Inhibition by oxalomalate of rat liver mitochondrial and extramitochondrial aconitate hydratase

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.

Download Full-text

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

Biochemical Journal ◽

10.1042/bj1400413 ◽

1974 ◽

Vol 140 (3) ◽

pp. 413-422 ◽

Cited By ~ 15

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.

Download Full-text

Fluorocitrate inhibition of aconitate hydratase and the tricarboxylate carrier of rat liver mitochondria

Biochemical Journal ◽

10.1042/bj1340217 ◽

1973 ◽

Vol 134 (1) ◽

pp. 217-224 ◽

Cited By ~ 23

Author(s):

M. D. Brand ◽

Susan M. Evans ◽

J. Mendes-Mourão ◽

J. B. Chappell

Keyword(s):

Rat Liver ◽

Inhibitory Action ◽

Liver Mitochondria ◽

Rat Liver Mitochondria ◽

Exchange Reactions ◽

Aconitate Hydratase ◽

Isolated Rat Liver ◽

Tricarboxylate Carrier ◽

Hydratase Activity ◽

Isolated Rat

1. The effect of biologically synthesized and purified fluorocitrate on the metabolism of tricarboxylate anions by isolated rat liver mitochondria was investigated, in relation to the claim by Eanes et al. (1972) that this fluoro compound inhibits the tricarboxylate carrier at concentrations at which it has little effect on the aconitate hydratase activity. 2. That the inhibitory action of fluorocitrate is at the level of the aconitate hydratase and not at the level of the tricarboxylate carrier is indicated by the following findings. Although the oxidation of citrate and cis-aconitate, but not that of isocitrate, was inhibited by fluorocitrate, the exchange of internal citrate for external citrate or l-malate was not. Had the tricarboxylate carrier been affected, these latter exchange reactions would have been inhibited. 3. By using aconitate hydratase solubilized from mitochondria it was found that with citrate as substrate the inhibition by fluorocitrate was partially competitive (Ki=3.4×10−8m), whereas with cis-aconitate as substrate the inhibition was partially non-competitive (Ki=3.0×10−8m).

Download Full-text

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

Biochemical Journal ◽

10.1042/bj2060419 ◽

1982 ◽

Vol 206 (2) ◽

pp. 419-421 ◽

Cited By ~ 27

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.

Download Full-text

Time-dependence of inhibition of carnitine palmitoyltransferase I by malonyl-CoA in mitochondria isolated from livers of fed or starved rats. Evidence for transition of the enzyme between states of low and high affinity for malonyl-CoA

Biochemical Journal ◽

10.1042/bj2180379 ◽

1984 ◽

Vol 218 (2) ◽

pp. 379-386 ◽

Cited By ~ 21

Author(s):

V A Zammit

Keyword(s):

