Biomedical subjects

G Kispal

Publications and source records attributed to G Kispal.

At least 37 records · Page 2Linked to original sources

PubMed1997

The ABC transporter Atm1p is required for mitochondrial iron homeostasis.

The function of the ABC transporter Atm1p located in the mitochondrial inner membrane is not yet known. To study its cellular role, we analyzed a mutant in which ATM1 was disrupted. Delta atm1 cells are deficient in the holoforms, but not the apoforms of heme-carrying proteins both within and outside mitochondria, yet both synthesis and transport of heme are functional. Delta atm1 cells are hypersensitive for growth in the presence of oxidative reagents, and they contain increased levels of the antioxidant glutathione, in particular of its oxidized form. Mitochondria deficient in Atm1p accumulate 30-fold higher levels of free iron as compared to wild-type organelles, i.e. three-fold more than mitochondria deficient in frataxin, the protein mutated in Friedreich's ataxia. The increased mitochondrial iron content may be causative of the oxidative damage of heme-containing proteins in delta atm1 cells. Our data assign an important function to Atm1p in mitochondrial iron homeostasis.

ATP-Binding Cassette Transporters↗

PubMed1996

Heme binding to a conserved Cys-Pro-Val motif is crucial for the catalytic function of mitochondrial heme lyases.

Covalent attachment of heme to the apoforms of mitochondrial cytochromes c and c1 requires the activity of cytochrome c heme lyase (CCHL) and cytochrome c1 heme lyase (CC1HL), respectively. The two enzymes differ in their cytochrome specificity, but they are related in sequence, and both contain conserved Cys-Pro-Val (CPV) motifs. By using various in vitro assays we investigated whether heme can bind directly to heme lyases and whether the CPV motif may be involved in heme binding. Heme stabilized CC1HL, as a model protein, in a folded, protease-resistant conformation, stimulated the refolding of CC1HL after urea denaturation, and inhibited the import of the CC1HL precursor into mitochondria. These effects were not observed with a point mutant, CC1HLSPV, in which cysteine was replaced by serine, and with CC1HLDeltaCPV, in which the motif was deleted. These results show that heme lyases can bind heme directly, and they identify the CPV sequence as a structural element important for this interaction. The phenotype of a yeast mutant expressing CC1HLSPV is in good agreement with such a role of the CPV motif. The mutant cells accumulate the heme-free intermediate form of cytochrome c1 and display a severe deficiency in the holo form. We suggest that the CPV motif forms a crucial part of the substrate binding site for heme.

Amino Acid Sequence↗

PubMed1996

Mitochondrial and cytosolic branched-chain amino acid transaminases from yeast, homologs of the myc oncogene-regulated Eca39 protein.

We have isolated a high copy suppressor of a temperature-sensitive mutation in ATM1, which codes for an ABC transporter of Saccharomyces cerevisiae mitochondria. The suppressor, termed BAT1, encodes a protein of 393 amino acid residues with an NH2-terminal extension that directs Bat1p to the mitochondrial matrix. A highly homologous protein, Bat2p, of 376 amino acid residues was found in the cytosol. Both Bat proteins show striking similarity to the mammalian protein Eca39, which is one of the few known targets of the myc oncogene. Deletion of a single BAT gene did not impair growth of yeast cells. In contrast, deletion of both genes resulted in an auxotrophy for branched-chain amino acids (Ile, Leu, and Val) and in a severe growth reduction on glucose-containing media, even after supply of these amino acids. Mitochondria and cytosol isolated from bat1 and bat2 deletion mutants, respectively, contained largely reduced activities for the conversion of branched-chain 2-ketoacids to their corresponding amino acids. Thus, the Bat proteins represent the first known isoforms of yeast branched-chain amino acid transaminases. The severe growth defect of the double deletion mutant observed even in the presence of branched-chain amino acids suggests that the Bat proteins, in addition to the supply of these amino acids, perform another important function in the cell.

Amino Acid Sequence↗

PubMed1993

Targeting and translocation of the phosphate carrier/p32 to the inner membrane of yeast mitochondria.

We analyzed the submitochondrial location and biogenesis pathway of the phosphate carrier (PiC), also termed p32, of Saccharomyces cerevisiae mitochondria, PiC/p32 was found to behave as an integral membrane protein that cofractionated with the ADP/ATP carrier of the inner membrane. Import of the precursor of PiC/p32 required a membrane potential across the inner membrane, supporting its localization to the inner membrane. This makes it unlikely that the major function of PiC/p32 is that of an import receptor on the surface of the mitochondrial outer membrane. Furthermore, we found that both receptors MOM72 and MOM19 were involved in the import pathway of the precursor of PiC/p32 with MOM72 being responsible for the bulk of import. Yeast PiC/p32 is thus not only structurally homologous to the ADP/ATP carrier, but has a similar targeting mechanism and submitochondrial location, supporting its classification as a member of the inner membrane carrier family.

