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M Neuburger

Publications and source records attributed to M Neuburger.

72 records · Page 4Linked to original sources
PubMed1990

A nuclear-encoded potato (Solanum tuberosum) mitochondrial tRNA(Leu) and its cytosolic counterpart have identical nucleotide sequences.

Sequencing of potato mitochondrial (mt) tRNA(Leu)(NAA) and of its cytosolic (cyt) counterpart revealed that these tRNAs are identical, except for a post-transcriptional modification: a Gm is present at position 18 in mt tRNA(Leu), instead of a G in cyt tRNA(Leu). Hybridization studies have shown that potato mt tRNA(Leu)(NAA) has a nuclear origin and must therefore be imported from the cytosol.

Base Sequence↗
PubMed1990

Intraorganelle localization and substrate specificities of the mitochondrial acyl-CoA: sn-glycerol-3-phosphate O-acyltransferase and acyl-CoA: 1-acyl-sn-glycerol-3-phosphate O-acyltransferase from potato tubers and pea leaves.

The mitochondrial sn-glycerol-3-phosphate and 1-acyl-sn-glycerol-3-phosphate O-acyltransferases from potato tubers and pea leaves were investigated with respect to their intraorganelle localization, their positional and substrate specificities, and their fatty acid selectivities. In mitochondria from potato tubers both enzymes were found to be located in the outer membrane. The 1-acyl-sn-glycerol-3-phosphate O-acyltransferase of pea mitochondria showed the same intraorganelle localization whereas the sn-glycerol-3-phosphate O-acyltransferase behaved like a soluble protein of the intermembrane space. The sn-glycerol-3-phosphate O-acyltransferase of both potato and pea mitochondria used sn-glycerol-3-phosphate but not dihydroxyacetone phosphate as acyl acceptor and exclusively catalyzed the formation of 1-acyl-sn-glycerol-3-phosphate which subsequently served as substrate for the second acylation reaction at its C-2 position. Both acyltransferases of potato as well as pea mitochondria showed higher activities with acyl-CoA than with the corresponding acyl-(acyl carrier protein) thioesters. When different acyl-CoA thioesters were offered separately, the sn-glycerol-3-phosphate O-acyltransferase of potato mitochondria displayed no fatty acid specificity whereas the enzyme of pea mitochondria revealed one for saturated acyl groups. On the other hand, the mitochondrial 1-acyl-sn-glycerol-3-phosphate O-acyltransferases from both potato tubers and pea leaves were more active on unsaturated than on saturated acyl-CoA thioesters. Furthermore, these enzymes preferentially used oleoyl- and linoleoyl-CoA when they were offered in a mixture with saturated ones, although the fatty acid selectivity of the pea enzyme was less pronounced than that of the potato enzyme. The sn-glycerol-3-phosphate O-acyltransferase of potato mitochondria displayed a slight preference for saturated acyl groups.

1-Acylglycerol-3-Phosphate O-Acyltransferase↗
PubMed1990

Interaction between the Component Enzymes of the Glycine Decarboxylase Multienzyme Complex.

The glycine decarboxylase multienzyme complex comprises about one-third of the soluble protein of the matrix of pea (Pisum sativum) leaf mitochondria where it exists at a concentration of approximately 130 milligrams protein/milliliter. Under these conditions the complex is stable with an approximate subunit ratio of 2 P-protein dimers:27 H-protein monomers:9 T-protein monomers:1 L-protein dimer. When the complex is diluted it tends to dissociate into its component enzymes. This prevents the purification of the intact complex by gel filtration or ultracentrifugation. In the dissociated state the H-protein acts as a mobile cosubstrate that commutes between the other three enzymes and shows typical substrate kinetics. When the complex is reformed, the H-protein no longer acts as a substrate but as an integrated part of the enzyme complex.

Journal Article↗
PubMed1988

Resolution and characterization of the glycine-cleavage reaction in pea leaf mitochondria. Properties of the forward reaction catalysed by glycine decarboxylase and serine hydroxymethyltransferase.

