scholarly journals Acetyl-coenzyme A hydrolase, an artifact? The conversion of acetyl-coenzyme A into acetate by the combined action of carnitine acetyltransferase and acetylcarnitine hydrolase

1975 ◽  
Vol 152 (2) ◽  
pp. 167-172 ◽  
Author(s):  
N. D. Costa ◽  
A. M. Snoswell
Keyword(s):  
Rat Liver ◽  
Mitochondrial Membrane ◽  
Hydrolase Activity ◽  
Carnitine Acetyltransferase ◽  
Inner Mitochondrial Membrane ◽  
Acetyl Coa ◽  
Acetyl Coenzyme A ◽  
Sheep Liver ◽  
Liver Homogenates ◽  
Coa Hydrolase

1. The nature of the acetyl-CoA hydrolase (EC 3.1.2.1) reaction in rat and sheep liver homogenates was investigated. 2. The activity determined in an incubated system was 5.10 and 3.28nmol/min per mg of protein for rat and sheep liver homogenate respectively. This activity was not affected by the addition of l-carnitine, but was decreased by the addition of d-carnitine. 3. No acetyl-CoA hydrolase activity could be detected in rat or sheep liver homogenates first treated with Sephadex G-25. This treatment decreased the carnitine concentrations of the homogenates to about one-twentieth. Subsequent addition of l-carnitine, but not d-carnitine, restored the apparent acetyl-CoA hydrolase activity. 4. Sephadex treatment did not affect acetyl-carnitine hydrolase activity of the homogenates, which was 5.8 and 8.1nmol/min per mg of protein respectively for rat and sheep liver. 5. Direct spectrophotometric assay of acetyl-CoA hydrolase, based on the reaction of CoA released with 5,5′-dithiobis-(2-nitrobenzoic acid), clearly demonstrated that after Sephadex treatment no activity could be measured. 6. Carnitine acetyltransferase (EC 2.3.1.7) activity measured in the same assay system in response to added l-carnitine was very low in normal rat liver homogenates, owing to the apparent high acetyl-CoA hydrolase activity, but was increased markedly after Sephadex treatment. The Vmax. for this enzyme in rat liver homogenates was increased from 3.4 to 14.8nmol/min per mg of protein whereas the Km for l-carnitine was decreased from 936 to 32μm after Sephadex treatment. 7. Acetyl-CoA hydrolase activity could be demonstrated in disrupted rat liver mitochondria but not in separated outer or inner mitochondrial membrane fractions. Activity could be demonstrated after recombination of outer and inner mitochondrial membrane fractions. The outer mitochondrial membrane fraction showed acetylcarnitine hydrolase activity and the inner mitochondrial membrane fraction showed carnitine acetyltransferase activity. 8. The results presented here demonstrate that acetyl-CoA hydrolase activity in rat and sheep liver is an artifact and the activity is due to the combined activity of carnitine acetyltransferase and acetylcarnitine hydrolase.

scholarly journals Deacylation of acetyl-coenzyme A and acetylcarnitine by liver preparations

1978 ◽  
Vol 171 (2) ◽  
pp. 299-303 ◽  
Author(s):  
A M Snoswell ◽  
P K Tubbs
Keyword(s):  
Gel Filtration ◽  
Mitochondrial Fraction ◽  
Liver Mitochondria ◽  
Carnitine Acetyltransferase ◽  
Acetyl Coa ◽  
Acetyl Coenzyme A ◽  
Sheep Liver ◽  
Chain Acyl ◽  
Coa Hydrolase ◽  
Hydrolysis Of

The breakdown of acetylcarnitine catalysed by extracts of rat and sheep liver was completely abolished by Sephadex G-25 gel filtration, whereas the hydrolysis of acetyl-CoA was unaffected. Acetyl-CoA and CoA acted catalytically in restoring the ability of Sephadex-treated extracts to break down acetylcarnitine, which was therefore not due to an acetylcarnitine hydrolase but to the sequential action of carnitine acetyltransferase and acetyl-CoA hydrolase. Some 75% of the acetyl-CoA hydrolase activity of sheep liver was localized in the mitochondrial fraction. Two distinct acetyl-CoA hydrolases were partially purified from extracts of sheep liver mitochondria. Both enzymes hydrolysed other short-chain acyl-CoA compounds and succinyl-CoA (3-carboxypropionyl-CoA), but with one acetyl-CoA was the preferred substrate.

