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R J Wanders

Publications and source records attributed to R J Wanders.

At least 487 records · Page 27Linked to original sources
PubMed1984

Modern theories of metabolic control and their applications (review).

Existing, qualitative notions with respect to the way in which enzyme properties control metabolism are discussed in the light of the control analysis developed by H. Kacser and J. A. Burns ( (1973) in: Rate Control of Biological Processes, Davies DD, ed., Cambridge University Press, pp. 65-104) and R. Heinrich and T. A. Rapoport ( (1974) Eur. J. Biochem. 42, 89-95), and recent experimental data. Points at which the existing notions should be adjusted are: Metabolic control is shared by enzymes rather than confined to one rate-limiting enzyme per pathway. Whether an enzyme exercises strong control on a flux cannot be deduced solely from its own properties, nor is it directly related to its distance from equilibrium. With respect to metabolic control, enzymes should be classified into four groups, rather than two (reversible versus irreversible). The distribution of control among the enzymes depends on the metabolic conditions. Control structures of metabolic pathways probably differ with the function of that pathway.

Adenosine Diphosphate↗
PubMed1984

[Diagnosis of Zellweger's cerebrohepatorenal syndrome].

The cerebro-hepato-renal syndrome of Zellweger is an autosomal recessive inborn error of metabolism. Clinically the disease is characterised by craniofacial malformations, a lack of muscle tone, disturbances in liver function, renal cysts and mental retardation. The disease is characterised biochemically by the absence of peroxisomes (microbodies) in liver and kidney and variable abnormalities in mitochondria. This results in elevated concentrations of pipecolic acid and of tri(di)hydroxycoprostanoic acid in urine, CSF and/or duodenal fluid, an elevation of the concentration of very long chain (greater than C22) fatty acids in plasma and fibroblasts and a deficiency of plasmalogens in tissues, erythrocytes and fibroblasts. Moreover, we recently found that in Zellweger patients the activity of dihydroxyacetone phosphate acyltransferase is deficient in tissues, fibroblasts and cultured amniotic fluid cells and that the incorporation of a radioactive precursor of plasmalogens into phospholipids is impaired in fibroblasts and amniocytes. These recent findings allow specific prenatal and postnatal diagnosis of this disease.

Abnormalities, Multiple↗
PubMed1983

Properties of carbamoyl-phosphate synthetase (ammonia) in rat-liver mitochondria made permeable with toluene.

Some properties of carbamoyl-phosphate synthetase (ammonia) were studied in rat-liver mitochondria made selectively permeable by pretreatment with toluene. The Michaelis constants for NH3, MgATP and HCO-3 were 0.7, 1.2 and 2 mM respectively. N-Acetylglutamate activated the enzyme with a Ka of about 0.1 mM. At saturating concentrations of substrates and effectors the enzyme was inhibited by 50% by carbamoyl phosphate at a concentration of 13 mM. Binding of N-acetylglutamate to carbamoyl-phosphate synthetase required the presence of both free Mg2+ ions and MgATP, and was inhibited by Ca2+ ions and by N-carbamoylglutamate. The known activation of carbamoyl-phosphate synthetase by free Mg2+ is due to an increased affinity of the enzyme for N-acetylglutamate. Binding of N-acetylglutamate to carbamoyl-phosphate synthetase was a slow process: at N-acetylglutamate concentrations below 0.5 mM maximal binding was not completed within 30 min. The rate of binding increased with increasing N-acetylglutamate concentrations. Dissociation of N-acetylglutamate from the enzyme was relatively fast, with a half-time of about 5 min. Under all conditions studied there was a close relationship between carbamoyl-phosphate synthetase activity and the amount of N-acetylglutamate bound to the enzyme. The data are discussed in relation to the control of carbamoyl-phosphate synthetase in the intact hepatocyte.

Animals↗
PubMed1983

Bicarbonate and the pathway of glutamate oxidation in isolated rat-liver mitochondria.

