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.

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 Biosynthesis of acetyl-coenzyme A in the electric organ of Torpedo marmorata in relation to acetylcholine metabolism

1977 ◽  
Vol 166 (3) ◽  
pp. 447-453 ◽  
Author(s):  
M F Diebler ◽  
Y Morot-Gaudry
Keyword(s):  
Choline Acetyltransferase ◽  
Gel Filtration ◽  
Electric Organ ◽  
Nerve Endings ◽  
Tissue Extract ◽  
Torpedo Marmorata ◽  
Forward Reaction ◽  
Acetyl Coa ◽  
Acetyl Coenzyme A ◽  
Acetylcholine Metabolism

Formation of acetyl-CoA through acetyl-CoA synthetase (forward reaction) and through choline acyltransferase (backward reaction) was investigated in tissue extract from the electric organ of Torpedo marmorata. When the tissue extract was submitted to gel filtration on Sephadex G-25, the formation of acetyl-CoA by acetyl-CoA synthetase appeared fully dependent on ATP and CoA and partially dependent on acetate (an endogenous supply of acetate is discussed). Choline acetyltransferase was a potent source of acetyl-CoA, only requiring acetylcholine and CoA, and was much more efficient than acetyl-CoA synthetase for concentrations of acetylcholine likely to be present in nerve endings.

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 Acetyl-coenzyme A deacylase activity in liver is not an artifact. Subcellular distribution and substrate specificity of acetyl-coenzyme A deacylase activities in rat liver

1979 ◽  
Vol 177 (1) ◽  
pp. 71-79 ◽  
Author(s):  
Klaus-P. Grigat ◽  
Klaus Koppe ◽  
Claus-D. Seufert ◽  
Hans-D Söling
Keyword(s):  
Rat Liver ◽  
Coenzyme A ◽  
Liver Mitochondria ◽  
Acid Oxidation ◽  
Matrix Space ◽  
Acetyl Coa ◽  
Acetyl Coenzyme A ◽  
Lysosomal Fraction ◽  
Total Enzyme Activity ◽  
Acetyl Coenzyme

Whole liver and isolated liver mitochondria are able to release free acetate, especially under conditions of increased fatty acid oxidation. In the present paper it is shown that rat liver contains acetyl-CoA deacylase (EC 3.1.2.1) activity (0.72μmol/min per g wet wt. of liver at 30°C and 0.5mm-acetyl-CoA). At 0.5mm-acetyl-CoA 73% of total enzyme activity was found in the mitochondria, 8% in the lysosomal fraction and 19% in the postmicrosomal supernatant. Mitochondrial subfractionation shows that mitochondrial acetyl-CoA deacylase activity is restricted to the matrix space. Mitochondrial acetyl-CoA deacylase showed almost no activity with either butyryl- or hexanoyl-CoA. Acetyl-CoA hydrolase activity from purified rat liver lysosomes exhibited a very low affinity for acetyl-CoA (apparent Km>15mm compared with an apparent Km value of 0.5mm for the mitochondrial enzyme) and reacted at about the same rate with acetyl-, n-butyryl- and hexanoyl-CoA. We could not confirm the findings of Costa & Snoswell [(1975) Biochem. J.152, 167–172] according to which mitochondrial acetyl-CoA deacylase was considered to be an artifact resulting from the combined actions of acetyl-CoA–l-carnitine acetyltransferase (EC 2.3.1.7) and acetylcarnitine hydrolase. The results are in line with the concept that free acetate released by the liver under physiological conditions stems from the intramitochondrial deacylation of acetyl-CoA.

scholarly journals Hint2, the mitochondrial nucleoside 5'-phosphoramidate hydrolase; properties of the homogeneous protein from sheep (Ovis aries) liver.