Antibodies, Monoclonal↗

PubMed1993

Cloning and sequencing of a cDNA encoding Saccharomyces cerevisiae carnitine acetyltransferase. Use of the cDNA in gene disruption studies.

cDNA encoding for carnitine acetyltransferase (CAT) of yeast S. cerevisiae was isolated by screening a yeast cDNA lambda gt11 library with antibody. The whole coding sequence was obtained from the cDNA and from a YEP 13 DNA clone identified using the cDNA as probe. The coding sequence consists of 670 residues, which amounts to a molecular mass of 77,300 kDa. This cDNA was used successfully to disrupt the gene for the mitochondrial isoenzyme of CAT, which was shown by measuring the enzyme activity and by immunoblot. The acetylcarnitine content of these cells decreased significantly. A search in the PIR protein data base revealed that besides the known carnitine acyltransferases, choline acyltransferases are highly homologous to yeast CAT. The mitochondrial CAT-deficient (CAT-) cells were able to grow on different fermentable and nonfermentable carbon sources, even on acetate at the same rate as the parental strain. In contrast to these, 13C NMR studies revealed significant differences between parental and CAT- cells. In CAT-cells [3-13C]pyruvate was converted mainly to lactate and acetate, whereas in the parental cells alanine and tricarboxylic acid cycle intermediates were found as the main products of pyruvate metabolism beside acetate. These results suggest diminished flux through the pyruvate dehydrogenase complex in the absence of mitochondrial CAT in yeast cells.

Amino Acid Sequence↗

PubMed1993

Functional independence of the protein translocation machineries in mitochondrial outer and inner membranes: passage of preproteins through the intermembrane space.

The protein translocation machineries of the outer and inner mitochondrial membranes usually act in concert during translocation of matrix and inner membrane proteins. We considered whether the two machineries can function independently of each other in a sequential reaction. Fusion proteins (pF-CCHL) were constructed which contained dual targeting information, one for the intermembrane space present in cytochrome c heme lyase (CCHL) and the other for the matrix space contained in the signal sequence of the precursor of F1-ATPase beta-subunit (pF1 beta). In the absence of a membrane potential, delta psi, the fusion proteins moved into the intermembrane space using the CCHL pathway. In contrast, in the presence of delta psi they followed the pF1 beta pathway and eventually were translocated into the matrix. The fusion protein pF51-CCHL containing 51 amino acids of pF1 beta, once transported into the intermembrane space in the absence of a membrane potential, could be further chased into the matrix upon re-establishing delta psi. The sequential and independent movement of the fusion protein across the two membranes demonstrates that the translocation machineries act as distinct entities. Our results support a model in which the two translocation machineries can function independently of each other, but generally interact in a dynamic fashion to achieve simultaneous translocation across both membranes. In addition, the results provide information about the targeting sequences within CCHL. The protein does not contain a signal for retention in the intermembrane space; rather, it lacks matrix targeting information, and therefore is unable to undergo delta psi-dependent interaction with the protein translocation apparatus in the inner membrane.

Biological Transport↗

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

Studies on yeast peroxisomal citrate synthase.

Peroxisomal (nonmitochondrial) citrate synthase (CS2) has been purified from a Saccharomyces cerevisiae strain in which the gene for the mitochondrial citrate synthase (CS1) had been disrupted and no CS1 protein is produced. The enzyme, CS2, the sequence of which had been previously determined from its DNA, behaved differently from CS1 in its purification, kinetics, stability, and binding to the inner surface of mitochondrial inner membranes.

Animals↗

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↗

PubMed1989

Metabolic studies on citrate synthase mutants of yeast. A change in phenotype following transformation with an inactive enzyme.

We have studied the growth on acetate, the metabolism of acetate enzymes, and respiration of a series of citrate synthase mutants of Saccharomyces cerevisiae. The results confirmed and extended our previous observation that cytosolic citrate synthase is not necessary for growth on acetate. Deletion of mitochondrial citrate synthase (CS1) protein resulted in changes in metabolites, decrease in the amounts of pyruvate and alpha-ketoglutarate dehydrogenase complexes, reduced mitochondrial respiration of citrate and isocitrate, and an inability to grow on acetate. Using site-directed mutagensis, we constructed two separate CS1 proteins with mutations in the enzyme's active site. The mitochondria of cells carrying either site-directed mutagenized CS1 contained the inactive citrate synthase protein. With one mutant in which His313 was replaced with a glycine (CS1/H313G), growth on acetate was restored, and mitochondrial respiration of citrate and isocitrate increased toward parental levels as did the levels of several enzymes. With the other mutant CS1 in which Asp414 was replaced with a glycine (CS1/D414G), no growth on acetate or changes in other parameters was observed. We propose that the characteristics of the strain carrying the CS1 with a H313G mutation result from the formation of an intact Krebs cycle complex by the inactive but structurally unchanged H313G protein.

