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I Alkonyi

Publications and source records attributed to I Alkonyi.

At least 19 recordsLinked to original sources
PubMed1994

[Specific enzyme diagnosis in mitochondrial myopathies and encephalomyopathies].

Mitochondrial enzyme activities (cytochrome c-oxidase = COX, carnitine acyl-transferase = CAT, citrate synthase = CS, lipoamide dehydrogenase = lipDH from the pyruvate-dehydrogenase complex, lactate dehydrogenase = LDH, and malate-dehydrogenase = MDH) were measured from progressive myopathy/encephalomyopathy. Cytochrome oxidase (COX) deficiency was detected from muscle or liver tissues, adult type of COX defectus had been diagnosed in 1 case and infantile type in further 6 cases. The 3 familial atactic children showed decreased activity of carnitine acetyl-transferase, too.

Adolescent↗
PubMed1991

Isolation and characterization of carnitine acetyltransferase from S. cerevisiae.

Carnitine acetyltransferase was isolated from yeast Saccharomyces cerevisiae with an apparent molecular weight of 400,000. The enzyme contains identical subunits of 65,000 Da. The Km values of the isolated enzyme for acetyl-CoA and for carnitine were 17.7 microM and 180 microM, respectively. Carnitine acetyltransferase is an inducible enzyme, a 15-fold increase in the enzyme activity was found when the cells were grown on glycerol instead of glucose. Carnitine acetyltransferase, similarly to citrate synthase, has a double localization (approx. 80% of the enzyme is mitochondrial), while acetyl-CoA synthetase was found only in the cytosol. In the mitochondria carnitine acetyltransferase is located in the matrix space. The incorporation of 14C into CO2 and in lipids showed a similar ratio, 2.9 and 2.6, when the substrate was [1-14C]acetate and [1-14C]acetylcarnitine, respectively. Based on these results carnitine acetyltransferase can be considered as an enzyme necessary for acetate metabolism by transporting the activated acetyl group from the cytosol into the mitochondrial matrix.

Acetylcarnitine↗
PubMed1991

Kinetic advantage of the interaction between the fatty acid beta-oxidation enzymes and the complexes of the respiratory chain.

Respiration-linked oxidation of 3-hydroxybutyryl-CoA, crotonyl-CoA and saturated fatty acyl (C4, C8 and C14)-CoA esters was studied in different mitochondrial preparations. Oxidation of acyl-CoA esters was poor in intact mitochondria; however, it was significant, as well as, NAD+ and CoA-dependent in gently and in vigorously sonicated mitochondria. The respiration-linked oxidation of crotonyl-CoA and 3-hydroxybutyryl-CoA proceeded at much higher rates (over 700%) in gently disrupted mitochondria than in completely disrupted mitochondria. The redox dye-linked oxidation of crotonyl-CoA (with inhibited respiratory chain) was also higher in gently disrupted mitochondria (149%) than in disrupted ones. During the respiration-linked oxidation of 3-hydroxybutyryl-CoA the steady-state NADH concentrations in the reaction chamber were determined, and found to be 8 microM in gently sonicated and 15 microM in completely sonicated mitochondria in spite of the observation that the gently sonicated mitochondria oxidized the 3-hydroxybutyryl-CoA much faster than the completely sonicated mitochondria. The NAD(+)-dependence of 3-hydroxybutyryl-CoA oxidation showed that a much smaller NAD+ concentration was enough to half-saturate the reaction in gently disrupted mitochondria than in completely disrupted ones. Thus, these observations indicate the positive kinetic consequence of organization of beta-oxidation enzymes in situ. Respiration-linked oxidation of butyryl-, octanoyl- and palmitoyl-CoA was also studied and these CoA intermediates were oxidized at approx. 50% of the rate of crotonyl- and 3-hydroxybutyryl-CoA in the gently disrupted mitochondria. In vigorously disrupted mitochondria the oxidation rate of these saturated acyl-CoA intermediates was hardly detectable indicating that the connection between the acyl-CoA dehydrogenase and the respiratory chain had been disrupted.

Acyl Coenzyme A↗
PubMed1990

Surplus acylcarnitines in the plasma of starved rats derive from the liver.

