scholarly journals Factors influencing the inactivation of phosphate-dependent glutaminase in the matrix fraction of rat liver mitochondria

1991 ◽  
Vol 274 (1) ◽  
pp. 109-114
Author(s):  
J D McGivan ◽  
F A Doyle ◽  
K Boon
Keyword(s):  
Liver Mitochondria ◽  
Polyclonal Antiserum ◽  
Rat Liver Mitochondria ◽  
Mitochondrial Matrix ◽  
Factors Influencing ◽  
Low Concentrations ◽  
The Matrix ◽  
High Concentrations ◽  
Matrix Fraction ◽  
Mitochondrial Extract

1. The activity of phosphate-dependent glutaminase was measured in a matrix extract of essentially lysosome-free liver mitochondria. 2. ATP, GTP or a non-hydrolysable analogue of ATP stimulated the rapid inactivation of glutaminase, but not of other matrix enzymes. 3. Glutaminase was protected against inactivation if high concentrations of glutamine or low concentrations of NH3 were present. 4. Inactivation of glutaminase in the presence of ATP did not markedly affect the reaction of the enzyme with a specific polyclonal antiserum. 5. These results in a mitochondrial extract are similar to the characteristics of glutaminase inactivation in intact hepatocytes, suggesting a similar mechanism in each case. 6. The presence of a specific ATP-activated protease in the mitochondrial matrix is suggested to be responsible for glutaminase inactivation.

scholarly journals MORPHOLOGY AND ATP-ASE OF ISOLATED MITOCHONDRIA

1955 ◽  
Vol 1 (2) ◽  
pp. 127-138 ◽  
Author(s):  
Robert F. Witter ◽  
Michael L. Watson ◽  
Mary A. Cottone
Keyword(s):  
Electron Microscope ◽  
Liver Mitochondria ◽  
Rat Liver Mitochondria ◽  
Substantial Part ◽  
Mitochondrial Matrix ◽  
Isolated Mitochondria ◽  
The Matrix ◽  
First Time ◽  
Methods Of Isolation

Changes in the morphology of rat liver mitochondria brought about by different methods of isolation and the concomitant changes in ATP-ase activity were studied. The morphology was investigated with the electron microscope. It was found that the ATP-ase activity of the isolated mitochondria cannot be readily correlated with the morphology of the mitochondria. The ATP-ase found in these preparations was latent, resembling the enzyme described in mitochondria prepared in 0.25 M sucrose. In confirmation of earlier results the use of 0.88 M sucrose yielded preparations with a higher initial ATP-ase than did other methods. Preparation in 0.25 M sucrose resulted in round, swollen mitochondria of which 30 to 40 per cent appeared to have lost a substantial part of the mitochondrial matrix. Preparations in 0.44 to 0.88 M sucrose contained mainly rod-shaped mitochondria plus a small amount of another type of swollen mitochondria. The matrix of mitochondria isolated in 0.88 M sucrose was highly condensed. By the use of 0.44 M sucrose adjusted to pH 6.2 with citric acid, it was possible to isolate, for the first time, mitochondria closely resembling those in situ and containing latent ATP-ase.

scholarly journals Inhibition by oxalomalate of rat liver mitochondrial and extramitochondrial aconitate hydratase

1971 ◽  
Vol 125 (2) ◽  
pp. 557-562 ◽  
Author(s):  
A. Adinolfi ◽  
V. Guarriera-Bobyleva ◽  
S. Olezza ◽  
A. Ruffo
Keyword(s):  
Rat Liver ◽  
Initial Rate ◽  
Competitive Inhibition ◽  
Liver Mitochondria ◽  
Rat Liver Mitochondria ◽  
Mitochondrial Enzyme ◽  
Aconitate Hydratase ◽  
Inhibited Oxidation ◽  
Low Concentrations ◽  
High Concentrations

