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PubMed1978

Cysteine conjugate beta-lyase in rat liver. A novel enzyme catalyzing formation of thiol-containing metabolites of drugs.

A novel enzyme catalyzing cleavage of the thioether linkage in cysteine conjugates of aromatic compounds, such as 2,4-dinitrobenzene and p-bromobenzene, has been purified about 500-fold from rat liver cytosol. Incubation of S-(2,4-dinitrophenyl)cysteine with the enzyme preparation yielded 2,4-dinitrobenzenethiol, pyruvic acid, and NH3 at equimolar ratios, indicating that the thioether cleavage probably proceeds via an alpha,beta elimination reaction. The thiol product was methylated and the methylated derivative 1-methylthio-2,4-dinitrobenzene, was identified by mass spectrometry and proton NMR spectroscopy. The Km value of S-(2,4-dinitrophenyl)cysteine was 0.5 mM at pH 7.4 in phosphate buffer. The enzyme activity was inhibited by hydroxylamine. No cofactor requirement was observed. A combination of the partially purified enzyme and hepatic microsomes that contain thiol methyltransferase (EC 2.1.1.9) converted cysteine conjugates of 2,4-dinitrobenzene and p-bromobenzene to the corresponding methylthio-containing metabolites; S-adenosylmethionine was required. An important role of this novel beta-lyase enzyme in the formation of methylthio-containing metabolites of various drugs is indicated.

Animals↗

PubMed1981

[Characteristics of myocardial metabolic disorders in rats as affected by alternating magnetic fields of varying parameters].

It was shown in experiments on albino rats that repeated exposure to an alternating field of 32 kA/m voltage leads to the reduction in the content of glycogen, lactic and pyruvic acid and the reduced form of nicotinamide coenzymes and to delayed processes of oxidation and phosphorylation without changing the degree of their coupling and the level of macroergic phosphates. An alternating magnetic field with a voltage of 7.5 kA/m leads to an increase in the level of glycogen and glutamine in half a month, causes accumulation of ammonia in three months, increases the content of glutamine and reduces the glucose level after six months of exposure.

Animals↗

PubMed1981

[Metabolic shifts in the myocardium in the long-term effect of atmospheric pollution on the body].

Metabolic shifts in the myocardium under conditions of long-term exposure of the body to air pollution were studied in experiments on albino rats. Study of the content of protein, RNA, DNA, glycogen, ATP, lactic and pyruvic acids and the activity of lactate dehydrogenase and creatine kinase in the heart muscle allowed the conclusion that the glycolytic processes are intensified, the bioenergic processes are inhibited and the synthesis of protein and RNA are increased in the myocardium of the experimental animals.

Air Pollutants↗

PubMedDate not supplied

[Estimation of serine- and threonine dehydratase activity of blood serum].

A procedure for estimation of serine dehydratase (SDH) and threonine dehydratase (TDH) activities in blood serum was based on the colorimetric measurement of the products, increasing after deamination of 1-serine (pyruvic acid) and 1-threonine (alpha-ketobutyric acid) using the reaction with 2,4-dinitrophenyl hydrazine in an alkaline medium. Conditions for the enzymatic reaction were developed. Activity of enzymes was expressed as micrometer of the reaction products per 1 L of blood serum within 1 hr of incubation at 37 degrees. Blood serum from normal donors did not exhibit the SDH and TDH activities. The enzymatic activity was estimated in 126 patients with various impairments of liver tissue. The procedure developed was shown to be highly sensitive and informative. It enabled to detect both the earliest periods of the cytolytic syndrome and transition of the syndrome into its irreversible step.

Clinical Enzyme Tests↗

PubMed1980

[Diaplacental inhibition of fetal blood coagulation in pregnant sheep (author's transl)].

We investigated the effect of the synthetic low-molecular thrombin inhibitor 4-amidinophenyl-pyruvic acid (APPA) on the maternal and fetal coagulation at 10 far advanced in pregnancy sheep. APPA inhibits as well the maternal as by diaplacental passage the fetal hemostasis in dependence on the dosage till the incoagulability. The effect in the fetal organism occurs after a delay of some minutes and wars of a bit earlier.

Animals↗

PubMed1993

Rat models of protein and protein-energy malnutrition.

