but augmented 14CO2 output from islets prelabelled or incubated with L-[U-14C]glutamine. 6. In the presence of L-glutamine, 3-phenylpyruvate augmented the ATP/ADP ratio and NAD(P)H islet content, and caused a rapid and sustained decrease in the outflow of radioactivity from islets prelabelled with [2-3H]adenosine. 7. These data support the view that the insulin-releasing capacity of 3-phenylpyruvate coincides with an increase in the catabolism of endogenous amino acids acting as 'partners' in transamination reactions leading to the conversion of 3-phenylpyruvate into phenylalanine.

Adenine Nucleotides↗
PubMed1995

Hexose metabolism in pancreatic islets: regulation of the mitochondrial NADH/NAD+ ratio.

In rat pancreatic islets, D-glucose in concentrations exceeding 5.6 mM caused a concentration-related decrease of the mitochondrial NADH/NAD+ ratio, as judged from the changes in the islet content of glutamate, NH4+, and 2-ketoglutarate, and assuming that the glutamate dehydrogenase reaction is near equilibrium with the mitochondrial NAD system. The concentration dependency of the response to D-glucose was vastly different in islet and parotid cells, respectively. L-Leucine, 2-ketoisocaproate, BCH (a nonmetabolized but insulinotropic analog of L-leucine) and 3-phenylpyruvate also lowered the mitochondrial NADH/NAD+ ratio. In the presence of D-glucose, the latter ratio was also decreased by NH4+ or the absence of extracellular Ca2+, but dramatically increased by aminooxyacetate. Taking into account prior metabolic findings, the nutrient-induced fall in the mitochondrial redox state is thought to reflect an increased clearance of mitochondrial NADH through both the respiratory chain and malate-aspartate shuttle. The nutrient-induced decrease in the mitochondrial NADH/NAD+ ratio might favor both the circulation of metabolites in the Krebs cycle and the exit of Ca2+ from the mitochondria.

Amino Acids↗
PubMed1972

The effect of phenylpyruvate on pyruvate metabolism in rat brain.

1. The effect of phenylalanine and phenylpyruvate on the metabolism of pyruvate by isolated mitochondria from rat brain was investigated. 2. Phenylpyruvate inhibited the fixation of H(14)CO(3) (-) in the presence of pyruvate by intact rat brain mitochondria, whereas phenylalanine and other metabolites of this amino acid had no inhibitory effect on this process. 3. Pyruvate carboxylase activity in freeze-dried rat brain mitochondrial preparations was also inhibited only by phenylpyruvate, and a ;mixed type' inhibition was observed. 4. The K(m) for pyruvate of rat brain pyruvate carboxylase was about 0.2mm. 5. The concentration of phenylpyruvate required for a 50% inhibition of H(14)CO(3) (-) fixation by the intact mitochondria and of pyruvate carboxylase activity was dependent on the concentration of pyruvate used in the incubation medium. 6. The possible significance of inhibition of pyruvate carboxylase activity by phenylpyruvate in the brains of phenylketonuric patients is discussed.

Animals↗
PubMed1981

Behavioral deficit in phenylketonuric rats: role of aromatic acid metabolites of phenylalanine.

To investigate the relationship between the biochemical and behavioral deficits in phenylketonuria (PKU), we treated rats from Postnatal Days 2 through 21 with p-chlorophenylalanine plus L-phenylalanine to simulate PKU or with one of the following aromatic acid metabolites of phenylalanine phenylacetate, phenylpyruvate, phenyllactate, and mandelate. Behavioral tests were begun at about 9 weeks of age. Differences between experimental and control animals were found only in the water maze and in the open field. In the former, PKU rats required significantly more trials to reach criterion than controls. None of the single metabolite-treated groups exhibited a similar learning deficit. In the open field, PKU and mandelate-treated rats were hypoactive compared with controls, whereas phenylacetate- and phenylpyruvate-treated rats wee hyperactive. These results demonstrate a lack of congruence between morphological and behavioral effects of treatment, suggesting that performance deficits in PKU rats may be due to interactive effects of 2 or more metabolites.

Age Factors↗
PubMed1981

Metabolism of 2-oxoacid analogues of leucine, valine and phenylalanine by heart muscle, brain and kidney of the rat.

[1-14C]-Labelled 2-oxoacid analogues of leucine, valine and phenylalanine were used to study the metabolism of these 2-oxoacids in the brain, kidney and heart muscle of rats. By following the 14CO2 release during 30-60 min of incubation at 37 degrees C the decarboxylation rate was determined and measurement of the 14C-incorporation into the corresponding amino acid yielded the transamination rate. From these rates, decarboxylation/transamination ratios could be calculated which are indicative for the metabolic fate of the 2-oxoacid in the various organs. The results obtained show that all three tissues are capable of utilizing the 2-oxoacid analogues of leucine, valine and phenylalanine, however, to a different extent: kidney greater than heart muscle greater than brain. The decarboxylation/transamination ratios reveal that the branched-chain 2-oxoacids are predominantly decarboxylated in kidney and heart muscle while in brain they are mainly transaminated. The ratios calculated for phenylpyruvate in all tissues are within 0.19 and 0.36, indicating that this 2-oxoacid is preferentially transaminated. The results are discussed with respect to possible dietary alterations of enzymes involved in 2-oxoacid metabolism in order to improve transamination of these compounds.

Aminooxyacetic Acid↗
PubMed1979

Enzyme-enzyme interaction and the biosynthesis of aromatic amino acids in Escherichia coli.

The technique of affinity chromatography has been used to demonstrate that enzymes involved in the biosynthesis of tyrosine and phenylalanine in Escherichia coli undergo reversible interactions. Thus it has been shown that the aromatic amino acid aminotransferase (aromatic-amino-acid: 2-oxoglutarate amino-transferase, EC 2.6.1.57) reacts specifically with chorismate mutaseprephenate dehydrogenase (chorismate pyruvate mutase, EC 5.4.99.5 and prephenate: NAD+ oxidoreductase (decarboxylating), EC 1.3.1.12) in the absence of reactants and with chorimate mutase-prephenatedehydratase (prephenate hydro-lyase (decarboxylating), EC 4.2.1.51) in the presence of phyenylpyruvate. Tyrosine causes dissociation of the aminotransferase: mutasedehydrogenase complex while dissociation of the aminotransferase-mutasedehydratase complex occurs on omission of phenylpyruvate. Only the active form of chorismate mutase-prephenate dehydrogenase participates in complex formation.

Amino Acids↗