Rat Liver ◽

Initial Rate ◽

Liver Mitochondria ◽

Time Dependent ◽

Rat Liver Mitochondria ◽

High Affinity ◽

Malonyl Coa ◽

Rate Of Increase ◽

Cpt I ◽

Zero Time

The degree of inhibition of CPT I (carnitine palmitoyltransferase, EC 2.3.1.21) in isolated rat liver mitochondria by malonyl-CoA was studied by measuring the activity of the enzyme over a short period (15s) after exposure of the mitochondria to malonyl-CoA for different lengths of time. Inhibition of CPT I by malonyl-CoA was markedly time-dependent, and the increase occurred at the same rate in the presence or absence of palmitoyl-CoA (80 microM), and in the presence of carnitine, such that the time-course of acylcarnitine formation deviated markedly from linearity when CPT I activity was measured in the presence of malonyl-CoA over several minutes. The initial rate of increase in degree of inhibition with time was independent of malonyl-CoA concentration. CPT I in mitochondria from 48 h-starved rats had a lower degree of inhibition by malonyl-CoA at zero time, but was equally capable of being sensitized to malonyl-CoA, as judged by an initial rate of increase of inhibition identical with that of the enzyme in mitochondria from fed rats. Double-reciprocal plots for the degree of inhibition produced by different malonyl-CoA concentrations at zero time for the enzyme in mitochondria from fed or starved animals indicated that the enzyme in the latter mitochondria was predominantly in a state with low affinity for malonyl-CoA (concentration required to give 50% inhibition, I0.5 congruent to 10 microM), whereas that in mitochondria from fed rats displayed two distinct sets of affinities: low (congruent to 10 microM) and high (less than 0.3 microM). Plots for mitochondria after incubation for 0.5 or 1 min with malonyl-CoA indicated that the increased sensitivity observed with time was due to a gradual increase in the high-affinity state in both types of mitochondria. These results suggest that the sensitivity of CPT I in rat liver mitochondria in vitro had two components: (i) an instantaneous sensitivity inherent to the enzyme which depends on the nutritional state of the animal from which the mitochondria are isolated, and (ii) a slow, malonyl-CoA-induced, time-dependent increase in sensitivity. It is suggested that the rate of malonyl-CoA-induced sensitization of the enzyme to malonyl-CoA inhibition is limited by a slow first-order process, which occurs after the primary event of interaction of malonyl-CoA with the mitochondria.(ABSTRACT TRUNCATED AT 400 WORDS)

Download Full-text

Comparative studies of CDP-diacylglycerol synthase in rat liver mitochondria and microsomes

Biochemistry and Cell Biology ◽

10.1139/o93-029 ◽

1993 ◽

Vol 71 (3-4) ◽

pp. 183-189 ◽

Cited By ~ 17

Author(s):

Amy Y. P. Mok ◽

Gordon E. McDougall ◽

William C. McMurray

Keyword(s):

Phosphatidic Acid ◽

Rat Liver ◽

Mitochondrial Fraction ◽

Liver Mitochondria ◽

Subcellular Fractions ◽

Rat Liver Mitochondria ◽

Mitochondrial Enzyme ◽

Egg Lecithin ◽

Microsomal Membranes ◽

Concentration Curves

CDP-diacylglycerol for polyglycerophosphatide biogenesis can be synthesized within rat liver mitochondria. Contamination by microsomal membranes cannot account for the CDP-diacylglycerol synthesis found in the mitochondria. Phosphatidic acid from egg lecithin was the best substrate for the synthesis of CDP-diacylglycerol in both subcellular fractions. Concentration curves for CTP and Mg2+ differed for the two subcellular fractions. Microsomal CDP-diacylglycerol synthase was specifically stimulated by the nucleotide GTP; this stimulatory effect by GTP was not observed in the mitochondrial fraction. By comparison, the microsomal enzyme was more sensitive towards sulfhydryl inhibitors than the mitochondrial enzyme. The enzymes could be solubilized from the membrane fractions using 3-[(cholamidopropyl)dimethylammonio]-1-propanesulfonate, and the detergent-soluble activity could be partially restored by addition of phospholipids. Based on the differences in properties, it was concluded that there are two distinct enzyme localizations for CDP-diacylglycerol synthesis in mitochondria and microsomes from rat liver.Key words: CDP-diacylglycerol, synthase, phosphatidic acid, mitochondria, microsomes, solubilization.

Download Full-text

Effects of lysophospholipids on Ca2+ transport in rat liver mitochondria incubated at physiological Ca2+ concentrations in the presence of Mg2+, phosphate and ATP at 37° C

Biochemical Journal ◽

10.1042/bj2240423 ◽

1984 ◽

Vol 224 (2) ◽

pp. 423-430 ◽

Cited By ~ 17

Author(s):

S Dalton ◽

B P Hughes ◽

G J Barritt

Keyword(s):