Two animal models of infantile malnutrition were produced where some aspects of carbohydrate metabolism can be studied: protein-depleted (PD, "kwashiorkor") rats, fed a protein-free diet for 18 days, starting 3 days before weaning, and chronically malnourished animals during suckling (PED, "marasmus") where 20-22 pups were fed by the same dam. PED and PD rats were sacrificed at 21 and 36 days of age, respectively. Body weight was 35% in PD, and 50% in PED groups when compared to their age-matched controls. After an overnight fast, some of the PD animals presented with low blood glucose levels, and the levels of lactic and pyruvic acids were depressed in PED, while lactic acid was increased in PD animals. Blood glucose utilization was reduced in PED, and increased in PD rats, while the opposite was true for the kidney gluconeogenic capacity. When compared to the respective control values, muscle glycogen was high in both groups, while the level of liver glycogen was unchanged. The rate of muscle glycolysis was decreased in both groups. These results are in agreement with clinical observations, suggesting that these models may be used to study some of the metabolic changes brought about by infantile malnutrition.

Animals↗

PubMed1995

[Clearance, reabsorption and excretion of free and bound L-carnitine in children].

Acylcarnitines are by-products of fatty acid, pyruvate and some xenobiotics metabolism excreted in urine. We compare the renal handling of free and acylcarnitine in a group of 15 healthy children in relation to sodium and urea excretion and to sodium fractional tubular reabsorption (FTR%) and fractional excretion (FE%). Urea and sodium were determined by routine laboratory method; free and acylcarnitines by enzymatic one. FTR% and FE% were determined as a % of filtration load. Linear regression was used for calculation of correlation coefficient "r" from paired data. Low correlation (r = 0.25; p = 0.42) between urea and free carnitine excretion was observed; for excretion of acylcarnitine and urea correlation was much higher (r = 0.61; p < 0.03). Excretion of free correlation was negatively correlated with sodium FTR (r = -0.86; p < 0.001) and there was no correlation at all for acylcarnitine excretion and sodium FTR. It can be assumed:-neither excretion nor reabsorption of acylcarnitines are Na-dependent;-renal handling of free and total carnitines proceeds on different mechanisms.

Adolescent↗

PubMedDate not supplied

[Effectiveness of the use of synthetic opioid peptide in rats with traumatic shock].

Experiments were conducted on 80 male Wistar rats. Traumatic shock was simulated by applying clamps to lower extremity soft tissue for 6 hours. After removing the clamps, experimental animals were intraperitoneally injected the synthetic leucine-enkephalin dalargin. The blood ionogram, the levels of urea, glucose, phosphorus, creatinine, lactic and pyruvic acids, blood plasma osmolarity, LDH activity were determined. In the animals untreated with dalargin, plasma concentrations of ions of potassium, phosphorus, creatinine, urea, osmolarity, LDH activity increased and the levels of glucose decreased when shock developed. At the same time the rat death rates were 70-80% 24 hours later. The administration of dalargin corrected the majority of the above-mentioned parameters and the death rates were decreased to 20%. It is concluded that dalargin has an early antishock effect.

Animals↗

PubMedDate not supplied

[Characteristics of the pentose phosphate pathway of carbohydrate metabolism in the gastric mucosa of persons with peptic ulcer].

Activity of the main enzymes of pentose phosphate pathway of carbohydrate metabolism was studied in human mucosa of the stomach. In the mucosa glucose-6-phosphate dehydrogenase in gastric ulcer and 6-phosphogluconate dehydrogenase in normal stomach were shown to be less active as compared with these enzymatic activities in duodenal ulcer. A distinct decrease in the activity of glucose-6-phosphate dehydrogenase was observed in the ulcerous region, independently of the ulcer localization either in corpus ventriculi or in duodenum, as compared with the parts of the mucosa away from the impairment; the lowest enzymatic activity was estimated in the gastric ulcer zone. Activities of 6-phosphogluconate dehydrogenase and transketolase were the same in the ulcerous and normal mucosa and did not depend on the localization of the impairment. Contents of lactic and pyruvic acids were similar in mucosa of the stomach both in gastric and duodenal ulcer.

Duodenal Ulcer↗

PubMed1997

Structural and immunochemical studies on the O-specific polysaccharide of Proteus penneri strain 15.

On the basis of sugar analysis and 1H- and 13C-NMR spectroscopy, it was shown that the O-specific polysaccharide of Proteus penneri strain 15 has a trisaccharide repeating unit, including an acetal-linked pyruvic acid residue, and is structurally identical to the capsular polysaccharide of Proteus vulgaris strain ATCC 49990. Serological studies supported this conclusion and demonstrated the presence in the homological antiserum of both anti-core and anti-O chain antibodies reacting with a lipopolysaccharide (LPS) epitope containing N-acetylglucosamine and galactose residues.

Animals↗

PubMedDate not supplied

[Glycolytic metabolites and adenosine triphosphoric acid in the herpes-infected eye].

