scholarly journals Comparison of the effects of Ca2+, adenine nucleotides and pH on the kinetic properties of mitochondrial NAD+-isocitrate dehydrogenase and oxoglutarate dehydrogenase from the yeast Saccharomyces cerevisiae and rat heart

1994 ◽  
Vol 303 (2) ◽  
pp. 461-465 ◽  
Author(s):  
B J Nichols ◽  
M Rigoulet ◽  
R M Denton
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Isocitrate Dehydrogenase ◽  
Rat Heart ◽  
Adenine Nucleotides ◽  
Amino Acid Sequences ◽  
Kinetic Properties ◽  
Yeast Saccharomyces Cerevisiae ◽  
Rat Heart Mitochondria ◽  
Oxoglutarate Dehydrogenase ◽  
Regulatory Properties

The regulatory properties of NAD(+)-isocitrate dehydrogenase and oxoglutarate dehydrogenase in extracts of yeast and rat heart mitochondria were studied under identical conditions. Yeast NAD(+)-isocitrate dehydrogenase exhibits a low K0.5 for isocitrate and is activated by AMP and ADP, but is insensitive to ATP and Ca2+. In contrast, the rat heart NAD(+)-isocitrate dehydrogenase was insensitive to AMP, but was activated by ADP and by Ca2+ in the presence of ADP or ATP. Both yeast and rat heart oxoglutarate dehydrogenase were stimulated by ADP, but only the heart enzyme was activated by Ca2+. All the enzymes studied were activated by decreases in pH, but to differing extents. The effects of Ca2+, adenine nucleotides and pH were through K0.5 for isocitrate or 2-oxoglutarate. These observations are discussed with reference to the deduced amino acid sequences of the constituent subunits of the enzymes, where they are available.

scholarly journals Regulation of NAD+-linked isocitrate dehydrogenase and 2-oxoglutarate dehydrogenase by Ca2+ ions within toluene-permeabilized rat heart mitochondria. Interactions with regulation by adenine nucleotides and NADH/NAD+ ratios

1988 ◽  
Vol 252 (1) ◽  
pp. 181-189 ◽  
Author(s):  
G A Rutter ◽  
R M Denton
Keyword(s):  
Isocitrate Dehydrogenase ◽  
Rat Heart ◽  
Adenine Nucleotides ◽  
Heart Mitochondria ◽  
Maximal Effect ◽  
Dehydrogenase System ◽  
Rat Heart Mitochondria ◽  
Oxoglutarate Dehydrogenase ◽  
Intact Heart

1. Toluene-permeabilized rat heart mitochondria have been used to study the regulation of NAD+-linked isocitrate dehydrogenase and 2-oxoglutarate dehydrogenase by Ca2+, adenine and nicotinamide nucleotides, and to compare the properties of the enzymes in situ, with those in mitochondrial extracts. 2. Although K0.5 values (concn. giving half-maximal effect) for Ca2+ of 2-oxoglutarate dehydrogenase were around 1 microM under all conditions, corresponding values for NAD+-linked isocitrate dehydrogenase were in the range 5-43 microM. 3. For both enzymes, K0.5 values for Ca2+ observed in the presence of ATP were 3-10-fold higher than those in the presence of ADP, with values increasing over the ADP/ATP range 0.0-1.0. 4. 2-Oxoglutarate dehydrogenase was less sensitive to inhibition by NADH when assayed in permeabilized mitochondria than in mitochondrial extracts. Similarly, the Km of NAD+-linked isocitrate dehydrogenase for threo-Ds-isocitrate was lower in permeabilized mitochondria than in extracts under all the conditions investigated. 5. It is concluded that in the intact heart Ca2+ activation of NAD+-linked isocitrate dehydrogenase may not necessarily occur in parallel with that of the other mitochondrial Ca2+-sensitive enzymes, 2-oxoglutarate dehydrogenase and the pyruvate dehydrogenase system.

scholarly journals The control of isocitrate oxidation by rat liver mitochondria

1969 ◽  
Vol 114 (2) ◽  
pp. 215-225 ◽  
Author(s):  
D. G. Nicholls ◽  
P. B. Garland
Keyword(s):  
Respiratory Chain ◽  
Rat Liver ◽  
Isocitrate Dehydrogenase ◽  
Adenine Nucleotides ◽  
Kinetic Studies ◽  
Liver Mitochondria ◽  
Rat Liver Mitochondria ◽  
Kinetic Properties ◽  
Dependent Inhibition ◽  
Nad 4