High-molecular-mass proteins from pea (Pisum sativum) mitochondrial matrix retained on an XM-300 Diaflo membrane ('matrix extract') exhibited high rates of glycine oxidation in the presence of NAD+ and tetrahydropteroyl-L-glutamic acid (H4 folate) as long as the medium exhibited a low ionic strength. Serine hydroxymethyltransferase (SHMT) (4 x 53 kDa) and the four proteins of the glycine-cleavage system, including a pyridoxal phosphate-containing enzyme ('P-protein'; 2 x 97 kDa), a carrier protein containing covalently bound lipoic acid ('H-protein'; 15.5 kDa), a protein exhibiting lipoamide dehydrogenase activity ('L-protein'; 2 x 61 kDa) and an H4 folate-dependent enzyme ('T-protein'; 45 kDa) have been purified to apparent homogeneity from the matrix extract by using gel filtration, ion-exchange and phenyl-Superose fast protein liquid chromatography. Gel filtration on Sephacryl S-300 in the presence of 50 mM-KCl proved to be the key step in disrupting this complex. During the course of glycine oxidation catalysed by the matrix extract a steady-state equilibrium in the production and utilization of 5,10-methylene-H4 folate was reached, suggesting that glycine cleavage and SHMT are linked together via a soluble pool of H4 folate. The rate of glycine oxidation catalysed by the matrix extract was sensitive to the NADH/NAD+ molar ratios, because NADH competitively inhibited the reaction catalysed by lipoamide dehydrogenase.

Amino Acid Oxidoreductases↗
PubMed1985

Transport of NAD in Percoll-Purified Potato Tuber Mitochondria: Inhibition of NAD Influx and Efflux by N-4-Azido-2-nitrophenyl-4-aminobutyryl-3'-NAD.

A mechanism by which intact potato (Solanum tuberosum) mitochondria may regulate the matrix NAD content was studied in vitro. If mitochondria were incubated with NAD(+) at 25 degrees C in 0.3 molar mannitol, 10 millimolar phosphate buffer (pH 7.4), 5 millimolar MgCl(2), and 5 millimolar alpha-ketoglutarate, the NAD pool size increased with time. In the presence of uncouplers, net uptake was not only inhibited, but NAD(+) efflux was observed instead. Furthermore, the rate of NAD(+) accumulation in the matrix space was strongly inhibited by the analog N-4-azido-2-nitrophenyl-4-aminobutyryl-3'-NAD(+). When suspended in a medium that avoided rupture of the outer membrane, intact purified mitochondria progressively lost their NAD(+) content. This led to a slow decrease of NAD(+)-linked substrates oxidation by isolated mitochondria The rate of NAD(+) efflux from the matrix space was strongly temperature dependent and was inhibited by the analog inhibitor of NAD(+) transport indicating that a carrier was required for net flux in either direction. It is proposed that uptake and efflux operate to regulate the total matrix NAD pool size.

Journal Article↗
PubMed1984

Activation of NAD-linked malic enzyme in intact plant mitochondria by exogenous coenzyme A.

O2 uptake by potato and cauliflower bud mitochondria oxidizing malate was progressively inhibited as the pH of the external medium was increased, in response to accumulation of oxaloacetate. Adding 0.5 mM coenzyme A to the medium reversed this trend by stimulating intramitochondrial NAD-linked malic enzyme at alkaline pH. In intact potato mitochondria, coenzyme A stimulation of malic enzyme was not observed when the external pH was above 7.5; in cauliflower mitochondria, coenzyme A stimulated even at pH 8. This difference in the response of intact mitochondria was attributed to an inherent difference in the properties of malic enzyme from the two tissues. Malic enzyme solubilized from potato mitochondria was inactive at pH values above 7.8, while that from cauliflower mitochondria retained its activity at pH 8 in the presence of coenzyme A. In potato mitochondria, coenzyme A stimulation of O2 uptake at alkaline pH was only observed when NAD+ was also provided exogenously. The results show that coenzyme A can be taken up by intact mitochondria and that pH, NAD+, and coenzyme A levels in the matrix act together to regulate malate oxidation.