Plasma Membrane ◽

Rat Liver ◽

Isolated Hepatocytes ◽

Liver Mitochondria ◽

Ruthenium Red ◽

Rat Liver Mitochondria ◽

Intact Cells ◽

Phosphate Ions ◽

Low Concentrations ◽

Significant Impairment

Lysophospholipids caused the release of 45Ca2+ from isolated rat liver mitochondria incubated at 37 degrees C in the presence of low concentrations of free Ca2+, ATP, Mg2+, and phosphate ions. The concentrations of lysophosphatidylethanolamine, lysophosphatidylcholine, lysophosphatidic acid and lysophosphatidylinositol which gave half-maximal effects were 5, 26, 40 and 56 microM, respectively. The effects of lysophosphatidylethanolamine were not associated with a significant impairment of the integrity of the mitochondria as monitored by measurement of membrane potential and the rate of respiration. Lysophosphatidylethanolamine did not induce the release of Ca2+ from a microsomal fraction, or enhance Ca2+ inflow across the plasma membrane of intact cells, but did release Ca2+ from an homogenate prepared from isolated hepatocytes and incubated under the same conditions as isolated mitochondria. The proportion of mitochondrial 45Ca2+ released by lysophosphatidylethanolamine was not markedly affected by altering the total amount of Ca2+ in the mitochondria, the concentration of extramitochondrial Mg2+, by the addition of Ruthenium Red, or when oleoyl lysophosphatidylethanolamine was employed instead of the palmitoyl derivative. The effects of 5 microM-lysophosphatidylethanolamine were reversed by washing the mitochondria. The possibility that lysophosphatidylethanolamine acts to release Ca2+ from mitochondria in intact hepatocytes following the binding of Ca2+-dependent hormones to the plasma membrane is briefly discussed.

Download Full-text

The transport of sulphate and sulphite in rat liver mitochondria

Biochemical Journal ◽

10.1042/bj1420127 ◽

1974 ◽

Vol 142 (1) ◽

pp. 127-137 ◽

Cited By ~ 46

Author(s):

M. Crompton ◽

F. Palmieri ◽

Michela Capano ◽

E. Quagliariello

Keyword(s):

Rat Liver ◽

Liver Mitochondria ◽

Mitochondrial Swelling ◽

Rat Liver Mitochondria ◽

Additional Mechanism ◽

Low Concentrations ◽

Hydroxyl Ion ◽

Osmotic Solution

1. The mechanism of sulphite and sulphate permeation into rat liver mitochondria was investigated. 2. Extramitochondrial sulphite and sulphate elicit efflux of intramitochondrial phosphate, malate, succinate and malonate. The sulphate-dependent effluxes and the sulphite-dependent efflux of dicarboxylate anions are inhibited by butylmalonate, phenylsuccinate and mersalyl. Inhibition of the phosphate efflux produced by sulphite is caused by mersalyl alone and by N-ethylmaleimide and butylmalonate when present together. 3. External sulphite and sulphate cause efflux of intramitochondrial sulphate, and this is inhibited by butylmalonate, phenylsuccinate and mersalyl. 4. External sulphite and sulphate do not cause efflux of oxoglutarate or citrate. 5. Mitochondria swell when suspended in an iso-osmotic solution of ammonium sulphite; this is not inhibited by N-ethylmaleimide or mersalyl. 6. Low concentrations of sulphite, but not sulphate, produce mitochondrial swelling in iso-osmotic solutions of ammonium malate, succinate, malonate, sulphate, or phosphate in the presence of N-ethylmaleimide. 7. It is concluded that both sulphite and sulphate may be transported by the dicarboxylate carrier of rat liver mitochondria and also that sulphite may permeate by an additional mechanism; the latter may involve the permeation of sulphurous acid or SO2 or an exchange of the sulphite anion for hydroxyl ion(s).

