scholarly journals Changes in the ability of malonyl-CoA to inhibit carnitine palmitoyltransferase I activity and to bind to rat liver mitochondria during incubation in vitro. Differences in binding at 0°C and 37°C with a fixed concentration of malonyl-CoA

1984 ◽  
Vol 222 (2) ◽  
pp. 335-342 ◽  
Author(s):  
V A Zammit ◽  
C G Corstorphine ◽  
S R Gray
Keyword(s):  
Liver Mitochondria ◽  
Carnitine Palmitoyltransferase ◽  
Time Dependent ◽  
Rat Liver Mitochondria ◽  
Temperature Dependent ◽  
Fixed Concentration ◽  
Malonyl Coa ◽  
Time Courses ◽  
Cpt I

Time courses for inhibition of carnitine palmitoyltransferase (CPT) I activity in, and [14C]malonyl-CoA binding to, liver mitochondria from fed or 48 h-starved rats were obtained at 37 degrees C by using identical incubation conditions and a fixed concentration of malonyl-CoA (3.5 microM), which represents the middle of the physiological range observed previously [Zammit (1981) Biochem. J. 198, 75-83] Incubation of mitochondria in the absence of malonyl-CoA resulted in a time-dependent decrease in the ability of the metabolite instantaneously to inhibit CPT I and to bind to the mitochondria. Both degree of inhibition and binding were restored in parallel over a period of 6-8 min on subsequent addition of malonyl-CoA to the incubation medium. However, the increased inhibition of CPT I activity on addition of mitochondria directly to malonyl-CoA-containing medium was not accompanied by an increase in the amount of [14C]malonyl-CoA bound to mitochondria at 37 degrees C. Time courses for binding of [14C]malonyl-CoA performed at 0 degree C were different from those obtained at 37 degrees C. There was little loss of ability of [14C]malonyl-CoA to bind to mitochondria on incubation in the absence of the metabolite, but there was a time-dependent increase in binding on addition of mitochondria to malonyl-CoA-containing medium. It is suggested that these temperature-dependent differences between the time courses obtained may be due to the occurrence of different changes at 37 degrees C and at 0 degree C in the relative contributions of different components (with different affinities) to the binding observed at 3.5 microM-malonyl-CoA. Evidence for multi-component binding was obtained in the form of strongly curvilinear Scatchard plots for instantaneous (5s) binding of malonyl-CoA to mitochondria. Such multi-component binding would be expected from previous results on the different affinities of CPT I for malonyl-CoA with respect to inhibition [Zammit (1984) Biochem. J. 218, 379-386]. Mitochondria obtained from starved rats showed qualitatively the same time courses as those described above, with notable quantitative differences with respect both to the absolute extents of CPT I inhibition and [14C]malonyl-CoA binding achieved as well as to the time taken to attain them.

scholarly journals Role of carnitine palmitoyltransferase I in the regulation of hepatic ketogenesis during the onset and reversal of chronic diabetes

1988 ◽  
Vol 249 (2) ◽  
pp. 409-414 ◽  
Author(s):  
B D Grantham ◽  
V A Zammit
Keyword(s):  
Insulin Treatment ◽  
Regular Insulin ◽  
Liver Mitochondria ◽  
Diabetic Rats ◽  
Carnitine Palmitoyltransferase ◽  
Rat Liver Mitochondria ◽  
Kinetic Properties ◽  
Malonyl Coa ◽  
Time Courses ◽  
Cpt I

1. The kinetic properties of overt carnitine palmitoyltransferase (CPT I, EC 2.3.1.21) were studied in rat liver mitochondria isolated from untreated, diabetic and insulin-treated diabetic animals. A comparison was made of the time courses required for the changes in these properties of CPT I to occur and for the development of ketosis during the induction of chronic diabetes and its reversal by insulin treatment. 2. The development of hyperketonaemia over the first 5 days of insulin withdrawal from streptozotocin-treated rats was accompanied by parallel increases in the activity of CPT I and in the I0.5 (concentration required to produce 50% inhibition) of the enzyme for malonyl-CoA. 3. The rapid reversal of the ketotic state by treatment of chronically diabetic rats with 6 units of regular insulin was not accompanied by any change in the properties of CPT I over the first 4 h. Higher doses of insulin (15 units), delivered throughout a 4 h period, resulted in an increase in the affinity of CPT I for malonyl-CoA, but the sensitivity of the enzyme to the inhibitor was still significantly lower than in mitochondria from normal animals. 4. Conversely, when insulin treatment was continued over a 24 h period, full restoration of the sensitivity of the enzyme to malonyl-CoA was achieved. However, the activity of the enzyme was only decreased marginally. 5. These results are discussed in terms of the possibility that the major regulatory sites of the rate of hepatic oxidation may vary in different phases of the induction and reversal of chronic diabetes.

