scholarly journals Enzymatically inactive forms of acetyl-CoA carboxylase in rat liver mitochondria

1988 ◽  
Vol 251 (3) ◽  
pp. 881-885 ◽  
Author(s):  
J B Allred ◽  
C R Roman-Lopez
Keyword(s):  
Rat Liver ◽  
Microsomal Fraction ◽  
Specific Activity ◽  
Polyacrylamide Gel Electrophoresis ◽  
Mitochondrial Fraction ◽  
Liver Mitochondria ◽  
Rat Liver Mitochondria ◽  
Outer Mitochondrial Membrane ◽  
Acetyl Coa Carboxylase ◽  
Acetyl Coa

Biotinyl proteins were labelled by incubation of SDS-denatured preparations of subcellular fractions of rat liver with [14C]methylavidin before polyacrylamide-gel electrophoresis. Fluorographic analysis showed that mitochondria contained two forms of acetyl-CoA carboxylase [acetyl-CoA:carbon dioxide ligase (ADP-forming) EC 6.4.1.2], both of which were precipitated by antibody to the enzyme. When both forms were considered, almost three-quarters of the total liver acetyl-CoA carboxylase was found in the mitochondrial fraction of liver from fed rats while only 3.5% was associated with the microsomal fraction. The remainder was present in cytosol, either as the intact active enzyme or as a degradation product. The actual specific activity of the cytosolic enzyme was approx. 2 units/mg of acetyl-CoA carboxylase protein while that of the mitochondrial enzyme was about 20-fold lower, indicating that mitochondrial acetyl-CoA carboxylase was relatively inactive. Fractionation of mitochondria with digitonin showed that acetyl-CoA carboxylase was associated with the outer mitochondrial membrane. The available evidence suggests that mitochondrial acetyl-CoA carboxylase represents a reservoir of enzyme which can be released and activated under lipogenic conditions.

scholarly journals Re-evaluation of the interaction of malonyl-CoA with the rat liver mitochondrial carnitine palmitoyltransferase system by using purified outer membranes

1990 ◽  
Vol 267 (1) ◽  
pp. 85-90 ◽  
Author(s):  
M P Kolodziej ◽  
V A Zammit
Keyword(s):  
Rat Liver ◽  
Specific Activity ◽  
Liver Mitochondria ◽  
Subsequent Treatment ◽  
Carnitine Palmitoyltransferase ◽  
Rat Liver Mitochondria ◽  
Outer Membranes ◽  
High Affinity ◽  
Malonyl Coa ◽  
Order Of Magnitude

1. The interaction of malonyl-CoA with the outer carnitine palmitoyltransferase (CPT) system of rat liver mitochondria was re-evaluated by using preparations of highly purified outer membranes, in the light of observations that other subcellular structures that normally contaminate crude mitochondrial preparations also contain malonyl-CoA-sensitive CPT activity. 2. In outer-membrane preparations, which were purified about 200-fold with respect to the inner-membrane-matrix fraction, malonyl-CoA binding was largely accounted for by a single high-affinity component (KD = 0.03 microM), in contrast with the dual site (low- and high-affinity) previously found with intact mitochondria. 3. There was no evidence that the decreased sensitivity of CPT to malonyl-CoA inhibition observed in outer membranes obtained from 48 h-starved rats (compared with those from fed animals) was due to a decreased ratio of malonyl-CoA binding to CPT catalytic moieties. Thus CPT specific activity and maximal high-affinity [14C]malonyl-CoA binding (expressed per mg of protein) were increased 2.2- and 2.0-fold respectively in outer membranes from 48 h-starved rats. 4. Palmitoyl-CoA at a concentration that was saturating for CPT activity (5 microM) decreased the affinity of malonyl-CoA binding by an order of magnitude, but did not alter the maximal binding of [14C]malonyl-CoA. 5. Preincubation of membranes with either tetradecylglycidyl-CoA or 2-bromopalmitoyl-CoA plus carnitine resulted in marked (greater than 80%) inhibition of high-affinity binding, concurrently with greater than 95% inhibition of CPT activity. These treatments also unmasked an effect of subsequent treatment with palmitoyl-CoA to increase low-affinity [14C]malonyl-CoA binding. 6. These data are discussed in relation to the possible mechanism of interaction between the malonyl-CoA-binding site and the active site of the enzyme.

scholarly journals Hepatic ketogenesis in newborn pigs is limited by low mitochondrial 3-hydroxy-3-methylglutaryl-CoA synthase activity

1994 ◽  
Vol 298 (1) ◽  
pp. 207-212 ◽  
Author(s):  
P H Duée ◽  
J P Pégorier ◽  
P A Quant ◽  
C Herbin ◽  
C Kohl ◽  
...  
Keyword(s):  
Rat Liver ◽  
Liver Mitochondria ◽  
Oxidation Products ◽  
Rat Liver Mitochondria ◽  
Acid Oxidation ◽  
Acetyl Coa ◽  
Pig Liver ◽  
Adult Rat ◽  
Malonyl Coa ◽  
Cpt I