Cytochemical Localization of Transferase Activities: Carnitine Acetyltransferase

1970 ◽  
Vol 6 (1) ◽  
pp. 29-50
Author(s):  
JOAN A. HIGGINS ◽  
R. J. BARRNETT
Keyword(s):  
Mitochondrial Membrane ◽  
Carnitine Acetyltransferase ◽  
Inner Mitochondrial Membrane ◽  
Acetyl Coa ◽  
Cytochemical Localization ◽  
Fixed Tissue ◽  
Structural Localization ◽  
Outer Membranes ◽  
Sh Group ◽  
Ferrocyanide Method

Two methods for the cytochemical detection of free CoA and their utilization in the fine-structural localization of carnitine acetyltransferase in rat heart are described. The first utilizes the reducing property of the SH group of CoA to reduce potassium ferricyanide to potassium ferrocyanide, which in the presence of uranyl ions forms an electron-dense precipitate of uranyl ferrocyanide. The second utilizes the mercaptide-forming property of the free SH group of CoA, which forms a precipitate with cadmium ions. Using the uranyl-ferrocyanide method, reaction product due to endogenous enzymic activity was found on and between the cristae and between the inner and outer membranes of the mitochondria in fresh heart muscle. In aldehyde-fixed tissue activity was recorded only between the inner and outer membranes. Endogenous activity was removed by preincubation of the tissue in a solution of ferricyanide. On addition of acetyl CoA and carnitine to the incubation medium, fresh tissue, which had been preincubated in ferricyanide, showed reaction product between and on the cristae and between the inner and outer membranes of the mitochondria, while fixed tissue showed reaction product in the latter position only. In both cases the activity between the outer and inner mitochondrial membranes was dependent on both acetyl CoA and carnitine, while the cristae reaction occurred in the absence of carnitine, but required acetyl CoA. All activity was inhibited by mercuric chloride. Acetyl carnitine reduced the activity in the fixed tissue and had severe effects on the structure of fresh mitochondria. These results suggest the presence of carnitine acetyltransferase, which survives aldehyde fixation, on the inner surface of the outer mitochondrial membrane and/or the outer surface of the inner mitochondrial membrane. A second enzyme which released CoA from acetyl CoA occurred in relation to the cristae of unfixed mitochondria. The cadmium method was less satisfactory than the uranyl-ferrocyanide method but with fixed tissue gave confirmatory results.

scholarly journals Uptake of protoporphyrin IX by isolated rat liver mitochondria

1980 ◽  
Vol 188 (2) ◽  
pp. 329-335 ◽  
Author(s):  
M E Koller ◽  
I Romslo
Keyword(s):  
Rat Liver ◽  
Mitochondrial Membrane ◽  
Protoporphyrin Ix ◽  
Liver Mitochondria ◽  
Rat Liver Mitochondria ◽  
Inner Mitochondrial Membrane ◽  
Erythropoietic Protoporphyria ◽  
Inner Membrane ◽  
Isolated Rat Liver ◽  
Isolated Rat

Rat liver mitochondria accumulate protoporphyrin IX from the suspending medium into the inner membrane in parallel with the magnitude of the transmembrane K+ gradient (K+in/K+out). Only protoporphyrin IX taken up in parallel with the transmembrane K+ gradient is available for haem synthesis. Coproporphyrins (isomers I and III) are not taken up by the mitochondria. The results support the suggestion by Elder & Evans [(1978) Biochem. J. 172, 345-347] that the prophyrin to be taken up by the inner mitochondrial membrane belongs to the protoporphyrin(ogen) IX series. Protoporphyrin IX at concentrations above 15 nmol/mg of protein has detrimental effects on the structural and functional integrity of the mitochondria. The relevance of these effects to the hepatic lesion in erythropoietic protoporphyria is discussed.

Phospholipid topology of the inner mitochondrial membrane of rat liver

Biochemistry ◽  
1979 ◽  
Vol 18 (11) ◽  
pp. 2407-2414 ◽  
Author(s):  
Richard C. Crain ◽  
Guido V. Marinetti
Keyword(s):  
Rat Liver ◽  
Mitochondrial Membrane ◽  
Inner Mitochondrial Membrane

scholarly journals The maturation of the inner membrane of foetal rat liver mitochondria. An example of a positive-feedback mechanism

1975 ◽  
Vol 150 (3) ◽  
pp. 477-488 ◽  
Author(s):  
J K Pollak
Keyword(s):  
Oxidative Phosphorylation ◽  
Rat Liver ◽  
Mitochondrial Membrane ◽  
Respiratory Control ◽  
Liver Mitochondria ◽  
Neonatal Rat ◽  
Rat Liver Mitochondria ◽  
Inner Mitochondrial Membrane ◽  
Mature Adult ◽  
Foetal Rat

