scholarly journals Evidence for distinct functional molecular sizes of carnitine palmitoyltransferases I and II in rat liver mitochondria

1988 ◽  
Vol 250 (2) ◽  
pp. 415-420 ◽  
Author(s):  
V A Zammit ◽  
C G Corstorphine ◽  
M G Kelliher
Keyword(s):  
Rat Liver ◽  
Residual Activity ◽  
Liver Mitochondria ◽  
High Energy ◽  
Size Analysis ◽  
Rat Liver Mitochondria ◽  
Molecular Sizes ◽  
Cpt I ◽  
Carnitine Palmitoyltransferases ◽  
Logarithm Function

1. Estimates of the functional sizes of the molecular species responsible for the overt (I) and latent (II) activities of carnitine palmitoyltransferase (CPT) in 48 h-starved rat liver mitochondria were obtained from radiation inactivation experiments. 2. The decay in the activity of total CPT and that of CPT II only (after inhibition of CPT I) was measured in mitochondrial samples exposed to different doses of high-energy ionizing radiation. 3. The decay curves obtained by plotting residual activity of total CPT as a logarithm function of irradiation dose suggested the contribution of more than one target towards total CPT activity. 4. By contrast, in mitochondria in which CPT I activity was approximately 95% inhibited, the activity of CPT decayed in a simple mono-exponential manner. Target-size analysis yielded an approximate Mr of 69,700 for this component (CPT II). 5. This information, as well as that on the relative non-irradiated activities of CPT I and CPT II, was used in graphical and statistical methods to obtain the parameters of the decay curve for CPT I. These analyses yielded an approximate Mr of 96,700 for CPT I.

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.

scholarly journals Enrichment of carnitine palmitoyltransferases I and II in the contact sites of rat liver mitochondria

1998 ◽  
Vol 329 (2) ◽  
pp. 225-229 ◽  
Author(s):  
Fiona FRASER ◽  
A. Victor ZAMMIT
Keyword(s):  
Rat Liver ◽  
Liver Mitochondria ◽  
Rat Liver Mitochondria ◽  
Energy Status ◽  
Contact Sites ◽  
Carnitine Acyltransferases ◽  
Site Fraction ◽  
Lipid Environment ◽  
Cpt I ◽  
Carnitine Palmitoyltransferases

The submitochondrial distribution of the overt and latent carnitine palmitoyltransferases (CPT I and II respectively) of rat liver mitochondria were studied. Separation of outer and inner membranes, as well as of a fraction of intermediate density consisting of contact sites between the two membranes, was achieved, as judged by the distribution of marker enzymes. Both CPT I and CPT II were found to be enriched within the contact- site fraction of mitochondria. These data show that the two carnitine acyltransferases are distributed non-uniformly within their respective membranes, and that subpopulations of the two enzymes occur in close proximity within the mitochondrial membrane structure, while retaining their different accessibilities to cytosolic and matrix pools of metabolites. As the number of contact sites is known to vary with changes in the energy status of mitochondria, the possibility that such changes may acutely affect the proportion of CPT I within the distinctive lipid environment of the contact sites, and thus its overall kinetic characteristics, is discussed.

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 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 Modification by calcium ions of adenine nucleotide translocation in rat liver mitochondria

1972 ◽  
Vol 129 (2) ◽  
pp. 355-365 ◽  
Author(s):  
T. Spencer ◽  
F. L. Bygrave
Keyword(s):  
Rat Liver ◽  
Calcium Ions ◽  
Adenine Nucleotide ◽  
Cell Metabolism ◽  
Liver Mitochondria ◽  
High Energy ◽  
Small Degree ◽  
Metabolic Inhibitors ◽  
Rat Liver Mitochondria ◽  
Stimulation Of

1. Added Ca2+stimulates the translocation of ATP by isolated rat liver mitochondria. 2. The apparent Km for added Ca2+in stimulating the translocation of 200μm-ATP is approx. 160μm (75μm ‘free’ Ca2+). 3. The greatest stimulation of ATP translocation by Ca2+occurs at the lower concentrations of ATP. 4. Sr2+(and to a lesser extent Ba2+) can replace Ca2+whereas Mg2+and Mn2+have only little ability to stimulate ATP translocation. 5. Translocation of dATP is also stimulated by Ca2+whereas that of ADP is stimulated to only a relatively small degree. 6. Studies with metabolic inhibitors and uncouplers provide evidence that stimulation by Ca2+and by uncouplers is additive and that the mechanism of Ca2+stimulation does not seem to involve the high-energy intermediate of oxidative phosphorylation. 7. In the presence of Ca2+, ATP is able to effectively compete with ADP for translocation. 8. Added K+further enhances the ability of Ca2+to stimulate ATP translocation. 9. These findings are discussed in relation to the potential involvement of Ca2+in modifying enzymic reactions involved in the regulation of cell metabolism.

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