Coenzyme A↗
PubMed1984

Transport of coenzyme A in plant mitochondria.

Oxoglutarate oxidation by purified potato mitochondria which had been stored at low temperature for 48 h or longer was stimulated by added coenzyme A. Exogenous coenzyme A was accumulated by potato mitochondria, both freshly prepared and aged, in a manner sensitive to uncouplers and low temperature. Coenzyme A was concentrated approximately 10-fold in the matrix under steady-state conditions. This coenzyme A uptake followed saturation kinetics with an apparent Km of 0.2 mM and a V of 4-6.5 nmol min-1 mg-1 protein, suggesting carrier-mediated transport. This transport was insensitive to an inhibitor of NAD+ transport. It is suggested that plant mitochondria possess a specific carrier for the net accumulation of coenzyme A.

Biological Transport, Active↗
PubMed1983

Slow passive diffusion of NAD+ between intact isolated plant mitochondria and suspending medium.

Isolated potato (Solanum tuberosum) tuber mitochondria purified by isopycnic centrifugation in density gradients of Percoll were found to be highly intact, to be devoid of extramitochondrial contaminations and to retain a high rate of O2 consumption. When suspended in a medium that avoided rupture of the outer membrane, intact purified mitochondria progressively lost their NAD+ content by passive diffusion. This led to a slow decrease of oxoglutarate-dependent O2 consumption by isolated mitochondria. Addition of NAD+ to the medium restored the initial State-3 rate of oxoglutarate oxidation. The rate of NAD+ accumulation in the matrix space was concentration-dependent, exhibited Michaelis-Menten kinetics and was strongly inhibited by the analogue N-4-azido-2-nitrophenyl-4-aminobutyryl-NAD+.

Azides↗
PubMed1983

Exogenous NAD Effects on Plant Mitochondria: A Reinvestigation of the Transhydrogenase Hypothesis.

Addition of NAD(+) to purified potato (Solanum tuberosum L.) mitochondria respiring alpha-ketoglutarate and malate in the presence of the electron transport inhibitor rotenone, stimulated O(2) uptake. This stimulation was prevented by incubating mitochondria with N-4-azido-2-nitrophenyl-aminobutyryl-NAD(+) (NAP(4)-NAD(+)), an inhibitor of NAD(+) uptake, but not by 1 mm EGTA, an inhibitor of external NADH oxidation. NAD(+)-stimulated malate-cytochrome c reductase activity, and reduction of added NAD(+) by intact mitochondria, could be duplicated by rupturing the mitochondria and adding a small quantity to the cuvette. The extent of external NAD(+) reduction was correlated with the amount of extra mitochondrial malate dehydrogenase present. Malate oxidation by potato mitochondria depleted of endogenous NAD(+) by storing on ice for 72 hours, was completely dependent on added NAD(+), and the effect of NAD(+) on these mitochondria was prevented by incubating them with NAP(4)-NAD(+). External NAD(+) reduction by these mitochondria was not affected by NAP(4)-NAD(+). We conclude that all effects of exogenous NAD(+) on plant mitochondrial respiration can be attributed to net uptake of the NAD(+) into the matrix space.

Journal Article↗
PubMed1981

Role of Glutamate-oxaloacetate Transaminase and Malate Dehydrogenase in the Regeneration of NAD for Glycine Oxidation by Spinach leaf Mitochondria.

During glycine oxidation by spinach leaf mitochondria, oxygen consumption showed a strong and transient inhibition upon addition of oxaloacetate or aspartate plus alpha-ketoglutarate. During the course of the inhibition, aspartate and alpha-ketoglutarate were stoichiometrically transformed into malate and glutamate.It is concluded that oxaloacetate formed by transamination is reduced by the malate dehydrogenase, which allows the regeneration of NAD(+) for glycine oxidation and, thus, by-passes the respiratory chain. Efficiency of a malate-glutamate/aspartate-alpha-ketoglutarate shuttle upon illumination and under in vivo conditions is discussed.