1. The factors affecting the pathway of glutamate oxidation were studied in isolated rat-liver mitochondria in incubations of 2-3 min. 2. It was found that bicarbonate at a physiological concentration has a profound effect on the pathway of glutamate oxidation. Ammonia formation via glutamate dehydrogenase is stimulated by bicarbonate [from 5.48 +/- 0.29 (n = 10) to 9.57 +/- 0.73 (n = 8) nmol X min-1 X mg protein-1], whereas aspartate formation via the transamination pathway is inhibited [from 38.41 +/- 2.24 (n = 9) to 24.56 +/- 3.28 (n = 6) nmol X min-1 X mg protein-1]. 3. Bicarbonate has no effect on the rate of transport of glutamate via the glutamate-hydroxyl translocator. 4. The interaction of bicarbonate with the pathway of glutamate oxidation occurs primarily at the level of succinate dehydrogenase, due to competitive inhibition of the enzyme by bicarbonate. 5. Inhibition by bicarbonate of the transamination pathway leads to a decrease in intramitochondrial 2-oxoglutarate, so that the deamination pathway is stimulated. 6. Using an equation which describes flux through glutamate dehydrogenase kinetically, it could be shown that the bicarbonate-induced decrease in intramitochondrial 2-oxoglutarate quantitatively accounts for the enhanced rate of deamination. 7. It is concluded that in the intact liver flux through glutamate dehydrogenase is sufficient to account for the ammonia formation required for urea synthesis from substrates such as alanine.

Adenosine Diphosphate↗
PubMed1983

Measurement of binding of adenine nucleotides and phosphate to cytosolic proteins in permeabilized rat-liver cells.

1. A method is described for measuring the binding of metabolites to cytosolic proteins in situ in isolated rat-liver cells treated with filipin to render the plasma membrane permeable to compounds of low molecular weight. 2. There is no binding of ATP or inorganic phosphate to cytosolic proteins, either in the presence or in the absence of Mg2+. 3. Binding of ADP to cytosolic proteins occurs both in the absence and in the presence of Mg2+. The concentration of binding sites was 0.68 and 0.52 mumol/g dry weight of cells (n = 3-4) in the absence and presence of Mg2+, respectively. The corresponding Kd values were 320 microM and 235 microM.

Adenosine Diphosphate↗
PubMed1983

Fluorimetric determination of carbamoyl phosphate.

A simple fluorimetric assay for the determination of carbamoyl phosphate in tissue extracts is described. In the assay, production of ATP from carbamoyl phosphate and ADP by carbamate kinase is coupled to the formation of NADPH, using glucose, hexokinase, NADP+, and glucose-6-phosphate dehydrogenase. Production of NADPH in this system proved to be equal to the amount of carbamoyl phosphate present.

Adenosine Diphosphate↗
PubMed1983

Control of mitochondrial respiration.

The control theory of Kacser and Burns [in: Rate Control of Biological Processes (Davies, D.D. ed) pp. 65-104, Cambridge University Press, London, 1973] and Heinrich and Rapoport [Eur. J. Biochem. (1974) 42, 97-105] has been used to quantify the amount of control exerted by different steps on mitochondrial oxidative phosphorylation in rat-liver mitochondria. Inhibitors were used to manipulate the amount of active enzyme. The control strength of the adenine nucleotide translocator was measured by carrying out titrations with carboxyatractyloside. In state 4, the control strength of the translocator was found to be zero. As the rate of respiration was increased by adding hexokinase, the control strength of the translocator increased to a maximum value of approximately 30% at approximately 80% of state 3 respiration. In state 3, control of respiration is distributed between a number of steps, including the adenine nucleotide translocator, the dicarboxylate carrier and cytochrome c oxidase. The measured values for the distribution of control agree very well with those calculated with the aid of a model for mitochondrial oxidative phosphorylation developed by Bohnensack et al. [Biochim. Biophys. Acta (1982) 680, 271-280].

Adenosine Diphosphate↗
PubMed1982

An evaluation of the metabolite indicator method for determining the cytosolic phosphate potential in rat liver cells.