1. The effect of oxalomalate on the oxidation of citrate and cis-aconitate in rat liver mitochondria, and on the activity of mitochondrial and cytoplasmic aconitate hydratase, has been investigated. 2. Oxalomalate that was added to intact rat liver mitochondria at high concentrations (2mm) produced complete inhibition of citrate and cis-aconitate oxidation, but lower concentrations (0.1–0.25mm) inhibited oxidation of citrate more than that of cis-aconitate. 3. Aconitate hydratase that was either extracted from mitochondria or soluble in the cytoplasm, was strongly inhibited by low concentrations of oxalomalate (0.01–0.2mm), the mitochondrial enzyme being more sensitive than the soluble one. 4. Oxalomalate, when added together with citrate, produced competitive inhibition; the Ki values calculated were 1×10−6m for the mitochondrial and 2.5×10−6m for the cytoplasmic enzyme. 5. With both the enzymic preparations oxalomalate added together with the substrates inhibited the initial rate of the reaction citrate→cis-aconitate more than that of the reaction isocitrate→cis-aconitate. 6. After 2min of preincubation of the inhibitor with either of the enzymic preparations the inhibition increased tenfold and became irreversible; under these conditions both the reactions were inhibited to the same extent. 7. The inhibition by oxalomalate of aconitate hydratase appeared to be similar in many respects to that produced by fluorocitrate on the same enzyme.

scholarly journals Inhibition by local anaesthetics of adenine nucleotide translocation in rat liver mitochondria

1974 ◽  
Vol 140 (3) ◽  
pp. 413-422 ◽  
Author(s):  
Terry L. Spencer ◽  
Fyfe L. Bygrave
Keyword(s):  
Rat Liver ◽  
Protein Interactions ◽  
Adenine Nucleotide ◽  
Adenine Nucleotides ◽  
Local Anaesthetics ◽  
Liver Mitochondria ◽  
Rat Liver Mitochondria ◽  
Low Concentrations ◽  
High Concentrations ◽  
Lipid Protein Interactions

1. The mechanism of adenine nucleotide translocation in mitochondria isolated from rat liver was further examined by using the local anaesthetics procaine, butacaine, nupercaine and tetracaine as perturbators of lipid–protein interactions. Each of these compounds inhibited translocation of ADP and of ATP; butacaine was the most effective with 50% inhibition occurring at 30μm for 200μm-ATP and at 10μm for 200μm-ADP. The degree of inhibition by butacaine of both adenine nucleotides was dependent on the concentration of adenine nucleotide present; with low concentrations of adenine nucleotide, low concentrations of butacaine-stimulated translocation, but at high concentrations (greater than 50μm) low concentrations of butacaine inhibited translocation. Butacaine increased the affinity of the translocase for ATP to a value which approached that of ADP. 2. Higher concentrations of nupercaine and of tetracaine were required to inhibit translocation of both nucleotides; 50% inhibition of ATP translocation occurred at concentrations of 0.5mm and 0.8mm of these compounds respectively. The pattern of inhibition of ADP translocation by nupercaine and tetracaine was more complex than that of ATP; at very low concentrations (less than 250μm) inhibition ensued, followed by a return to almost original rates at 1mm. At higher concentrations inhibition of ADP translocation resulted. 3. That portion of ATP translocation stimulated by Ca2+ was preferentially inhibited by each of the local anaesthetics tested. In contrast, inhibition by the anaesthetics of ADP translocation was prevented by low concentrations of Ca2+. 4. The data provide further support for our hypothesis that lipid–protein interactions are important determinants in the activity of the adenine nucleotide translocase in mitochondria.

scholarly journals Identification, purification and partial characterization of a carboxypeptidase from the matrix of rat liver mitochondria: a novel metalloenzyme