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 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.

scholarly journals Reversible sensitization and desensitization of carnitine palmitoyltransferase I to inhibition by malonyl-CoA in isolated rat liver mitochondria. Significance for the mechanism of malonyl-CoA-induced sensitization

1983 ◽  
Vol 214 (3) ◽  
pp. 1027-1030 ◽  
Author(s):  
V A Zammit
Keyword(s):  
Rat Liver ◽  
Reduced Glutathione ◽  
Thiol Group ◽  
Liver Mitochondria ◽  
Carnitine Palmitoyltransferase ◽  
Rat Liver Mitochondria ◽  
Nitrobenzoic Acid ◽  
Malonyl Coa ◽  
Carnitine Palmitoyltransferase I ◽  
Cpt I

Preincubation of rat liver mitochondria with 5,5′-dithiobis-(2-nitrobenzoic acid) (Nbs2) followed by removal of excess reagent by washing the mitochondria with 0.5 mM-reduced glutathione resulted in a desensitization of carnitine palmitoyltransferase (CPT) I activity to malonyl-CoA inhibition. The effect was not observed if mitochondria were washed with 0.5 mM-dithiothreitol. The desensitization effect of Nbs2 could be reversed by a second incubation in the presence of 8 microM-malonyl-CoA. In addition, malonyl-CoA, when present simultaneously with Nbs2, protected CPT I activity against the desensitization effect of the thiol-group reagent. These results suggest that malonyl-CoA exerts an effect on one or more thiol groups of the enzyme, and that this effect is related to the ability of the metabolite to sensitize CPT I to malonyl-CoA inhibition.

scholarly journals Effects of the mode of addition of acyl-CoA on the initial rate of formation of acylcarnitine in the presence of carnitine by intact rat liver mitochondria in vitro

1985 ◽  
Vol 229 (1) ◽  
pp. 273-275 ◽  
Author(s):  
V A Zammit
Keyword(s):  
Rat Liver ◽  
Initial Rate ◽  
Liver Mitochondria ◽  
Final Concentration ◽  
Rat Liver Mitochondria ◽  
Molar Ratios ◽  
Time Courses ◽  
Concentrated Solution ◽  
Cpt I

Time courses for the formation of palmitoylcarnitine from palmitoyl-CoA and carnitine, catalysed by the overt activity of carnitine palmitoyltransferase (CPT I) in rat liver mitochondria, were obtained. Significant initial non-linearity was observed only when reactions were started by addition of a concentrated solution of palmitoyl-CoA (4mM, to give a final concentration of 100 microM) uncomplexed to albumin. Minimal effects were observed when the reactions were started by addition of palmitoyl-CoA-albumin mixtures, even though the final palmitoyl-CoA/albumin molar ratios in the assay medium were identical in the two sets of experiments.

scholarly journals Binding of [14C]malonyl-CoA to rat liver mitochondria after blocking of the active site of carnitine palmitoyltransferase I. Displacement of low-affinity binding by palmitoyl-CoA

1986 ◽  
Vol 233 (2) ◽  
pp. 589-593 ◽  
Author(s):  
B D Grantham ◽  
V A Zammit
Keyword(s):  
Binding Site ◽  
Rat Liver ◽  
Active Site ◽  
Liver Mitochondria ◽  
Carnitine Palmitoyltransferase ◽  
Rat Liver Mitochondria ◽  
High Affinity ◽  
Malonyl Coa ◽  
High Affinity Site ◽  
Cpt I

The active site of the overt activity of carnitine palmitoyltransferase (CPT I) in rat liver mitochondria was blocked by the self-catalysed formation of the S-carboxypalmitoyl-CoA ester of (-)-carnitine, followed by washing of the mitochondria. CPT I activity in treated mitochondria was inhibited by 90-95%. Binding of [14C]malonyl-CoA to these mitochondria was not inhibited as compared with that of control mitochondria. When CPT I activity was inhibited, palmitoyl-CoA could markedly displace [14C]malonyl-CoA binding from the low-affinity site for the inhibitor [Zammit, Corstorphine & Gray (1984) Biochem. J. 222, 335-342], but not from the high-affinity site for malonyl-CoA binding. The saturation characteristics of the malonyl-CoA-binding component lost in the presence of palmitoyl-CoA were sigmoidal, and thus suggestive of co-operative binding at this site. It is suggested that the site hitherto considered to be a low-affinity malonyl-CoA-binding site may be effectively a second, allosteric, acyl-CoA-binding site on CPT I under conditions that prevail in vivo, whereas the high-affinity site for malonyl-CoA may be exclusive to the inhibitor. The possibility that the competitive-type interactions of malonyl-CoA and acyl-CoA on CPT I activity could arise from the effects of separate malonyl-CoA and acyl-CoA allosteric sites is considered. The possible significance of the large difference in the capacity of the two sites and their different saturation kinetics is also discussed.