The cytosolic phosphate potential was estimated in isolated rat liver parenchymal cells incubated with various gluconeogenic substrates. The value of the cytosolic [ATP]/[ADP][Pi] ratio was either estimated directly from measurements of ATP, ADP and Pi after digitonin fractionation of the cells, or calculated by the metabolite indicator method. When cells were incubated with lactate, pyruvate or alanine so that net flux through the indicator enzymes was in gluconeogenic direction, there was excellent agreement between the values obtained by the two methods over a wide range of fluxes. However, when the cells were incubated with substrates that could be converted both to glucose and to lactate so that net flux through the indicator enzymes was in the glycolytic direction, a large difference in the values of the cytosolic [ATP]/([ATP][Pi]) ratio as derived by the two methods was observed. It is concluded that the reaction catalyzed by glyceraldehyde-3-phosphate dehydrogenase plus 3-phosphoglycerate kinase is out of equilibrium when flux through the reaction is the glycolytic direction, and that use of the metabolite indicator method for the calculation of the cytosolic phosphate potential under these conditions leads to erroneous values.

Adenosine Diphosphate↗
PubMed1982

Transport of N-acetylglutamate in rat-liver mitochondria.

The permeability properties of the rat-liver mitochondrial membrane for N-acetylglutamate, the activator of carbamoyl-phosphate synthetase (ammonia), were studied. 1. Transport of N-acetylglutamate into the mitochondria was only observed in partially or fully de-energized mitochondria and when the extramitochondrial concentration was unphysiologically high (in the mM range). However, even under these conditions the intramitochondrial concentration of N-acetylglutamate was much lower than that outside. 2. Mitochondrial N-acetylglutamate efflux only occurs when the mitochondria are in an energized state. At 25 degrees C, at an intramitochondrial N-acetylglutamate concentration of 0.7-1.0 nmol/mg protein, efflux proceeds at a rate of about 0.05 nmol X min-1 X mg protein-1. This is 10-fold lower than the maximal rate of N-acetylglutamate synthesis in the mitochondria. 3. Homologous exchange between intramitochondrial N-[14C]acetylglutamate and extramitochondrial unlabelled N-acetylglutamate could not be demonstrated. 4. It is concluded that transport of N-acetylglutamate in vivo is effectively unidirectional, out of the mitochondria. This behaviour is in accordance with the physiological requirement for efflux of N-acetylglutamate from the mitochondria in order to be degraded in the cytosol.

Animals↗
PubMed1982

Quantification of the contribution of various steps to the control of mitochondrial respiration.

Using principles developed by Kacser and Burns ((1973) in Rate Control of Biological Processes (Davies, D. D., ed) pp. 65-104, Cambridge University Press, London) and Heinrich and Rapoport ((1974) Eur. J. Biochem. 42, 97-105), inhibitor titration studies were carried out in order to quantify the amount of control (control strength) exerted by different steps in oxidative phosphorylation on the rate of mitochondrial oxygen uptake. In the resting state of respiration, nearly all control is exerted by the passive permeability of the mitochondrial inner membrane to protons. In the intermediate states and even in the active state of respiration, control is distributed among different steps including the adenine nucleotide translocator and cytochrome c oxidase. It is therefore not possible to speak of the rate-limiting step in oxidative phosphorylation. Since both the adenine nucleotide translocator and cytochrome c oxidase appear to be rate-controlling, hypotheses suggesting that respiration is fully controlled by either of these two steps alone need to be modified.

Animals↗
PubMed1982

Activity of carbamoyl-phosphate synthetase (ammonia) in isolated rat-liver mitochondria: cycling of carbamoyl phosphate in the absence of ornithine.

1. When NH3 was added to isolated rat-liver mitochondria incubated with succinate and bicarbonate, oxidation of succinate was stimulated to a greater extent than could be accounted for by the net formation of carbamoyl phosphate. 2. Measurement of the rate of incorporation of [14C]bicarbonate into carbamoyl phosphate, after the mitochondria had been preincubated with NH3 and unlabelled bicarbonate, revealed that flux through carbamoyl-phosphate synthetase (ammonia) was much greater than the net formation of carbamoyl phosphate indicated. 3. It is concluded that part of the carbamoyl phosphate produced in the absence of ornithine is degraded. About 20% of the degradation can be accounted for by non-enzymatic reactions of carbamoyl phosphate outside the mitochondria. It is proposed that the remainder of the degradation of carbamoyl phosphate occurs by partial reversal of the reaction of carbamoyl-phosphate synthetase.