2013 ◽  
Vol 60 (2) ◽  
Author(s):  
Ewa Bretes ◽  
Anna M Wojdyła-Mamoń ◽  
Joanna Kowalska ◽  
Jacek Jemielity ◽  
Renata Kaczmarek ◽  
...  
Keyword(s):  
Molecular Mass ◽  
Simple Procedure ◽  
Mitochondrial Fraction ◽  
Protein Family ◽  
Ovis Aries ◽  
Molecular Properties ◽  
Kinetic Properties ◽  
Biological Functions ◽  
Sheep Liver ◽  
Hydrolysis Of

Adenosine 5'-phosphoramidate (NH2-pA) is a rare natural nucleotide and its biochemistry and biological functions are poorly recognized. All organisms have proteins that may be involved in the catabolism of NH2-pA. They are members of the HIT protein family and catalyze hydrolytic splitting of NH2-pA to 5'-AMP and ammonia. At least five HIT proteins have been identified in mammals; however, the enzymatic and molecular properties of only Fhit and Hint1 have been comprehensively studied. Our study focuses on the Hint2 protein purified by a simple procedure to homogeneity from sheep liver mitochondrial fraction (OaHint2). Hint1 protein was also prepared from sheep liver (OaHint1) and the molecular and kinetic properties of the two proteins compared. Both function as homodimers and behave as nucleoside 5'-phosphoramidate hydrolases. The molecular mass of the OaHint2 monomer is 16 kDa and that of the OaHint1 monomer 14.9 kDa. Among potential substrates studied, NH2-pA appeared to be the best; the Km and kcat values estimated for this compound are 6.6 μM and 68.3 s⁻¹, and 1.5 μM and 11.0 s⁻¹ per natively functioning dimer of OaHint2 and OaHint1, respectively. Studies of the rates of hydrolysis of different NH2-pA derivatives show that Hint2 is more specific towards compounds with a P-N bond than Hint1. The thermostability of these two proteins is also compared.

scholarly journals Aspects of carnitine ester metabolism in sheep liver

1970 ◽  
Vol 119 (1) ◽  
pp. 59-65 ◽  
Author(s):  
A. M. Snoswell ◽  
G. D. Henderson
Keyword(s):  
Rat Liver ◽  
Bovine Liver ◽  
Liver Mitochondria ◽  
Free Carnitine ◽  
Rat Liver Mitochondria ◽  
Carnitine Acetyltransferase ◽  
Total Acid ◽  
Sheep Liver ◽  
Ad Libitum ◽  
Carnitine Palmitoyltransferase Activity

1. Carnitine acetyltransferase (EC 2.3.1.7) activity in sheep liver mitochondria was 76nmol/min per mg of protein, in contrast with 1.7 for rat liver mitochondria. The activity in bovine liver mitochondria was comparable with that of sheep liver mitochondria. Carnitine palmitoyltransferase activity was the same in both sheep and rat liver mitochondria. 2. The [free carnitine]/[acetylcarnitine] ratio in sheep liver ranged from 6:1 for animals fed ad libitum on lucerne to approx. 1:1 for animals grazed on open pastures. This change in ratio appeared to reflect the ratio of propionic acid to acetic acid produced in the rumen of the sheep under the two dietary conditions. 3. In sheep starved for 7 days the [free carnitine]/[acetylcarnitine] ratio in the liver was 0.46:1. The increase in acetylcarnitine on starvation was not at the expense of free carnitine, as the amounts of free carnitine and total acid-soluble carnitine rose approximately fivefold on starvation. An even more dramatic increase in total acid-soluble carnitine of the liver was seen in an alloxan-diabetic sheep. 4. The [free CoA]/[acetyl-CoA] ratio in the liver ranged from 1:1 in the sheep fed on lucerne to 0.34:1 for animals starved for 7 days. 5. The importance of carnitine acetyltransferase in sheep liver and its role in relieving `acetyl pressure' on the CoA system is discussed.