The method used here to assess the contribution of liver to plasma acylcarnitine is based on the idea that in rat, shortly after administration of [3H]butyrobetaine the [3H]carnitine appearing in the plasma derives from the liver and so does the acyl moiety of [acyl-3H] carnitine. In the perchloric acid extracts of plasma and liver, the ester fraction of total carnitine was determined by enzymatic analysis and that of [3H]carnitines was determined by high performance liquid chromatography. The ester fraction of total carnitine in the plasma of fed rats was 32.6% while that of [3H]carnitines was 67.9%, 1 h following injection of [3H]butyrobetaine. For 48 h starved rats the equivalent values were 54.2 and 84.0%, respectively. 24 h after the administration of [3H]butyrobetaine, the ester content became the same in the total and [3H]carnitines. That the newly synthesized carnitine was more acylated (67.9 versus 32.6%, fed) indicates that liver exports acyl groups with carnitine as carrier. The observation that the ester fraction in the newly synthesized plasma carnitine increased with fasting (84.0 versus 67.9%) indicates that the surplus plasma acylcarnitine in fasting ketosis derives from the liver. Perfused livers, however, released carnitine with the same ester content (60-61%) whether they were from fed or fasted animals. Probably, the increased plasma [acylcarnitine] in fasting develops not by an increased ester output from the liver but by an altered handling in extrahepatic tissues.

Acylation↗
PubMed1990

Role of the liver in carnitine metabolism: the mechanism of development of carnitine-deficient status in guinea-pigs.

It was shown that carnitine deficiency and an impairment of the conversion of butyrobetaine into carnitine develops not only in ascorbic acid-deficient guinea-pigs but also in partially starved animals. We propose that the same mechanism, an absolute or relative ascorbic acid deficiency, is operating in both nutritional states. An increased urinary excretion greatly contributes to the development of carnitine deficiency in guinea-pigs, both in ascorbic acid deficiency and starvation. With respect to the greatly increased excretion, guinea-pig carnitine deficiency resembles the human disorder and may serve as model for it.

Animals↗
PubMed1987

Ester composition of carnitine in the perfusate of liver and in the plasma of donor rats.

When the carnitine pool of fed rats was labelled with tritium, in non-recirculating perfusate of their liver 44% of acid-soluble 3H activity was identified as free carnitine and 47% as short-chain acylcarnitine. Of the latter component acetylcarnitine accounted for 30% and propionylcarnitine for 10% of total acid-soluble. In plasma the contribution of short-chain acylcarnitines to total carnitine in fed, fasted and diabetic rats was 15.6%, 43.1% and 48.0%, respectively. Recirculating perfusion of livers from the same animals revealed that livers from fed rats released short-chain acylcarnitines as much as 56.2% of total and this proportion did not increase further in the other two groups. At the same time, ketone bodies in the perfusate increased gradually in the fed, fasted and diabetic group, paralleling the plasma ketone levels. Although liver supplies the organism with carnitine the increment of plasma short-chain acylcarnitines seen in ketosis is not a result of some extra output by the liver.

Animals↗
PubMed1987

Interaction between NAD-dependent isocitrate dehydrogenase, alpha-ketoglutarate dehydrogenase complex, and NADH:ubiquinone oxidoreductase.

Interaction between the alpha-ketoglutarate dehydrogenase complex and NAD+-dependent isocitrate dehydrogenase was detected with a variety of techniques including polyethylene glycol precipitation, ultracentrifugation, and centrifugal gel filtration on a Sepharose 6B column. The interaction was specific in that citrate synthase, cytosolic malate dehydrogenase, and NADP-dependent isocitrate dehydrogenase did not interact with alpha-ketoglutarate dehydrogenase complex. The interaction was not inhibited by either 0.1 M KCl or 0.4 M (NH4)2SO4, but was completely prevented by 5% glycerol. A new method for the preparation of NADH: ubiquinone oxidoreductase resulted in an enzyme having a protein subunit composition similar to that of classical complex I preparation. Evidence is given for the existence of ternary complexes containing NADH:ubiquinone oxidoreductase-alpha-ketoglutarate dehydrogenase complex-NAD-dependent isocitrate dehydrogenase and NADH: ubiquinone oxidoreductase-alpha-ketoglutarate dehydrogenase complex-succinate thiokinase. These data suggest that a part of the citric acid cycle may be located in the vicinity of NADH: ubiquinone oxidoreductase. These complexes may facilitate the transport of metabolites among these enzymes without their equilibrating with the whole compartment.