1. The factors capable of affecting the rate of isocitrate oxidation in intact mitochondria include the rate of isocitrate penetration, the activity of the NAD-specific and NADP-specific isocitrate dehydrogenases, the activity of the transhydrogenase acting from NADPH to NAD+, the rate of NADPH oxidation by the reductive synthesis of glutamate and the activity of the respiratory chain. A quantitative assessment of these factors was made in intact mitochondria. 2. The kinetic properties of the NAD-specific and NADP-specific isocitrate dehydrogenases extracted from rat liver mitochondria were examined. 3. The rate of isocitrate oxidation through the respiratory chain in mitochondria with coupled phosphorylation is approximately equal to the maximal of the NAD-specific isocitrate dehydrogenase but at least ten times as great as the transhydrogenase activity from NADPH to NAD+. 4. It is concluded that the energy-dependent inhibition of isocitrate oxidation by palmitoylcarnitine oxidation is due to an inhibition of the NAD-specific isocitrate dehydrogenase. 5. Kinetic studies of NAD-specific isocitrate dehydrogenase demonstrated that its activity could be inhibited by one or more of the following: an increased reduction of mitochondrial NAD, an increased phosphorylation of mitochondrial adenine nucleotides or a fall in the mitochondrial isocitrate concentration. 6. Uncoupling agents stimulate isocitrate oxidation by an extent equal to the associated stimulation of transhydrogenation from NADPH to NAD+. 7. A technique is described for continuously measuring with a carbon dioxide electrode the synthesis of glutamate from isocitrate and ammonia.

scholarly journals The mechanism for the ATP-induced uncoupling of respiration in mitochondria of the yeast Saccharomyces cerevisiae

1995 ◽  
Vol 307 (3) ◽  
pp. 657-661 ◽  
Author(s):  
S Prieto ◽  
F Bouillaud ◽  
E Rial
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Membrane Potential ◽  
Cytochrome C ◽  
Oxidative Phosphorylation ◽  
Cytochrome C Oxidase ◽  
Kinetic Properties ◽  
Yeast Saccharomyces Cerevisiae ◽  
Saccharomyces Cerevisiae Mitochondria ◽  
Low Efficiency ◽  
Respiratory Chain Activity

We have recently reported that ATP induces an uncoupling pathway in Saccharomyces cerevisiae mitochondria [Prieto, Bouillaud, Ricquier and Rial (1992) Eur. J. Biochem. 208, 487-491]. The presence of this pathway would explain the reported low efficiency of oxidative phosphorylation in S. cerevisiae, and may represent one of the postulated energy-dissipating mechanisms present in these yeasts. In this paper we demonstrate that ATP exerts its action in two steps: first, at low ATP/Pi ratios, it increases the respiratory-chain activity, probably by altering the kinetic properties of cytochrome c oxidase. Second, at higher ATP/Pi ratios, an increase in membrane permeability leads to a collapse in membrane potential. The ATP effect on cytochrome c oxidase corroborates a recent report showing that ATP interacts specifically with yeast cytochrome oxidase, stimulating its activity [Taanman and Capaldi (1993) J. Biol. Chem. 268, 18754-18761].

Two functional alpha-tubulin genes of the yeast Saccharomyces cerevisiae encode divergent proteins

1986 ◽  
Vol 6 (11) ◽  
pp. 3711-3721
Author(s):  
P J Schatz ◽  
L Pillus ◽  
P Grisafi ◽  
F Solomon ◽  
D Botstein
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Budding Yeast ◽  
Amino Acid Sequences ◽  
Yeast Cells ◽  
Nucleotide Sequencing ◽  
Cell Fractionation ◽  
Gene Products ◽  
Yeast Saccharomyces Cerevisiae ◽  
Alpha Tubulin ◽  
Tubulin Genes

Two alpha-tubulin genes from the budding yeast Saccharomyces cerevisiae were identified and cloned by cross-species DNA homology. Nucleotide sequencing studies revealed that the two genes, named TUB1 and TUB3, encoded gene products of 447 and 445 amino acids, respectively, that are highly homologous to alpha-tubulins from other species. Comparison of the sequences of the two genes revealed a 19% divergence between the nucleotide sequences and a 10% divergence between the amino acid sequences. Each gene had a single intervening sequence, located at an identical position in codon 9. Cell fractionation studies showed that both gene products were present in yeast microtubules. These two genes, along with the TUB2 beta-tubulin gene, probably encode the entire complement of tubulin in budding yeast cells.