Adenosine Triphosphate↗
PubMed1981

Relationship between the rate of citrulline synthesis and bulk changes in the intramitochondrial ATP/ADP ratio in rat-liver mitochondria.

1. The relationship between intramitochondrial and extramitochondrial ATP-utilizing systems and the intramitochondrial ATP/ADP ratio was studied in isolated rat-liver mitochondria. Citrulline synthesis was used as an intramitochondrial ATP-utilizing system, and glucose-6-phosphate synthesis as an extramitochondrial ATP-utilizing system. The intramitochondrial ATP/ADP ratio was manipulated in three ways: with succinate and different concentrations of malonate and/or hexokinase; with 2-oxoglutarate (plus oligomycin) and different concentrations of hexokinase; and with added ATP in uncoupled mitochondria (oligomycin present). 2. Under all conditions used, citrulline synthesis was strictly correlated with the bulk intramitochondrial ATP/ADP ratio. 3. The curve relating citrulline synthesis and intramitochondrial ATP/ADP was shifted towards lower ATP/ADP ratios when the activity of carbamoyl-phosphate synthetase was enhanced by increasing the mitochondrial content of N-acetylglutamate. 4. It is concluded that under the experimental conditions used the intramitochondrial adenine nucleotides behave as a homogeneous pool.

Adenosine Diphosphate↗
PubMed1981

Control mechanisms of energy-dependent metabolic pathways in hepatocytes.

The control of respiration was studied in isolated rat-liver cells and isolated rat-liver mitochondria. When isolated rat-liver cells are perfused with different concentrations of lactate at a constant lactate/pyruvate ratio, the rate of mitochondrial respiration is a linear function of the affinity for the respiratory chain; the affinity term contains the intramitochondrial redox state and the extramitochondrial phosphate potential. However, when the cells are perfused with ammonia in addition to lactate (+ pyruvate), the stimulation of the oxygen uptake is not accompanied by an increase in the affinity. In order to obtain further insight into the relationship between oxygen uptake and intramitochondrial and extramitochondrial phosphate potential, experiments were also carried out with isolate rat-liver mitochondria incubated with an extramitochondrial ATP-utilizing system (glucose plus hexokinase) or an intramitochondrial ATP-utilizing system (citrulline synthesis). The results suggest that the rate of oxygen consumption is controlled by the intramitochondrial phosphate potential.

Adenosine Triphosphate↗
PubMed1980

Origin of the ammonia found in protein-free extracts of rat-liver mitochondria and rat hepatocytes.

1. Protein-free extracts of isolated rat-liver mitochondria contain 5.17 +/- 0.19 nmol ammonia/mg protein [cf. Harris, E. J. and Bassett, D. J. (1971) FEBS Lett. 19, 214-217]. 2. The ammonia found in the protein-free extracts does not originate from lysosomes contaminating the mitochondrial preparation. 3. When isolated mitochondria are incubated with ornithine, 14CO2 and a source of ATP a small amount of citrulline is formed. This amount is stoichiometrically equivalent to the ammonia that disappears from the extramitochondrial space, whereas the amount of ammonia found in the protein-free extracts of the mitochondria remains unchanged. Similar results were obtained when the reductive amination of 2-oxoglutarate was used as an ammonia-consuming reaction. 4. When isolated mitochondria are incubated under conditions such that the glutamate dehydrogenase and 3-hydroxybutyrate dehydrogenase reactions reach equilibrium, the thermodynamically active concentration of ammonia is not equal to the concentration measured in the protein-free extracts. 5. About 80% of the ammonia found in protein-free extracts of rat-liver mitochondria is derived from a component or components with a molecular weight of greater than or equal to 50,000. 6. Protein-free extracts of isolated rat-liver cells contain considerable amounts of ammonia. After digitonin fractionation this ammonia is found in the protein-free extract of the particulate fraction. 7. It is concluded that the ammonia found in protein-free extracts of rat-liver tissue is derived from a component or components in the mitochondria and is released during deproteinization.

Ammonia↗