Cytosol↗
PubMed1987

Enhanced uptake of carnitine by perfused rat liver following starvation.

Previously, the release of carnitine from the perfused rat liver was found to be protein-mediated, dependent on the nutritional state but not on metabolic energy. Further, it was shown to exceed the physiological demand by about 10-fold (Sandor et al. (1985) Biochim. Biophys. Acta 835, 83-91). In the present study the uptake of carnitine by perfused rat liver has been investigated. The liver tissue and the perfusate were in equilibrium when the carnitine concentration in the perfusate was close to 45 microM, physiological in the rat plasma. Under this condition, when no net carnitine transport occurred, an unidirectional uptake of L-[3H]carnitine was observed. Quantitatively, the uptake rate was 355 +/- 60 (S.D.) nmol/h per 100 g body weight at 45-50 microM perfusate concentration. This uptake capacity balances the previously reported excessive release (Sandor et al., op. cit.). On this basis we propose that a futile release/uptake cycle operates in carnitine transport across the liver cell membrane. Liverse of 24-h starved rats took up L-[3H]carnitine at 56% higher rate from the perfusate (75 microM) than livers of fed rats. Kinetic analysis revealed that fasting caused a decrease in Km value from 4.22 mM to 2.59 mM, whereas Vmax remained practically unchanged, average 0.95 mumol/min per 100 g body weight. D-[3H]Carnitine was transported at the same rate as L-carnitine and underwent the effect of fasting as well. The uptake was partially inhibited by 1 mM 2,4-dinitrophenol and 5 mM KCN, showing its dependency on metabolic energy. If Li+ replaced Na+ a strong inhibitory effect (to 20% of control) was observed, which suggests a co-transport of carnitine with Na+. Mersalyl, an SH reagent, had no effect on the uptake, whereas it practically abolished the release of carnitine from the perfused livers. This observation suggests that the inward and outward transport of carnitine are mediated by two different proteins.

2,4-Dinitrophenol↗
PubMed1986

Isolation and characterization of 3-hydroxyacyl coenzyme A dehydrogenase-binding protein from pig heart inner mitochondrial membrane.

3-Hydroxyacyl coenzyme A (CoA) dehydrogenase-binding protein was solubilized from inner mitochondrial membrane by using taurodeoxycholate at high ionic strength. The binding protein was isolated from the suspension using 3-hydroxyacyl-CoA dehydrogenase affinity chromatography. The protein eluted from the affinity column had a molecular weight of approximately 150,000, as determined by gel filtration. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed that the protein is a dimer consisting of 69,000 and 71,000 molecular weight subunits. The enzyme binding capacity of this protein was tested with a polyethylene glycol precipitation method: 0.5 mg of enzyme could be precipitated together with 1 mg of binding protein, showing that 1 mol of binding protein binds 1 mol of enzyme. This protein had no affinity toward malic dehydrogenase, citrate synthase, and fumarase. The approximately 2-fold increase in the 3-hydroxyacyl-CoA dehydrogenase activity when it was measured in the presence of the binding protein is additional evidence of enzyme-binding protein interaction. When incorporated into liposomes, the binding protein retained its ability to bind 3-hydroxyacyl-CoA dehydrogenase, but did not bind malic dehydrogenase, citrate synthase, and fumarase. These results suggest that the protein isolated by us has a specific function in anchoring a beta-oxidation enzyme to the matrix surface of the mitochondrial membrane.

3-Hydroxyacyl CoA Dehydrogenases↗
PubMed1985

Release of carnitine from the perfused rat liver.