scholarly journals Calcium ions and the regulation of NAD+-linked isocitrate dehydrogenase from the mitochondria of rat heart and other tissues

1978 ◽  
Vol 176 (3) ◽  
pp. 899-906 ◽  
Author(s):  
R M Denton ◽  
D A Richards ◽  
J G Chin
Keyword(s):  
Adipose Tissue ◽  
White Adipose Tissue ◽  
Calcium Ions ◽  
Isocitrate Dehydrogenase ◽  
Rat Heart ◽  
Maximum Velocity ◽  
Brown Adipose ◽  
Rat Heart Mitochondria ◽  
Fold Activation

The effects of Ca2+ on the activity of isocitrate dehydrogenase (NAD+) in extracts of rat heart mitochondria were explored in the presence of MgCl2 by using EGTA buffers. In the absence of ADP, Ca2+ (about 30 micrometer) resulted in a slight increase in apparent Km for threo-Ds-isocitrate; in the presence of ADP, Ca2+ (about 25 micrometer) greatly lowered the apparent Km for threo-Ds-isocitrate from 227 micrometer to 53 micrometer without changing the maximum velocity. At 100 micrometer-threo-Ds-isocitrate and 1 mM-ADP, there was an 8-fold activation by Ca2+, with a Km for Ca2+ of 1.2 micrometer. This activation was also observed with Sr2+ (Km 3.1 micrometer), but not with Mn2+ (at concentrations below 2.5 micrometer). Similar effects of Ca2+ were also observed on isocitrate dehydrogenase (NAD+) activity in extracts of mitochondria from liver, kidney, brown adipose tissue and white adipose tissue of the rat. The possible regulatory role of changes in the intramitochondrial concentration of Ca2+ is discussed.

Molecular cloning, gene expression and functional expression of a phosphoenolpyruvate carboxylase Osppc1 in developing rice seeds: implication of involvement in nitrogen accumulation

2014 ◽  
Vol 24 (1) ◽  
pp. 23-36 ◽  
Author(s):  
Naoki Yamamoto ◽  
Tatsuya Kubota ◽  
Takehiro Masumura ◽  
Naomasa Shiraishi ◽  
Kunisuke Tanaka ◽  
...  
Keyword(s):  
Expression Patterns ◽  
Grain Filling ◽  
Functional Expression ◽  
Amino Acid Sequences ◽  
Kinetic Properties ◽  
Nitrogen Accumulation ◽  
Consecutive Reaction ◽  
Gramineous Plant ◽  
Rice Seeds ◽  
Regulatory Properties

AbstractWe isolated two cDNAs of phosphoenolpyruvate carboxylase (PEPC) from developing rice seeds, Osppc1 and Osppc3. The deduced amino acid sequences of both cDNAs share several conserved motifs with other non-photosynthetic PEPCases, and these common motifs are known to be functionally important to their regulatory properties. The deduced protein sequence of Osppc1 was clustered into a monocotyledonous plant-specific clade, and Osppc3 was clustered into a gramineous plant-specific clade in the phylogenetic tree of plant PEPCases. The mRNA accumulations of Osppc1 and Osppc3 were found in developing rice seeds throughout the grain-filling stages, although their expression patterns differed: Osppc1 was strongly expressed at 7 d after flowering, and Osppc3 was strongly expressed at 4 d after flowering. The kinetic properties of the Osppc1 recombinant protein were quite similar to those of maize root-type PEPCase, except that the sensitivity for malate at pH 7.3 was weaker. Mining rice microarray data, we observed that Osppc1 was co-expressed with aspartate aminotransferase and alanine aminotransferase, which are involved in seed nitrogen metabolism. Moreover, reannotation of the co-expressed genes revealed that Osppc1, the two aminotransferases and the enolase were mapped on to the consecutive reaction from 2-phosphoglycerate to glutamate and pyruvate in the cytosol. These results imply that Osppc1 functions cooperatively with the two aminotransferases in the synthesis of amino acids that are used for storage protein synthesis in developing rice seeds.

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