Download Full-text

Transamination of Ring-Substituted L-Phenylalanines by an Extract from Rat Liver Mitochondria

Canadian Journal of Biochemistry ◽

10.1139/o73-047 ◽

1973 ◽

Vol 51 (4) ◽

pp. 407-411 ◽

Cited By ~ 6

Author(s):

J. H. Tong ◽

B. A. Stoochnoff ◽

A. D'Iorio ◽

N. Leo Benoiton

Keyword(s):

Amino Acids ◽

Rat Liver ◽

Liver Mitochondria ◽

Tyrosine Aminotransferase ◽

Rat Liver Mitochondria ◽

Amino Group ◽

Mitochondrial Enzyme ◽

Soluble Enzyme ◽

Keto Acids ◽

Mitochondrial Extract

The L- and D-isomers of m-tyrosine, o-tyrosine, p-chlorophenylalanine (p-CP), and p-fluorophenylalanine (p-FP) were tested as substrates for the soluble tyrosine aminotransferase and a mitochondrial extract of rat liver by measuring the amino acids formed with 2-oxoglutarate, oxaloacetate, and pyruvate as acceptors. None of the above were substrates for the soluble enzyme. L-m-Tyrosine, L-p-CP, and L-p-FP were transaminated at substantial rates (16–25% of the rate for L-tyrosine) by the mitochondrial enzyme with all three keto acids as amino group acceptors. A slow but definite transamination of L-o-tyrosine by the mitochondrial enzyme was demonstrated using labeled 2-oxoglutarate as acceptor.

Download Full-text

Hormone-initiated maturation of rat liver mitochondria after birth

Biochemical Journal ◽

10.1042/bj1860361 ◽

1980 ◽

Vol 186 (1) ◽

pp. 361-367 ◽

Cited By ~ 21

Author(s):

R Sutton ◽

J K Pollak

Keyword(s):

Cyclic Amp ◽

Rat Liver ◽

Beta Blocker ◽

Adenine Nucleotide ◽

Respiratory Control ◽

Liver Mitochondria ◽

Energy Transduction ◽

Rat Liver Mitochondria ◽

Maturation Process ◽

High Concentrations

1. The injection of adrenaline, glucagon or cyclic AMP into foetal rats in utero initiates the maturation of energy transduction in rat liver mitochondria before birth. 2. The injection of the beta-blocker, propranolol, prevents this maturation process. 3. The maturation of mitochondrial energy transduction is measured in terms of the increase in the respiratory control index and mitochondrial adenine nucleotide concentration. 4. It is postulated that the actions of the hormones, acting through cyclic AMP, affect glycogenolysis and glycolysis to give rise to transient localized high concentrations of ATP. 5. It is the ATP that acts as the molecular trigger, effecting mitochondrial maturation.

Download Full-text

Phosphatidic acid biosynthesis in rat liver mitochondria and microsomal fractions. Regulation of fatty acid positional specificity

Biochemical Journal ◽

10.1042/bj1580249 ◽

1976 ◽

Vol 158 (2) ◽

pp. 249-254 ◽

Cited By ~ 16

Author(s):

K S Bjerve ◽

L N W Daae ◽

J Bremer

Keyword(s):

Fatty Acid ◽

Phosphatidic Acid ◽

Rat Liver ◽

Microsomal Fraction ◽

Liver Mitochondria ◽

Rat Liver Mitochondria ◽

Acid Biosynthesis ◽

Positional Specificity ◽

Fatty Acid Specificity ◽

High Concentrations

The positional and fatty acid specificity of phosphatidic acid biosynthesis in rat liver mitochondria and microsomal fractions was studied by using acylcarnitines, CoA and an excess of carnitine palmitoyltransferase (EC 2.3.1.21) as the source of acyl-CoA. In the mitochondria, the preference for palmitic acid at the 1-position is increased at high acyl-CoA concentrations, whereas it is decreased in the microsomal fraction. There was no change in the fatty acid specificity at the 2-position with different acyl-CoA concentrations in any of the factions. The preference in mitochondria for linoleic acid at the 2-position is strongly increased at high concentrations of lysophosphatidic acid.

Download Full-text