In newborn-pig hepatocytes, the rate of oleate oxidation is extremely low, despite a very low malonyl-CoA concentration. By contrast, the sensitivity of carnitine palmitoyltransferase (CPT) I to malonyl-CoA inhibition is high, as suggested by the very low concentration of malonyl-CoA required for 50% inhibition of CPT I (IC50). The rates of oleate oxidation and ketogenesis are respectively 70 and 80% lower in mitochondria isolated from newborn-pig liver than from starved-adult-rat liver mitochondria. Using polarographic measurements, we showed that the oxidation of oleoyl-CoA and palmitoyl-L-carnitine is very low when the acetyl-CoA produced is channelled into the hydroxymethylglutaryl-CoA (HMG-CoA) pathway by addition of malonate. In contrast, the oxidation of the same substrates is high when the acetyl-CoA produced is directed towards the citric acid cycle by addition of malate. We demonstrate that the limitation of ketogenesis in newborn-pig liver is due to a very low amount and activity of mitochondrial HMG-CoA synthase as compared with rat liver mitochondria, and suggest that this could promote the accumulation of acetyl-CoA and/or beta-oxidation products that in turn would decrease the overall rate of fatty acid oxidation in newborn- and adult-pig livers.

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

1993 ◽  
Vol 71 (3-4) ◽  
pp. 183-189 ◽  
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.

scholarly journals The kinetic properties of citrate synthase from rat liver mitochondria

1969 ◽  
Vol 114 (3) ◽  
pp. 597-610 ◽  
Author(s):  
D. Shepherd ◽  
P. B. Garland
Keyword(s):  
Rat Liver ◽  
Citrate Synthase ◽  
Adenine Nucleotide ◽  
Sedimentation Coefficient ◽  
Liver Mitochondria ◽  
Rat Liver Mitochondria ◽  
Kinetic Properties ◽  
Acetyl Coa ◽  
Ph Optimum ◽  
Michaelis Constants

1. Citrate synthase (EC 4.1.3.7) was purified 750-fold from rat liver. 2. Measurements of the Michaelis constants for the substrates of citrate synthase gave values of 16μm for acetyl-CoA and 2μm for oxaloacetate. Each value is independent of the concentration of the other substrate. 3. The inhibition of citrate synthase by ATP, ADP and AMP is competitive with respect to acetyl-CoA. With respect to oxaloacetate the inhibition by AMP is competitive, but the inhibition by ADP and ATP is mixed, being partially competitive. 4. At low concentrations of both substrates the inhibition by ATP is sigmoidal and a Hill plot exhibits a slope of 2·5. 5. The pH optimum of the enzyme is 8·7, and is not significantly affected by ATP. 6. Mg2+ inhibits citrate synthase slightly, but relieves the inhibition caused by ATP in a complex manner. 7. At constant total adenine nucleotide concentration made up of various proportions of ATP, ADP and AMP, the activity of citrate synthase is governed by the concentration of the sum of the energy-rich phosphate bonds of ADP and ATP. 8. The sedimentation coefficient of the enzyme, as measured by activity sedimentation, is 6·3s, equivalent to molecular weight 95000.

scholarly journals Purification and Partial Characterization of Acetyl-CoA Synthetase in Rat Liver Mitochondria.

2002 ◽  
Vol 48 (5) ◽  
pp. 359-364 ◽  
Author(s):  
Hiromi YAMASHITA ◽  
Akemi FUKUURA ◽  
Tomomi NAKAMURA ◽  
Takao KANEYUKI ◽  
Masumi KIMOTO ◽  
...  
Keyword(s):  
Rat Liver ◽  
Liver Mitochondria ◽  
Partial Characterization ◽  
Rat Liver Mitochondria ◽  
Acetyl Coa

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 Carnitine palmitoyltransferase activities (EC 2.3.1.–) of rat liver mitochondria

1970 ◽  
Vol 119 (3) ◽  
pp. 547-552 ◽  
Author(s):  
D. W. Yates ◽  
P. B. Garland
Keyword(s):  
Rat Liver ◽  
Specific Activity ◽  
Liver Mitochondria ◽  
Carnitine Palmitoyltransferase ◽  
Rat Liver Mitochondria ◽  
Type I ◽  
Type Ii ◽  
Inner Mitochondrial Membrane ◽  
Group Transfer ◽  
Carnitine Palmitoyltransferase Activity

1. A continuously recording and sensitive fluorimetric assay is described for carnitine palmitoyltransferase. This assay has been applied to whole or disintegrated mitochondria and to soluble protein fractions. 2. When rat liver mitochondria had been disintegrated by ultrasound, the specific activity of carnitine palmitoyltransferase was 15–20m-units/mg of protein. Only one-fifth of this activity was assayable (with added substrates) before mitochondrial disintegration. 3. It is concluded that there are two carnitine palmitoyltransferase activities in rat liver mitochondria, of which one (type I) is relatively superficial in location and catalyses an acyl-group transfer between added CoA and carnitine, whereas the other (type II) is less superficial and catalyses an acyl-group transfer in unbroken mitochondria between added carnitine and intramitochondrial CoA. The existence of two distinct carnitine palmitoyltransferases was predicted by Fritz & Yue (1963). 4. In unbroken mitochondria, type I transferase is accessible to the inhibitor 2-bromostearoyl-CoA whereas the type II transferase is inaccessible. 5. A major part of the total carnitine palmitoyltransferase activity of rat liver mitochondria is membrane-bound and of type II. 6. These observations, when considered in conjunction with the penetration of mitochondria by CoASH or carnitine, indicate that the type II transferase is attached to the inner mitochondrial membrane.