Hepatic Ketogenesis During Development

1971 ◽  
Vol 49 (5) ◽  
pp. 599-605 ◽  
Author(s):  
Leo P. K. Lee ◽  
Irving B. Fritz
Keyword(s):  
Fetal Development ◽  
Ketone Body ◽  
Liver Mitochondria ◽  
Acid Oxidation ◽  
Carnitine Acetyltransferase ◽  
Acetyl Coa ◽  
Outer Membranes ◽  
Mitochondrial Fractions ◽  
Fetal Rats ◽  
Generating System

Livers from fetal rats were shown to have lower rates of ketogenesis from acetate, acetylcarnitine, pyruvate, octanoate, and palmitate than liver preparations from adult animals. The enzymes required for ketogenesis from acetyl-CoA were demonstrated to be nonlimiting in fetal livers. The maximal ketogenic activity by disrupted mitochondria incubated with an acetyl-CoA-generating system was one-third or more of that observed in liver mitochondrial fractions prepared from adult rats.The enzymes required for fatty acid oxidation were also shown to be present in liver mitochondria from fetal rats. Although rates of ketogenesis from octanoate and palmitate were low, ketogenesis from octanoylcarnitine was over 60% of that observed in liver mitochondria from adult rats.During late fetal development and shortly after birth, the maximal hepatic ketogenic-forming activity increased rapidly, with the increase occurring completely in mitochondrial and not in cytosol fractions. The enzymes involved with ketone body formation were shown to remain within mitochondrial particles which had been stripped of their outer membranes. Levels of carnitine acetyltransferase were measured in livers from developing rats, and results were compared with previous observations on changes in activities of carnitine palmitoyltransferase.

Complete β-oxidation of valproate: cleavage of 3-oxovalproyl-CoA by a mitochondrial 3-oxoacyl-CoA thiolase

2002 ◽  
Vol 362 (3) ◽  
pp. 755-760 ◽  
Author(s):  
Margarida F. B. SILVA ◽  
Jos P. N. RUITER ◽  
Henk OVERMARS ◽  
Albert H. BOOTSMA ◽  
Albert H. van GENNIP ◽  
...  
Keyword(s):  
Valproic Acid ◽  
Liver Mitochondria ◽  
Rat Liver Mitochondria ◽  
Tandem Ms ◽  
Carnitine Acetyltransferase ◽  
Reverse Phase Hplc ◽  
Reverse Phase ◽  
On Line ◽  
Hydrolysis Of

The β-oxidation of valproic acid (VPA; 2-n-propylpentanoic acid) was investigated in vitro in intact rat liver mitochondria incubated with 3H-labelled VPA. The metabolism of [4,5-3H2]VPA and [2-3H]VPA was studied by analysing the different acyl-CoA intermediates formed by reverse-phase HPLC with radiochemical detection. Valproyl-CoA, Δ2(E)-valproyl-CoA,3-hydroxyvalproyl-CoA and 3-oxovalproyl-CoA (labelled and non-labelled) were determined using continuous on-line radiochemical and UV detection. The formation of these intermediates was investigated using the two tritiated precursors in respiratory states 3 and 4. Valproyl-CoA was present at highest concentrations under both conditions. Two distinct labelled peaks were found, which were identified as 3H2O and [4,5-3H2]3-oxo-VPA. The formation of 3H2O strongly suggested that VPA underwent complete β-oxidation and that [4,5-3H2]3-oxo-VPA was formed by hydrolysis of the corresponding thioester. The hypothesis that 3-oxovalproyl-CoA undergoes thiolytic cleavage was investigated further. For this purpose a mito chondrial lysate was incubated with synthetic 3-oxovalproyl-CoA, carnitine and carnitine acetyltransferase for subsequent monitoring of the formation of propionylcarnitine and pentanoylcarnitine using electrospray ionization tandem MS. The detection of these compounds demonstrated unequivocally that the intermediate 3-oxovalproyl-CoA is a substrate of a mitochondrial thiolase, producing propionyl-CoA and pentanoyl-CoA, thus demonstrating the complete β-oxidation of VPA in the mitochondrion. Our data should lead to a re-evaluation of the generally accepted concept that the biotransformation of VPA by mitochondrial β-oxidation is incomplete.

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