1994 ◽  
Vol 300 (1) ◽  
pp. 15-19 ◽  
Author(s):  
E Figueiredo ◽  
M C Duque-Magalhães
Keyword(s):  
Rat Liver ◽  
Specific Activity ◽  
Liver Mitochondria ◽  
Rat Liver Mitochondria ◽  
Metal Affinity ◽  
Apparent Homogeneity ◽  
Sds Page ◽  
The Matrix ◽  
Carboxypeptidase Inhibitor ◽  
Matrix Fraction

A novel carboxypeptidase has been purified to apparent homogeneity from the matrix fraction of rat liver mitochondria by using a procedure mainly based on immobilized-metal-affinity chromatography (IMAC). This carboxypeptidase has been named mCP-III, since it represents the third major peak of carboxypeptidase activity after the IMAC step of purification. mCP-III hydrolyses a number of N-blocked dipeptides, with preference for Cbz-Phe-Ala, and shows no degrading activity towards 125I-casein. The optimal pH of its activity is 7.6, the apparent Km for Cbz-Phe-Ala is 0.12 mM and the specific activity is 145.5 mumol/min per mg of protein. The enzyme is a typical metalloproteinase, is inhibited by 1,10-phenanthroline and carboxypeptidase inhibitor and re-activated by added Zn2+ and Co2+. The molecular mass estimated by molecular-sieve h.p.l.c. was approx. 115 kDa with two protein bands of 61 and 50 kDa shown by SDS/PAGE analysis, indicating that the enzyme is active as a dimer. This is the first clearly identified carboxypeptidase within mitochondria.

scholarly journals Proton translocation by the mitochondrial cytochrome b-c1 complex is inhibited by NN′-dicyclohexylcarbodi-imide

1982 ◽  
Vol 206 (2) ◽  
pp. 419-421 ◽  
Author(s):  
B D Price ◽  
M D Brand
Keyword(s):  
Electron Transport ◽  
Cytochrome Oxidase ◽  
Cytochrome B ◽  
Rat Liver ◽  
Proton Translocation ◽  
Liver Mitochondria ◽  
Rat Liver Mitochondria ◽  
Mitochondrial Cytochrome ◽  
Low Concentrations ◽  
Mitochondrial Cytochrome B

NN'-Dicyclohexylcarbodi-imide at low concentrations decreases the H+/2e ratio for rat liver mitochondria over the span succinate to oxygen from 5.9 +/- 0.3 (mean +/- S.E.M.) to 4.0 +/- 0.1 and for the cytochrome b-c1 complex from 3.8 +/- 0.2 to 1.9 +/- 0.1, but has little effect on the H+/2e ratio of cytochrome oxidase. The decrease in stoicheiometry is due, not to uncoupling or inhibition of electron transport, but to inhibition of proton translocation. NN'-Dicyclohexylcarbodi-imide thus ‘decouples’ proton translocation in the cytochrome b-c1 complex.

scholarly journals Rat liver mitochondrial ADP-ribose pyrophosphatase in the matrix space with low Km for free ADP-ribose

1994 ◽  
Vol 299 (3) ◽  
pp. 679-682 ◽  
Author(s):  
D Bernet ◽  
R M Pinto ◽  
M J Costas ◽  
J Canales ◽  
J C Cameselle
Keyword(s):  
Rat Liver ◽  
Gradient Centrifugation ◽  
Liver Mitochondria ◽  
Rat Liver Mitochondria ◽  
Matrix Space ◽  
The Matrix ◽  
Submitochondrial Fractions

A study involving markers of subcellular and submitochondrial fractions, gradient centrifugation, latency measurements and extraction with digitonin, demonstrates the association of a specific ADP-ribose pyrophosphatase with rat liver mitochondria and its localization in the matrix space. The enzyme hydrolyses ADP-ribose to AMP, with a Km of 2-3 microM. The results support the occurrence of a specific turnover pathway for free ADP-ribose and its relevance in mitochondria.

scholarly journals Stable enhancement of calcium retention in mitochondria isolated from rat liver after the administration of glucagon to the intact animal