In the tissues of the herpes-infected eye of a rabbit the content of adenosine triphosphoric acid (ATP), glucose and pyroracemic acid decreases and the level of lactic acid rises, i.e. anaerobic glycolysis prevails over the aerobic decomposition of glucose. A sharp decrease in the amount of ATP in the cornea and the iris in herpetic keratitis is due to the increased consumption of ATP for the synthesis of DNA and polypeptides of herpes simplex virus, as well as to disturbances in the oxidation transformations of glucose linked with the increased proportion of anaerobic glycolysis.

Adenosine Triphosphate↗

PubMed1976

[Effects of aromatic bisamidines on blood coagulation and fibrinolysis].

The effect of the aromatic diamidine derivative 2,6-bis (4-amidinobenzyl)-cyclohexanon-(1) on blood coagulation and fibrinolysis in vitro and in vivo was compared with that of the benzamidine derivative 4-amidinophenyl pyruvic acid and the aromatic diamidine derivative 4,4'-diamidinophenoxypentane. 2,6-Bis(4-amidinobenzyl)-cyclohexanon-(1) was found to be a strong inhibitor of the clotting enzyme thrombin. Because of the toxic side effects and pharmacokinetic properties of both diamidine derivatives their in vivo use as anticoagulants is limited.

Amidines↗

PubMed2003

Pyruvate and citric acid cycle carbon requirements in isolated skeletal muscle mitochondria.

Carbohydrate depletion precipitates fatigue in skeletal muscle, but, because pyruvate provides both acetyl-CoA for mainline oxidation and anaplerotic carbon to the citric acid cycle (CAC), the mechanism remains obscure. Thus pyruvate and CAC kinetic parameters were independently quantified in mitochondria isolated from rat mixed skeletal muscle. Mitochondrial oxygen consumption rate (Jo) was measured polarographically while either pyruvate or malate was added stepwise in the presence of a saturating concentration of the other substrate. These substrate titrations were carried out across a physiological range of fixed extramitochondrial ATP free energy states (DeltaGP), established with a creatine kinase energy clamp, and also at saturating [ADP]. The apparent Km,malate for mitochondrial Jo ranged from 21 to 32 microM, and the apparent Km,pyruvate ranged from 12 to 26 microM, with both substrate Km values increasing as DeltaGP declined. Vmax for both substrates also increased as DeltaGP fell, reflecting thermodynamic control of Jo. Reported in vivo skeletal muscle [malate] are >10-fold greater than the Km,malate determined in this study. In marked contrast, the K(m,pyruvate) determined is near the [pyruvate] reported in muscle approaching exhaustion associated with glycogen depletion. When data were evaluated in the context of a linear thermodynamic force-flow (DeltaGP-Jo) relationship, the DeltaGP-Jo slope was essentially insensitive to changes in [malate] in the range observed in vivo but decreased markedly with declining [pyruvate] across the physiological range. Mitochondrial respiration is particularly sensitive to variations in [pyruvate] in the physiological range. In contrast, physiological [malate] exerts very little, if any, influence on mitochondrial pyruvate oxidation measured in vitro.

Animals↗

PubMed1992

Malate- and pyruvate-dependent Fatty Acid synthesis in leucoplasts from developing castor endosperm.

Leucoplasts were isolated from the endosperm of developing castor (Ricinis communis) endosperm using a discontinuous Percoll gradient. The rate of fatty acid synthesis was highest when malate was the precursor, at 155 nanomoles acetyl-CoA equivalents per milligram protein per hour. Pyruvate and acetate also were precursors of fatty acid synthesis, but the rates were approximately 4.5 and 120 times less, respectively, than when malate was the precursor. When acetate was supplied to leucoplasts, exogenous ATP, NADH, and NADPH were required to obtain maximal rates of fatty acid synthesis. In contrast, the incorporation of malate and pyruvate into fatty acids did not require a supply of exogenous reductant. Further, the incorporation of radiolabel into fatty acids by leucoplasts supplied with radiolabeled malate, pyruvate, or acetate was reduced upon coincubation with cold pyruvate or malate. The data suggest that malate and pyruvate may be good in vivo sources of carbon for fatty acid synthesis and that, in these preparations, leucoplast fatty acid synthesis may be limited by activity at or downstream of the acetyl-CoA carboxylase reaction.

Journal Article↗

PubMed1983

The effects of cyclopropane carboxylic acid on hepatic pyruvate metabolism.