Perfused rat liver was shown to be the proper model for studies on hepatic cellular transport of carnitine. During recirculating perfusion the livers kept equilibrium with 45 nmol/ml total carnitine in perfusate, exhibited concentrative uptake and there was no sign of artificial leakage. The release side of the carnitine transport was characterized by utilizing outflow perfusions. The livers from fed rats exported daily 9.93 mumol per 100 g body weight total carnitine. This release rate is 4- or 10-fold higher than the estimated daily turnover in vivo or the measured urinary excretion. Therefore, the major part of the released carnitine has to re-enter the liver. The outward carnitine transport does not depend on energy or the Na+-K+ pump, since it did not respond to metabolic poisons and ouabain. However, the release rate was strongly inhibited by mersalyl and showed saturability in function of tissue carnitine levels. The Vmax of the saturable outward transport system was 2.47 nmol . min-1 . g-1 liver, the apparent Km was 0.27 mM tissue level (both as compared to total carnitine). These data showed the outward transport of carnitine from the liver to be protein mediated. The contribution of a diffusion (nonsaturable) component was estimated to be 20-25% in the range of tissue levels occurring in vivo. The rate of carnitine release from the liver decreased as an effect of 24 h starvation from the daily 9.92 mumol release to 6.55 mumol on 100 g body weight basis. This decrease is more pronounced when the release rates are expressed on the basis of tissue carnitine levels. The resulting value can be called rate constant (at the linear part of the saturation curve, Fig. 5) and it decreased to 5.00 min-1 from 8.41 min-1 as an effect of starvation. We have concluded that the altered parameters of carnitine transport across the liver cell is decisive in developing the higher hepatic carnitine concentration in the fasted state.

2,4-Dinitrophenol↗
PubMed1983

A study on the physical interaction between the pyruvate dehydrogenase complex and citrate synthase.

In this paper, physicochemical evidence is given for the association between the pyruvate dehydrogenase complex (EC 1.2.4.1) and citrate synthase (EC 4.1.3.7) with two gel chromatographic techniques with poly(ethylene glycol) co-precipitation and with ultracentrifugation. Experiments with active enzyme gel chromatography indicate that citrate synthase also associates with pyruvate dehydrogenase complex in its functioning state. Citrate synthase binds to the isolated transacetylase core of pyruvate dehydrogenase complex, but in the binding to the whole pyruvate dehydrogenase complex the two other components of the complex are also involved. One pyruvate dehydrogenase complex can bind 10-11 citrate synthase dimers, and the dissociation constant is about 5.7-6.0 microM as determined by two independent methods. The association between the pyruvate dehydrogenase complex and citrate synthase raises the possibility of the dynamic compartmentation of acetyl-CoA in the mitochondria which results in the direction of acetyl-CoA from pyruvate towards citrate.

Animals↗
PubMed1983

Association between the alpha-ketoglutarate dehydrogenase complex and succinate thiokinase.

The kinetic parameters of the individual reaction of pig heart alpha-ketoglutarate dehydrogenase complex, succinate thiokinase and the alpha-ketoglutarate dehydrogenase complex-succinate thiokinase coupled system were studied. The KCoAm of alpha-ketoglutarate dehydrogenase complex and the K-succinyl CoAm of succinate thiokinase decreased in the coupled system when compared to those of the individual enzyme reactions. This phenomenon can be explained by the interaction between the alpha-ketoglutarate dehydrogenase complex and succinate thiokinase. By means of poly(ethylene glycol) precipitation, ultracentrifugation and gel chromatography we were able to detect a physical interaction between the alpha-ketoglutarate dehydrogenase complex and succinate thiokinase. Of the seven investigated proteins only succinate thiokinase showed association with alpha-ketoglutarate dehydrogenase complex. On the other hand, succinate thiokinase did not associate with other high molecular weight mitochondrial enzymes such as pyruvate dehydrogenase complex and glutamate dehydrogenase. On this basis, the interaction between succinate thiokinase and alpha-ketoglutarate dehydrogenase complex was assumed to be specific. These in vitro data raise the possibility that a portion of the citric acid cycle enzymes exists as a large multienzyme complex in the mitochondrial matrix.

Animals↗
PubMed1983

Elementary steps in the reaction of the pyruvate dehydrogenase complex from pig heart. Kinetics of thiamine diphosphate binding to the complex.