Journal Article↗
PubMed1980

Effect of bicarbonate and oxaloacetate on malate oxidation by spinach leaf mitochondria.

Mitochondria isolated from spinach leaves oxidized malate by both a NAD+-linked malic enzyme and malate dehydrogenase. In the presence of sodium arsenite the accumuation of oxaloacetate and pyruvate during malate oxidation was strongly dependent on the malate concentration, the pH in the reaction medium and the metabolic state condition. Bicarbonate, especially at alkaline pH, inhibited the decarboxylation of malate by the NAD+-linked malic enzyme in vitro and in vivo. Analysis of the reaction products showed that with 15 mM bicarbonate, spinach leaf mitochondria excreted almost exclusively oxaloacetate. The inhibition by oxaloacetate of malate oxidation by spinach leaf mitochondria was strongly dependent on malate concentration, the pH in the reaction medium and on the metabolic state condition. The data were interpreted as indicating that: (a) the concentration of oxaloacetate on both sides of the inner mitochondrial membrane governed the efflux and influx of oxaloacetate; (b) the NAD+/NADH ratio played an important role in regulating malate oxidation in plant mitochondria; (c) both enzymes (malate dehydrogenase and NAD+-linked malic enzyme) were competing at the level of the pyridine nucleotide pool, and (d) the NAD+-linked malic enzyme provided NADH for the reversal of the reaction catalyzed by the malate dehydrogenase.

Bicarbonates↗
PubMed1980

Effect of NAD on Malate Oxidation in Intact Plant Mitochondria.

Potato tuber mitochondria oxidizing malate respond to NAD(+) addition with increased oxidation rates, whereas mung bean hypocotyl mitochondria do not. This is traced to a low endogenous content of NAD(+) in potato mitochondria, which prove to take up added NAD(+). This mechanism concentrates NAD(+) in the matrix space. Analyses for oxaloacetate and pyruvate (with pyruvate dehydrogenase blocked) are consistent with regulation of malate oxidation by the internal NAD(+)/NADH ratio.

Journal Article↗
PubMed1977

[Oxidation of malate, NADH and glycine in C3 and C4 plant mitochondria].

Spinach leaf mitochondria (C3 plant) were capable of oxidizing Glycine. This oxidation was linked to the mitochondrial electron transport chain, was coupled to three phosphorylation sites and was sensitive to electron transport inhibitors. In marked contrast however, neither mitochondria from dark grown plants nor mitochondria from C4 leaf plants were capable of oxidizing this amino acid.

Darkness↗
PubMed1977

Strong binding of cytochrome C on the envelope of spinach chloroplasts.

Yeast cationic ferricytochrome c was able to bind to the spinach (Spinacia oleracea) chloroplast envelope with a low affinity (Kd = 1.1 mum). The total amount of low affinity binding sites was of the order of 50 nmol cytochrome c mg(-1) protein. We gave the evidence that binding of ferricytochrome c to the envelope was electrostatic and that the envelope membranes were strongly negatively charged. Addition of yeast ferricytochrome c to a preparation of intact washed chloroplasts (class I) induced a strong agglutination of chloroplasts.

Journal Article↗
PubMed1977

Isolation and oxidative properties of intact mitochondria isolated from spinach leaves.

A procedure was described for preparing intact mitochondria from spinach (Spinacia oleracea L.) leaves. These mitochondria oxidized succinate, malate, pyruvate, alpha-ketoglutarate, and NADH with good respiratory control and ADP/O ratios comparable to those observed with mitochondria from other plant tissues. Glycine was oxidized by the preparations. This oxidation linked to the mitochondrial electron transport chain, was coupled to three phosphorylation sites and was sensitive to electron transport and phosphorylation inhibitors.Cyanide completely inhibited the oxidation of NADH. The oxidation of succinate, malate, and glycine was only partially inhibited.

Journal Article↗