The effects of the hypoglycemic agent, cyclopropane carboxylate, on the metabolism of various substrates that enter the mitochondrion via the mitochondrial monocarboxylate transporter were investigated in perfused rat livers. Metabolism of pyruvate, branched-chain alpha-keto acids, acetoacetate and, to a lesser extent beta-hydroxybutyrate, were all inhibited by cyclopropane carboxylate. In addition, the stimulation of pyruvate decarboxylation by beta-hydroxybutyrate at low pyruvate concentrations because of exchange of extramitochondrial pyruvate with intramitochondrially generated acetoacetate, was abolished by cyclopropane carboxylate. Gluconeogenesis from alanine, which is not transported via the monocarboxylate translocator, was not inhibited by low concentrations of cyclopropane carboxylate. Cyclopropane carboxylate also inhibited fatty acid oxidation, as measured by ketone body production. These results support previous findings that the hypoglycemic agent, cyclopropane carboxylate, exerts at least some of its metabolic effects at the level of the mitochondrial monocarboxylate transporter.

Animals↗

PubMed2003

Engineering lactic acid bacteria with pyruvate decarboxylase and alcohol dehydrogenase genes for ethanol production from Zymomonas mobilis.

Lactic acid bacteria are candidates for engineered production of ethanol from biomass because they are food-grade microorganisms that can, in many cases, metabolize a variety of sugars and grow under harsh conditions. In an effort to divert fermentation from production of lactic acid to ethanol, plasmids were constructed to express pyruvate decarboxylase (PDC) and alcohol dehydrogenase (ADH), encoded by the pdc and adhB genes of Zymomonas mobilis, in lactic acid bacteria. Several strains were transformed with the plasmids, and transcription of pdc and adhB was confirmed by northern hybridization analysis of transformants. PDC and ADH enzyme activities were at least 5- to 10-fold lower in these bacteria compared to Escherichia coli transformed with the same plasmid. Glucose fermentations were carried out, and some, but not all, of the transformed strains produced more ethanol than the untransformed parent strains. However, lactic acid was the primary fermentation product formed by all of the transformants, indicating that ADH and PDC activities were insufficient to divert significant carbon flow towards ethanol.

Alcohol Dehydrogenase↗

PubMed1993

Adipose pyruvate carboxylase: amino acid sequence and domain structure deduced from cDNA sequencing.

The complete amino acid sequence of 3T3-L1 adipocyte pyruvate carboxylase (PC) [pyruvate:carbon-dioxide ligase (ADP-forming), EC 6.4.1.1] has been deduced from sequencing overlapping cDNA clones obtained from an adipocyte cDNA library constructed in the lambda Zap vector. The encoding mRNA for PC promoter contains 4067 nt, including a 3534-nt coding sequence and noncoding regions of 100 and 433 nt at the 5' and 3' ends, respectively. The biotinylated lysine of the encoded PC promoter (1178 amino acids with a calculated M(r) of apocarboxylase = 129,784) is located 35 residues from the COOH-terminal end and, as in most other biotin enzymes, is in the consensus sequence AMKM. The adipocyte PC is closely similar (53% identity) to the yeast enzyme and contains different segments that are homologous with regions from the biotin carboxylase component of Escherichia coli acetyl-CoA carboxylase, the keto acid-binding subunits of Propionibacterium shermanii oxaloacetate transcarboxylase and Klebsiella pneumoniae oxaloacetate decarboxylase, and to the biotin carboxyl-carrier protein of the bacterial biotin enzymes. In addition to the putative mitochondrial targeting signal, functional domains are readily identifiable in the sequence and are in the following order: biotin carboxylase-carboxyltransferase-biotin carboxyl-carrier protein, as proposed for yeast PC.

3T3 Cells↗

PubMed1997

Conversion of non-allosteric pyruvate kinase isozyme into an allosteric enzyme by a single amino acid substitution.

Pyruvate kinase M1, a non-allosteric isozyme, was converted into an allosteric enzyme by replacement of an amino acid in the intersubunit contact. The substitution of Ala-398 with Arg resulted in the pronounced allosteric enzyme. The Hill coefficient and the substrate concentration giving one-half of Vmax for the mutant with respect to phosphoenolpyruvate were 2.7 and 0.41 mM, respectively, whereas those values for the wild type were 1.0 and 0.049 mM. This mutation, however, gave rise to only minor effects on the apparent values of Km for ADP and on Vmax. Furthermore, in contrast to the wild-type enzyme, the mutant was activated by fructose 1, 6-bisphosphate. The Hill coefficient of the mutant was no longer increased by the allosteric inhibitor, L-phenylalanine, indicating that the equilibrium for the unligated enzyme is largely shifted toward the T-state. These results suggest that Ala-398 is one of the most critical residues allowing the enzyme to prefer the R-state and that allosteric regulation of pyruvate kinase involves amino acid residues in the intersubunit contact.

Allosteric Regulation↗