Acetates↗

PubMed1988

Metabolic changes in Saccharomyces cerevisiae strains lacking citrate synthases.

The yeast, Saccharomyces cerevisiae, contains two citrate synthase isoenzymes, mitochondrial (CS1) and cytosolic (CS2). In this study, we have examined the metabolic consequences of the absence of CS1, CS2, and both isoenzymes in the respective mutant strains CS1-, CS2-, and CS1-CS2-. No significant differences were found in the growth rates of the parental, CS1-, or CS2- strains when grown in the single carbon sources galactose, glycerol, lactate, pyruvate, or glutamate. However, in nonfermentable carbon sources, the lag period in growth of CS1- was approximately 4 times that of the parental strain and the CS2- mutant. This difference was found even in glutamate. The CS1- mutant failed to grow on acetate in either complete or minimal liquid medium. Total cellular citrate concentration in the CS1- compared to the parental strain was higher when the cells were grown in lactate or pyruvate. On these same substrates, the malate concentration was 2-fold higher in the CS1-mutant when compared to the parental or CS2- strains. The production of 14CO2 by CS1- from [1-14C]acetate was 36% and that from [2-14C]acetate was 9.2% of the amount from the parental or CS2- strains. The 14CO2 production from [1-14C]glutamate was 28% and 20% in CS1- and CS1-CS2-, respectively, compared to the parental strain. Since these results are not easily explained solely by the absence of mitochondrial citrate synthase enzyme, we also determined the activity of some other enzymes of the citric acid cycle and electron transport chain. We found decreased activity of pyruvate dehydrogenase complex, alpha-ketoglutarate dehydrogenase complex, and aconitase, while the rest of the citric acid cycle enzymes and oxidative enzymes did not change significantly. The same changes in enzyme activities were found in two different yeast strains carrying the same citrate synthase mutations.

Acetates↗

PubMed1988

Isolation, nucleotide sequence, and expression of a cDNA encoding pig citrate synthase.

Citrate synthase is a key enzyme of the Krebs tricarboxylic acid cycle and catalyzes the stereospecific synthesis of citrate from acetyl coenzyme A and oxalacetate. The amino acid sequence and three-dimensional structure of pig citrate synthase dimers are known, and regions of the enzyme involved in substrate binding and catalysis have been identified. A cloned complementary DNA sequence encoding pig citrate synthase has been isolated from a pig kidney lambda gt11 cDNA library after screening with a synthetic oligonucleotide probe. The complete nucleotide sequence of the 1.5-kilobase cDNA was determined. The coding region consists of 1395 base pairs and confirms the amino acid sequence of purified pig citrate synthase. The derived amino acid sequence of pig citrate synthase predicts the presence of a 27 amino acid N-terminal leader peptide whose sequence is consistent with the sequences of other mitochondrial signal peptides. A conserved amino acid sequence in the mitochondrial leader peptides of pig citrate synthase and yeast mitochondrial citrate synthase was identified. To express the pig citrate synthase cDNA in Escherichia coli, we employed the inducible T7 RNA polymerase/promoter double plasmid expression vectors pGP1-2 and pT7-7 [Tabor, S., & Richardson, C. C. (1985) Proc. Natl. Acad. Sci. U.S.A. 82, 1074-1078]. The pig citrate synthase cDNA was modified to delete the N-terminal leader sequence; then by use of a synthetic oligonucleotide linker, the modified cDNA was cloned into pT7-7 immediately following the initiator Met. A glutamate-requiring (citrate synthase deficient), recA- E. coli mutant, DEK15, was transformed with pGP1-2 and then pT7-7PCS. pT7-7PCS complemented the E. coli gltA mutation.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗

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

Effect of insulin and glucagon on the uptake of carnitine by perfused rat liver.

The possible direct effects of insulin and glucagon on carnitine uptake by perfused rat liver were studied with L-[3H]carnitine of an initial concentration of 50 microM in the perfusate. Insulin (10 nM) did not significantly affect the uptake by livers from fed animals. However, insulin could reverse the stimulated transport by livers from 24-h fasted animals, reducing the uptake rate from 852 +/- 54.1 to 480 +/- 39.9 (mean +/- S.E.), P less than 0.01 (rates are expressed as nmol per h per 100 g body wt). Glucagon (50 nM) stimulated the uptake rate when livers were either from fed (551 +/- 40.1 vs. 915 +/- 55.3, P less than 0.01) or from fasted animals (852 +/- 54.1 vs. 1142 +/- 88.1, P less than 0.02). Based on these and earlier observations, we propose that the carnitine concentration in rat liver is controlled by insulin and glucagon via cellular transport processes.

Animals↗

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

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↗