scholarly journals A spectrophotometric procedure for rapid and sensitive measurements of β-oxidation. Demonstration of factors that can be rate-limiting for β-oxidation

1977 ◽  
Vol 164 (3) ◽  
pp. 621-633 ◽  
Author(s):  
H Osmundsen ◽  
J Bremer
Keyword(s):  
Tricarboxylic Acid Cycle ◽  
Liver Mitochondria ◽  
Heart Mitochondria ◽  
Rat Liver Mitochondria ◽  
Beta Oxidation ◽  
Direct Reading ◽  
Kidney Mitochondria ◽  
Final Electron ◽  
Acyl Coa Dehydrogenases ◽  
Tricarboxylic Acid Cycle Intermediates

1. A spectrophotometric direct-reading assay for measurements of beta-oxidation by intact mitochondria is described. The procedure relies on the ability of ferricyanide to trap reducing equivalents generated by the acyl-CoA dehydrogenases (EC 1.3.99.3). The reduction of ferricyanide was recorded by using a dual-wavelength spectrophotometer. 2. Oxaloacetate or acetoacetate was used to stimulate the rate of beta-oxidation by rotenone-blocked mitochondria. Although both were effective with rat liver mitochondria, oxaloacetate gave about 75% more stimulation. With heart or kidney mitochondria, only oxaloacetate gave marked stimulation. Acetoacetate had no stimulatory effect with heart mitochondria, but a small stimulatory effect on the rate of beta-oxidation by kidney mitochondria. 3. The stoicheiometry of beta-oxidation-dependent reduction of ferricyanide was examined, and good correlations were found between experimental and theoretical amounts of ferricyanide reduced. 4. Ferricyanide appears as efficient a final electron acceptor as O2. With ferricyanide the rate of beta-oxidation by heart mitochondria can be measured without interference from the oxidation of tricarboxylic acid-cycle intermediates.

scholarly journals Biochemical effects of the hypoglycaemic compound pent-4-enoic acid and related non-hypoglycaemic fatty acids. Oxidative phosphorylation and mitochondrial oxidation of pyruvate, 3-hydroxybutyrate and tricarboxylic acid-cycle intermediates

1968 ◽  
Vol 110 (3) ◽  
pp. 499-509 ◽  
Author(s):  
A. E. Senior ◽  
H. S. A. Sherratt
Keyword(s):  
Oxidative Phosphorylation ◽  
Tricarboxylic Acid Cycle ◽  
Time Lag ◽  
Liver Mitochondria ◽  
Enoic Acid ◽  
Rat Liver Mitochondria ◽  
Pentanoic Acid ◽  
Low Concentrations ◽  
Cyclobutanecarboxylic Acid ◽  
Tricarboxylic Acid Cycle Intermediates

1. The effects of the hypoglycaemic compound pent-4-enoic acid, and of four structurally related non-hypoglycaemic compounds (pent-2-enoic acid, pentanoic acid, cyclopropanecarboxylic acid and cyclobutanecarboxylic acid), on several reactions in rat liver mitochondria were determined. 2. The use of manometric techniques for measurements of oxidations and of phosphorylation is critically discussed. 3. Pent-4-enoic acid and pentanoic acid uncoupled oxidative phosphorylation at low concentrations, but usually by not more than about 50%. 4. All the compounds, except cyclobutanecarboxylic acid, strongly inhibited the oxidation of pyruvate and 2-oxoglutarate, but the oxidations of succinate, citrate and 3-hydroxybutyrate were not strongly inhibited. 5. All the compounds, except cyclobutanecarboxylic acid, inhibited decarboxylation of [1−14C]pyruvate with ferricyanide as electron acceptor. 6. All the compounds, except pent-2-enoic acid, caused mitochondrial swelling after a time-lag.

scholarly journals Metabolism of pent-4-enoate in rat heart. Reduction of the double bond

1981 ◽  
Vol 194 (2) ◽  
pp. 427-432 ◽  
Author(s):  
J K Hiltunen ◽  
E J Davis
Keyword(s):  
Double Bond ◽  
Rat Heart ◽  
No Reduction ◽  
Tricarboxylic Acid Cycle ◽  
Liver Mitochondria ◽  
Tricarboxylic Acid ◽  
Beta Oxidation ◽  
Initial Oxidation ◽  
Acid Cycle ◽  
Tricarboxylic Acid Cycle Intermediates

1. Soluble extracts from rat heart and liver mitochondria were used to evaluate the early steps in the conversion of pent-4-enoyl-CoA into tricarboxylic acid-cycle intermediates. Hitherto the unresolved problem was the reduction of the double bond of pent-4-enoate. 2. Soluble extracts from heart mitochondria reduced pent-4-enoyl-CoA and penta-2,4-dienoyl-CoA in the presence of NADPH at rates (nmol/min per mg of protein) of 0.9 +/- 0.1 and 132 +/- 8 and from the liver mitochondria at the rates of 1.9 +/- 0.2 and 52 +/- 6 respectively. No reduction of acryloyl-CoA was found. 3. We show that primarily the double bond in position 4, not in position 2, of penta-2,4-dienoyl-CoA is reduced. 4. It is concluded that the principal metabolic pathway of penta-4-enoate is reduction of the double bond in position 4 after an initial oxidation of penta-2,4-dienoyl-CoA. The pent-2-enoyl-CoA thus formed can be further metabolized by the usual enzymes of beta-oxidation, and by the further metabolism of propionyl-CoA to tricarboxylic acid-cycle intermediates.