In the progress curve of the reaction of the pyruvate dehydrogenase complex, a lag phase was observed when the concentration of thiamin diphosphate was lower than usual (about 0.2-1 mM) in the enzyme assay. The length of the lag phase was dependent on thiamin diphosphate concentration, ranging from 0.2 min to 2 min as the thiamin diphosphate concentration varied from 800 nM to 22 nM. The lag phase was also observed in the elementary steps catalyzed by the pyruvate dehydrogenase component. A Km value of 107 nM was found for thiamin diphosphate with respect to the steady-state reaction rate following the lag phase. The pre-steady-state kinetic data indicate that the resulting lag phase was the consequence of a slow holoenzyme formation from apoenzyme and thiamin diphosphate. The thiamin diphosphate can bind to the pyruvate dehydrogenase complex in the absence of pyruvate, but the presence of 2 mM pyruvate increases the rate constant of binding from 1.4 X 10(4) M-1 S-1 to 1.3 X 10(5) M-1 S-1 and decreases the rate constant of dissociation from 2.3 X 10(-2) S-1 to 4.1 X 10(-3) S-1. On the other hand, the effect of pyruvate on the thiamin diphosphate binding revealed the existence of a thiamin-diphosphate-independent pyruvate-binding site in the pyruvate dehydrogenase complex. Direct evidence was also obtained with fluorescence techniques for the existence of this binding site and the dissociation constant of pyruvate was found to be 0.38 mM. On the basis of these data we have proposed a random mechanism for the binding of pyruvate and thiamin diphosphate to the complex. Binding of substrates to the enzyme complex caused an increase in the fluorescence of the dansylaziridine-labelled pyruvate dehydrogenase complex, showing that binding of substrates to the complex is accompanied by structural changes.

Animals↗
PubMed1983

Combined effect of ascorbic acid deficiency and underfeeding on the hepatic carnitine level in guinea-pigs.

Liver carnitine level decreased from 249 +/- 16.1 nmoles/g (mean +/- SEM) control value to 148 +/- 9.8 nmoles/g (59.4%) in ascorbic acid deficient guinea-pigs, while in the underfed ('pair-fed') group it decreased to 181 +/- 14.1 nmoles/g (72.6%). Underfeeding also resulted in lower ascorbic acid levels; the depression of carnitine in the underfed animals could be prevented by an overdose (200 mg daily) of ascorbic acid.

Animals↗
PubMed1983

Paracatalytic inactivation of pig heart pyruvate dehydrogenase complex.

The phenomenon of paracatalytic inactivation has been demonstrated and characterized with the pig heart pyruvate dehydrogenase complex. The enzyme became progressively inactive when it was preincubated in the presence of pyruvate, thiamine pyrophosphate, and extrinsic oxidative agent, 2,6-dichloroindophenol. Not only the overall reaction of enzyme complex but the individual reactions catalyzed by enzyme components, pyruvate dehydrogenase, and dihydrolipoamide acetyltrasferase, were reduced after preincubation. The observed inactivation is due to two factors: (i) covalent incorporation of the pyruvate atoms, and (ii) formation of a thiamine pyrophosphate analog. (i) A covalent incorporation of radioactivity from [2-14C]pyruvate into the enzyme complex, which was proportional to the inhibition of overall reaction was observed. Seventy-eight percent of the radioactivity incorporated into the dihydrolipoamide transacetylase but not into the lipoic acid. Thus, modification of the dihydrolipoamide acetyltransferase component can be attributed to the covalent incorporation of pyruvate atoms. (ii) During inactivation, the enzyme-bound thiamine pyrophosphate underwent a modification resulting in the formation of a thiamine pyrophosphate analog, presumably thiamine thiazolone pyrophosphate, which inhibited exclusively the pyruvate dehydrogenase component.

2,6-Dichloroindophenol↗
PubMed1982

On carnitine content of the human breast milk.

The concentration of total carnitine in human breast milk remained at a constant mean level near 62.9 range: 56.0-69.8/nmoles/ml during the first 21 days postpartum. The carnitine level fell significantly to 35.2 +/- 1.26 nmoles/ml until the 40-50th day. The carnitine concentrations did not depend on the secreted volume of milk. In comparison, fresh and commercial pasteurized cow's milk contained 206.2 (range: 192-269) nmoles/ml and 160.0 (range: 158-200) nmoles/ml carnitine, respectively. In the sera of nursing mothers, the concentration of total carnitine was lower on the first day after delivery (27.2 +/- 1.19 nmoles/ml) but returned to normal by the 21st day postpartum (38.8 +/- 2.97 nmoles/ml).

Animals↗
PubMed1981

Concurrent occurrence of elevated ketone body and depressed carnitine level in underfed guinea-pigs.

Food ingestion of male guinea-pigs was restricted to 10 g/day. Total carnitine content of liver of the underfed animals fell to 3.75 mumoles per liver (46.1% of control). Serum level of total carnitine was 35.0 nmoles/ml (64.4%) in the underfed animals while they developed high ketonemia, 384.2 nmoles/ml (555.2%). In "restricted' animals the carnitine levels also decreased in the muscles.

Animals↗