A new method was devised for the isolation of foetal and neonatal rat lvier mitochondria, giving higher yields than conventional methods. 2. During development from the perinatal period to the mature adult, the ratio of cytochrome oxidase/succinate-cytochrome c reductase changes. 3. The inner mitochondrial membrane of foetal liver mitochondria possesses virtually no osmotic activity; the permeability to sucrose decreases with increasing developmental age. 4. Foetal rat liver mitochondria possess only marginal respiratory control and do not maintain Ca2+-induced respiration; they also swell in respiratory-control medium in the absence of substrate. ATP enhances respiratory control and prevents swelling, adenylyl imidodiphosphate, ATP+atractyloside enhance the R.C.I. (respiratory control index), Ca2+-induced respiratory control and prevent swelling, whereas GTP and low concentrations of ADP have none of these actions. It is concluded that the effect of ATP depends on steric interaction with the inner mitochondrial membrane. 5. When 1-day pre-partum foetuses are obtained by Caesarean section and maintained in a Humidicrib for 90 min, mitochondrial maturation is ‘triggered’, so that their R.C.I. is enhanced and no ATP is required to support Ca2+-dependent respiratory control or to inhibit mitochondrial swelling. 6. It is concluded that foetal rat liver mitochondria in utero do not respire, although they are capable of oxidative phosphorylation in spite of their low R.C.I. The different environmental conditions which the neonatal rat encounters ex utero enable the hepatic mitochondria to produce ATP, which interacts with the inner mitochondrial membrane to enhance oxidative phosphorylation by an autocatalytic mechanism.

Carnitine acyltransferases and acyl-CoA hydrolases in human and rat liver

1987 ◽  
Vol 73 (1) ◽  
pp. 3-10 ◽  
Author(s):  
L. Agius ◽  
P. D. Wright ◽  
K. G. M. M. Alberti
Keyword(s):  
Rat Liver ◽  
Human Liver ◽  
Citrate Synthase ◽  
Carnitine Acetyltransferase ◽  
Diabetic Ketosis ◽  
Carnitine Metabolism ◽  
Carnitine Acyltransferases ◽  
Extrahepatic Tissues ◽  
Carnitine Palmitoyltransferase Activity ◽  
Coa Hydrolase

1. The activities of carnitine acyltransferases and acyl-CoA hydrolases were determined in human and rat liver to establish the validity of extrapolating from studies on rats to human metabolism. 2. In human liver, carnitine acetyltransferase activity was 10–14 times higher and carnitine octanoyltransferase 1.7–2.4 times higher than in rat liver, while carnitine palmitoyltransferase activity was similar in human and rat. 3. Acetyl-CoA hydrolase and octanoyl-CoA hydrolase activities were lower in human (42–57%) than in rat liver, but palmitoyl-CoA hydrolase activity was similar in both species. 4. The activity of citrate synthase was lower (44%) in human than in rat liver. The low citrate synthase activity and the high carnitine acetyltransferase in human liver suggest that in man acetylcarnitine might be more important as a vehicle for export of acetyl units from mitochondria than citrate. 5. The high activity of carnitine acetyltransferase in human liver is consistent with the observation that acetylcarnitine is the predominant acylcarnitine excreted in diabetic ketosis in man. 6. It is concluded that the rat may not be a valid model for carnitine metabolism in man, and that in human liver carnitine may have an important role in transfer of acetyl groups out of mitochondria and possibly also to extrahepatic tissues.

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.

scholarly journals Intramitochondrial localization of δ-aminolaevulate synthetase and ferrochelatase in rat liver

1969 ◽  
Vol 114 (3) ◽  
pp. 455-461 ◽  
Author(s):  
Roxane McKay ◽  
R. Druyan ◽  
G. S. Getz ◽  
M. Rabinowitz
Keyword(s):  
Glutamate Dehydrogenase ◽  
Density Gradient ◽  
Malate Dehydrogenase ◽  
Rat Liver ◽  
Mitochondrial Biogenesis ◽  
Mitochondrial Membrane ◽  
Density Gradient Centrifugation ◽  
Gradient Centrifugation ◽  
Differential Centrifugation ◽  
Inner Mitochondrial Membrane

Intramitochondrial loci for δ-aminolaevulate synthetase and ferrochelatase, the initial and final enzymes in haem synthesis, have been found in rat liver. Two different methods of fractionation were applied to mitochondria: (a) sonication and density-gradient centrifugation; (b) treatment with digitonin and differential centrifugation. Similar results were obtained with each technique. δ-Aminolaevulate synthetase is distributed similarly to two known matrix enzymes, malate dehydrogenase and glutamate dehydrogenase. Ferrochelatase is firmly bound to the the inner mitochondrial membrane. These results are considered in terms of the regulation of haem synthesis and in relation to mitochondrial biogenesis.

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