The inhibition of mammalian mitochondrial NADU oxidation by chloramphenicol and its isomers and analogues

1968 ◽  
Vol 46 (9) ◽  
pp. 1003-1008 ◽  
Author(s):  
K. B. Freeman ◽  
D. Haldar
Keyword(s):  
Cytochrome B ◽  
Respiratory Chain ◽  
Rat Liver ◽  
Nadh Dehydrogenase ◽  
Coenzyme Q ◽  
Liver Mitochondria ◽  
Heart Mitochondria ◽  
Rat Liver Mitochondria ◽  
Beef Heart ◽  
Beef Heart Mitochondria

Chloramphenicol and its isomers and analogues have been found to inhibit the oxidation of NADH, but not that of succinate, by beef heart mitochondria. They must therefore inhibit the NADH dehydrogenase segment of the respiratory chain. Chloramphenicol gave 50% inhibition at a concentration of 1 mM. The methylthio analogue of chloramphenicol inhibited NADH – coenzyme Q6 reductase but not NADH–ferricyanide reductase. Spectrophotometric observations suggest that these inhibitors act between NADH and flavin in coupled rat liver mitochondria and between flavin and cytochrome b in uncoupled beef heart mitochondria.

scholarly journals Inhibition in vitro of acyl-CoA dehydrogenases by 2-mercaptoacetate in rat liver mitochondria

1983 ◽  
Vol 215 (3) ◽  
pp. 457-464 ◽  
Author(s):  
F Bauché ◽  
D Sabourault ◽  
Y Giudicelli ◽  
J Nordmann ◽  
R Nordmann
Keyword(s):  
Rat Liver ◽  
Conformational Changes ◽  
Reaction Medium ◽  
Liver Mitochondria ◽  
Fatty Acyl ◽  
Rat Liver Mitochondria ◽  
Mitochondrial Matrix ◽  
Main Effect ◽  
Acyl Coa Dehydrogenases

In rat liver hypo-osmotically treated mitochondria, 2-mercaptoacetate inhibits respiration induced by palmitoyl-CoA, octanoate or butyryl-CoA only when the reaction medium is supplemented with ATP. Under this condition, NADH-stimulated respiration is not affected. In liver mitochondrial matrix, the presence of ATP is also required to observe a 2-mercaptoacetate-induced inhibition of acyl-CoA dehydrogenases tested with palmitoyl-CoA, butyryl-CoA or isovaleryl-CoA as substrate. As the oxidation of these substrates is also inhibited by the incubation medium resulting from the reaction of 2-mercaptoacetate with acetyl-CoA synthase, with conditions under which 2-mercaptoacetate has no effect, 2-mercaptoacetyl-CoA seems to be the likely inhibitory metabolite responsible for the effects of 2-mercaptoacetate. Kinetic experiments show that the main effect of the 2-mercaptoacetate-active metabolite is to decrease the affinities of fatty acyl-CoA dehydrogenases towards palmitoyl-CoA or butyryl-CoA and of isovaleryl-CoA dehydrogenase towards isovaleryl-CoA. Addition of N-ethylmaleimide to mitochondrial matrix pre-exposed to 2-mercaptoacetate results in the immediate reversion of the inhibitions of palmitoyl-CoA and isovaleryl-CoA dehydrogenations and in a delayed reversion of butyryl-CoA dehydrogenation. These results led us to conclude that (i) the ATP-dependent conversion of 2-mercaptoacetate into an inhibitory metabolite takes place in the liver mitochondrial matrix and (ii) the three fatty acyl-CoA dehydrogenases and isovaleryl-CoA dehydrogenase are mainly competitively inhibited by this compound. Finally, the present study also suggests that the inhibitory metabolite of 2-mercaptoacetate may bind non-specifically to, or induce conformational changes at, the acyl-CoA binding sites of these dehydrogenases.

scholarly journals Steady-state concentrations of coenzyme A, acetyl-coenzyme A and long-chain fatty acyl-coenzyme A in rat-liver mitochondria oxidizing palmitate

1965 ◽  
Vol 97 (2) ◽  
pp. 587-594 ◽  
Author(s):  
PB Garland ◽  
D Shepherd ◽  
DW Yates
Keyword(s):  
Rat Liver ◽  
Coenzyme A ◽  
Liver Mitochondria ◽  
Fatty Acyl ◽  
Rat Liver Mitochondria ◽  
Beta Oxidation ◽  
Acetyl Coa ◽  
Acetyl Coenzyme A ◽  
Acyl Coenzyme A ◽  
Long Chain

1. Fluorimetric assays are described for CoASH, acetyl-CoA and long-chain fatty acyl-CoA, and are sensitive to at least 50mumumoles of each. 2. Application of these assays to rat-liver mitochondria oxidizing palmitate in the absence and presence of carnitine indicated two pools of intramitochondrial CoA. One pool could be acylated by palmitate and ATP, and the other pool acylated by palmitate with ATP and carnitine, or by palmitoylcarnitine alone. 3. The intramitochondrial content of acetyl-CoA is increased by the oxidation of palmitate both in the absence and presence of l-malate. 4. The conversion of palmitoyl-CoA into acetyl-CoA by beta-oxidation takes place without detectable accumulation of acyl-CoA intermediates.

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