scholarly journals Heterogeneity of rat liver mitochondrial fractions and the effect of tri-iodothyronine on their protein turnover

1970 ◽  
Vol 118 (1) ◽  
pp. 111-121 ◽  
Author(s):  
S. S. Katyare ◽  
P. Fatterpaker ◽  
A. Sreenivasan
Keyword(s):  
Rat Liver ◽  
Mitochondrial Fraction ◽  
Liver Mitochondria ◽  
Heavy Fraction ◽  
Rat Liver Mitochondria ◽  
Turnover Rates ◽  
Loosely Coupled ◽  
Mitochondrial Fractions ◽  
Isoelectric Ph

1. Rat liver mitochondria were separated into heavy, light and fluffy fractions by differential centrifugation under standard conditions. 2. All mitochondrial fractions possessed soluble as well as membrane-bound enzymes typical of mitochondria. 3. The heavy fraction represented the stable mitochondrial structures and the fluffy particles appear to be loosely coupled. 4. The light mitochondrial fraction lacked the ability of coupled phosphorylation. 5. A study of mobility and isoelectric pH indicated a similarity in the basic membrane structure of all the mitochondrial fractions. 6. The turnover rates of proteins in the heavy and fluffy particles were almost identical; however, this rate was rapid for the light mitochondrial fraction. 7. On treatment with 3,3′,5-tri-iodo-l-thyronine, succinoxidase activity was maximally stimulated much earlier in the light mitochondrial fraction than in the heavy fraction. The activity of the fluffy particles, however, remained almost unaffected. 8. Malate dehydrogenase activity in all the mitochondrial fractions was stimulated only at 40h after tri-iodothyronine treatment. 9. The pattern of incorporation of dl-[1-14C]leucine in vivo in the tri-iodothyronine-treated animals indicated a rapid initial incorporation and high synthetic ability of the light mitochondrial fraction. 10. The turnover pattern of proteins of the mitochondrial fractions from animals receiving repeated doses of tri-iodothyronine was remarkably different from the normal pattern and suggested that preformed soluble protein units may be incorporated in the light mitochondrial fraction during maturation to form the stable heavy mitochondria. 11. The amount of light-mitochondrial proteins decreased by 40% on thyroidectomy and increased by 160% on treatment with tri-iodothyronine. 12. The possible significance of these results is discussed in relation to mitochondrial genesis.

scholarly journals Oxidative metabolism of long-chain fatty acids in mitochondria from sheep and rat liver. Evidence that sheep conserve linoleate by limiting its oxidation

1985 ◽  
Vol 225 (1) ◽  
pp. 233-237 ◽  
Author(s):  
J C W Reid ◽  
D R Husbands
Keyword(s):  
Rat Liver ◽  
Specific Activity ◽  
Liver Mitochondria ◽  
Rat Liver Mitochondria ◽  
Beta Oxidation ◽  
Sheep Liver ◽  
Carnitine Acyltransferase ◽  
Malonyl Coa ◽  
Tightly Coupled ◽  
Important Regulator

Mitochondria isolated from the livers of sheep and rats were shown to oxidize palmitate, oleate and linoleate in a tightly coupled manner, by monitoring the oxygen consumption associated with the degradation of these acids in the presence of 2mM-L-malate. Rat liver mitochondria oxidized linoleate and oleate at a rate 1.2-1.8 times that of palmitate. Sheep liver mitochondria had a specific activity for the oxidation of palmitate that was 50-80% of that of rats and a specific activity for the oxidation of oleate and linoleate that was 30-40% that of rats. This would indicate that sheep conserved linoleate by limiting its oxidation. Carnitine acyltransferase I (CAT I) actively esterified palmitoyl-CoA and linoleate to carnitine in both rat and sheep liver mitochondria, and in both cases the rate for linoleate was faster than for palmitate. The CAT I reaction in both rat and sheep liver was inhibited by micromolar amounts of malonyl-CoA. With 90 microM-palmitoyl-CoA as substrate, CAT I was inhibited by 50% with 2.5 microM-malonyl-CoA in rats, and in sheep, 50% inhibition was found with all malonyl-CoA concentrations tested (1-5 microM). With 90 microM-linoleate as substrate for CAT I, a much larger difference in response to malonyl-CoA was seen, the rat enzyme being 50% inhibited at 22 microM-malonyl-CoA, whereas sheep liver CAT I was 91% and 98% inhibited at 1 microM- and 5 microM-malonyl-CoA respectively. We propose that malonyl-CoA may act as an important regulator of beta-oxidation in sheep, discriminating against the use of linoleate as an energy-yielding substrate.

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