1978 ◽  
Vol 176 (3) ◽  
pp. 705-714 ◽  
Author(s):  
Veronica Prpić ◽  
Terry L. Spencer ◽  
Fyfe L. Bygrave
Keyword(s):  
Rat Liver ◽  
Molecular Mechanisms ◽  
Adenine Nucleotide ◽  
Adenine Nucleotides ◽  
Mitochondrial Protein ◽  
Liver Mitochondria ◽  
Rat Liver Mitochondria ◽  
Matrix Space ◽  
The Matrix

1. Mitochondria isolated from rat liver by centrifugation of the homogenate in buffered iso-osmotic sucrose at between 4000 and 8000g-min, 1h after the administration in vivo of 30μg of glucagon/100g body wt., retain Ca2+ for over 45min after its addition at 100nmol/mg of mitochondrial protein in the presence of 2mm-Pi. In similar experiments, but after the administration of saline (0.9% NaCl) in place of glucagon, Ca2+ is retained for 6–8min. The ability of glucagon to enhance Ca2+ retention is completely prevented by co-administration of 4.2mg of puromycin/100g body wt. 2. The resting rate of respiration after Ca2+ accumulation by mitochondria from glucagon-treated rats remains low by contrast with that from saline-treated rats. Respiration in the latter mitochondria increased markedly after the Ca2+ accumulation, reflecting the uncoupling action of the ion. 3. Concomitant with the enhanced retention of Ca2+ and low rates of resting respiration by mitochondria from glucagon-treated rats was an increased ability to retain endogenous adenine nucleotides. 4. An investigation of properties of mitochondria known to influence Ca2+ transport revealed a significantly higher concentration of adenine nucleotides but not of Pi in those from glucagon-treated rats. The membrane potential remained unchanged, but the transmembrane pH gradient increased by approx. 10mV, indicating increased alkalinity of the matrix space. 5. Depletion of endogenous adenine nucleotides by Pi treatment in mitochondria from both glucagon-treated and saline-treated rats led to a marked diminution in ability to retain Ca2+. The activity of the adenine nucleotide translocase was unaffected by glucagon treatment of rats in vivo. 6. Although the data are consistent with the argument that the Ca2+-translocation cycle in rat liver mitochondria is a target for glucagon action in vivo, they do not permit conclusions to be drawn about the molecular mechanisms involved in the glucagon-induced alteration to this cycle.

scholarly journals Stimulation of the respiration rate of rat liver mitochondria by sub-micromolar concentrations of extramitochondrial Ca2+

1987 ◽  
Vol 245 (1) ◽  
pp. 217-222 ◽  
Author(s):  
J D Johnston ◽  
M D Brand
Keyword(s):  
Respiration Rate ◽  
Rat Liver ◽  
Oxidative Metabolism ◽  
Mitochondrial Respiration ◽  
Liver Mitochondria ◽  
Ruthenium Red ◽  
Rat Liver Mitochondria ◽  
Mitochondrial Matrix ◽  
Stimulation Of

1. The respiration rate of rat liver mitochondria was stimulated by up to 70% when the extramitochondrial Ca2+ concentration was raised from 103 to 820 nM. This occurred when pyruvate, 2-oxoglutarate, or threo-(Ds)-isocitrate was employed as substrate, but not when succinate was used. 2. Ruthenium Red prevented the stimulation of mitochondrial respiration by extramitochondrial Ca2+, showing that the effect required Ca2+ uptake into the mitochondrial matrix. 3. Starvation of rats for 48 h abolished the stimulation of mitochondrial respiration by extramitochondrial Ca2+ when pyruvate was used as substrate, but did not affect the stimulation of 2-oxoglutarate oxidation by extramitochondrial Ca2+. 4. Our findings are in accord with proposals that oxidative metabolism in liver mitochondria may be stimulated by Ca2+ activation of intramitochondrial dehydrogenases.

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