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.

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 Sensitivity of carnitine acyltransferase I to malonyl-CoA inhibition in isolated rat liver mitochondria is quantitatively related to hepatic malonyl-CoA concentration in vivo

1982 ◽  
Vol 206 (1) ◽  
pp. 177-179 ◽  
Author(s):  
Ian N. Robinson ◽  
Victor A. Zammit
Keyword(s):  
Rat Liver ◽  
Acid Synthesis ◽  
Liver Mitochondria ◽  
Rat Liver Mitochondria ◽  
Reciprocal Regulation ◽  
Carnitine Acyltransferase ◽  
Malonyl Coa ◽  
Amplification Mechanism ◽  
Two Parameters

The sensitivity of carnitine acyltransferase I (EC 2.3.1.21) activity to malonyl-CoA inhibition in rat liver mitochondria isolated from animals in various physiological states was quantitatively proportional to the hepatic malonyl-CoA concentration in vivo. It is suggested that this relationship between the two parameters could result in a potent amplification mechanism for the reciprocal regulation of fatty acid synthesis and oxidation.

scholarly journals 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

1984 ◽  
Vol 218 (2) ◽  
pp. 379-386 ◽  
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)

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.

scholarly journals Importance of experimental conditions in evaluating the malonyl-CoA sensitivity of liver carnitine acyltransferase. Studies with fed and starved rats

1981 ◽  
Vol 200 (2) ◽  
pp. 217-223 ◽  
Author(s):  
J D McGarry ◽  
D W Foster
Keyword(s):  
Fatty Acid ◽  
Fatty Acid Oxidation ◽  
Rat Liver ◽  
Competitive Inhibition ◽  
Physiological Role ◽  
Rat Liver Mitochondria ◽  
Acid Oxidation ◽  
Carnitine Acyltransferase ◽  
Malonyl Coa

The experiments reconfirm the powerful inhibitory effect of malonyl-CoA on carnitine acyltransferase I and fatty acid oxidation in rat liver mitochondria (Ki 1.5 microM). Sensitivity decreased with starvation (Ki after 18 h starvation 3.0 microM, and after 42 h 5.0 microM). Observations by Cook, Otto & Cornell [Biochem. J. (1980) 192, 955--958] and Ontko & Johns [Biochem. J. (1980) 192, 959--962] have cast doubt on the physiological role of malonyl-CoA in the regulation of hepatic fatty acid oxidation and ketogenesis. The high Ki values obtained in the cited studies are shown to be due to incubation conditions that cause substrate depletion, destruction of malonyl-CoA or generation of excessively high concentrations of unbound acyl-CoA (which offsets the competitive inhibition of malonyl-CoA towards carnitine acyltransferase I). The present results are entirely consistent with the postulated role of malonyl-CoA as the primary regulatory of fatty acid synthesis and oxidation in rat liver.

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 Effects of incubation at physiological temperatures on the concentration-dependence of [2-14C]malonyl-CoA binding to rat liver mitochondria

1985 ◽  
Vol 231 (2) ◽  
pp. 343-347 ◽  
Author(s):  
V A Zammit ◽  
C G Corstorphine
Keyword(s):  
Rat Liver ◽  
Specific Binding ◽  
Liver Mitochondria ◽  
Rat Liver Mitochondria ◽  
Temperature Dependent ◽  
Malonyl Coa ◽  
Saturation Kinetics ◽  
Different Temperatures ◽  
Cpt I

Specific binding of [2-14C] malonyl-CoA to rat liver mitochondria was measured at different temperatures and after various periods of time of exposure of the mitochondria to the ligand. Incubation of mitochondria at 37 degrees C in the absence of malonyl-CoA resulted in a decrease in their ability to bind malonyl-CoA at all concentrations tested (up to 55 microM). However, incubation of mitochondria in the presence of malonyl-CoA resulted in the loss of the binding only by a low-affinity component. By contrast, there was an increase in the binding that occurred at low, physiological, concentrations of malonyl-CoA. These differences in the response of the two binding components to incubation conditions were used to obtain quantitative data about their respective saturation kinetics. Evidence was obtained that, whereas the high-affinity component approached saturation hyperbolically with respect to malonyl-CoA concentration, the low-affinity component had sigmoidal characteristics. The concentrations of malonyl-CoA required to half-saturate the two components were 2-3 microM and 30 microM for the high- and low-affinity components respectively. Evidence was also obtained for the involvement of a temperature-dependent transition, that occurred at around 25 degrees C, in the modulation of malonyl-CoA binding to the mitochondria. The possible physiological roles of the two components of malonyl-CoA binding in relation to the regulation of overt carnitine palmitoyltransferase (CPT I) activity in vivo are discussed.

scholarly journals Target size analysis by radiation inactivation of carnitine palmitoyltransferase activity and malonyl-CoA binding in outer membranes from rat liver mitochondria

1989 ◽  
Vol 263 (1) ◽  
pp. 89-95 ◽  
Author(s):  
V A Zammit ◽  
C G Corstorphine ◽  
M P Kolodziej
Keyword(s):  
Rat Liver ◽  
Molecular Size ◽  
Protein S ◽  
Liver Mitochondria ◽  
Carnitine Palmitoyltransferase ◽  
Size Analysis ◽  
Rat Liver Mitochondria ◽  
Radiation Inactivation ◽  
Malonyl Coa ◽  
Cpt I

The functional molecular sizes of the protein(s) mediating the carnitine palmitoyltransferase I (CPT I) activity and the [14C]malonyl-CoA binding in purified outer-membrane preparations from rat liver mitochondria were determined by radiation-inactivation analysis. In all preparations tested the dose-dependent decay in [14C]malonyl-CoA binding was less steep than that for CPT I activity, suggesting that the protein involved in malonyl-CoA binding may be smaller than that catalysing the CPT I activity. The respective sizes computed from simultaneous analysis for molecular-size standards exposed under identical conditions were 60,000 and 83,000 DA for malonyl-CoA binding and CPT I activity respectively. In irradiated membranes the sensitivity of CPT activity to malonyl-CoA inhibition was increased, as judged by malonyl-CoA inhibition curves for the activity in control and in irradiated membranes that had received 20 Mrad radiation and in which CPT activity had decayed by 60%. Possible correlations between these data and other recent observations on the CPT system are discussed.

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