scholarly journals Increased sensitivity of carnitine palmitoyltransferase I activity to malonyl-CoA inhibition after preincubation of intact rat liver mitochondria with micromolar concentrations of malonyl-CoA in vitro

1983 ◽  
Vol 210 (3) ◽  
pp. 953-956 ◽  
Author(s):  
V A Zammit
Keyword(s):  
Rat Liver ◽  
Liver Mitochondria ◽  
Carnitine Palmitoyltransferase ◽  
Rat Liver Mitochondria ◽  
Maximal Degree ◽  
Malonyl Coa ◽  
Degree Of Sensitization ◽  
Increased Sensitivity ◽  
Carnitine Palmitoyltransferase I

Carnitine palmitoyltransferase I in rat liver mitochondria preincubated with malonyl-CoA was more sensitive to inhibition by malonyl-CoA than was the enzyme in mitochondria preincubated in the absence of malonyl-CoA. For carnitine palmitoyltransferase I in mitochondria from starved animals this increase also resulted in the enzyme becoming significantly more sensitive than that in mitochondria assayed immediately after their isolation. Concentrations of malonyl-CoA that induced half the maximal degree of sensitization observed were 1-3 microM.

Reconstitution of purified, active and malonyl-CoA-sensitive rat liver carnitine palmitoyltransferase I: relationship between membrane environment and malonyl-CoA sensitivity

2000 ◽  
Vol 349 (1) ◽  
pp. 179-187 ◽  
Author(s):  
J. Denis MCGARRY ◽  
Nicholas F. BROWN
Keyword(s):  
Rat Liver ◽  
Liver Mitochondria ◽  
Carnitine Palmitoyltransferase ◽  
Rat Liver Mitochondria ◽  
Kinetic Properties ◽  
Suitable Model ◽  
Malonyl Coa ◽  
Carnitine Palmitoyltransferase I ◽  
Cpt I

Carnitine palmitoyltransferase I (CPT I) catalyses the initial step of fatty acid import into the mitochondrial matrix, the site of β-oxidation, and its inhibition by malonyl-CoA is a primary control point for this process. The enzyme exists in at least two isoforms, denoted L-CPT I (liver type) and M-CPT I (skeletal-muscle type), which differ in their kinetic characteristics and tissue distributions. A property apparently unique to L-CPT I is that its sensitivity to malonyl-CoA decreases in vivo with fasting or experimentally induced diabetes. The mechanism of this important regulatory effect is unknown and has aroused much interest. CPT I is an integral outer-membrane protein and displays little activity after removal from the membrane by detergents, precluding direct purification of active protein by conventional means. Here we describe the expression of a 6×His-tagged rat L-CPT I in Pichia pastoris and purification of the detergent-solubilized enzyme in milligram quantities. Reconstitution of the purified product into a liposomal environment yielded a 200-400-fold increase in enzymic activity and restored malonyl-CoA sensitivity. This is the first time that a CPT I protein has been available for study in a form that is both pure and active. Comparison of the kinetic properties of the reconstituted material with those of L-CPT I as it exists in mitochondria prepared from yeast over-expressing the enzyme and in livers from fed or fasted rats permitted novel insight into several aspects of the enzyme's behaviour. The malonyl-CoA response of the liposomal enzyme was found to be greater when the reconstitution procedure was carried out at 22 °C compared with 4 °C (IC50 ≈ 11 μM versus 30 μM, respectively). When the sensitivities of L-CPT I in each of the different environments were compared, they were found to decrease in the following order: fed liver > fasted liver≈ liposomes prepared at 22 °C≈ P. pastoris mitochondria > liposomes prepared at 4 °C. In addition, pre-treatment of L-CPT I liposomes with the membrane-fluidizing reagent benzyl alcohol caused densensitization to the inhibitor. In contrast with the variable response to malonyl-CoA, the liposomal L-CPT I displayed a pH profile and kinetics with regard to the carnitine and acyl-CoA substrates similar to those of the enzyme in fed or fasted liver mitochondria. However, despite a normal sensitivity to malonyl-CoA, L-CPT I in P. pastoris mitochondria displayed aberrant behaviour with regard to each of these other parameters. The kinetic data establish several novel points. First, even after stringent purification procedures in the presence of detergent, recombinant L-CPT I could be reconstituted in active, malonyl-CoA sensitive form. Second, the kinetics of the reconstituted, 6×His-tagged L-CPT I with regard to substrate and pH responses were similar to what is observed with rat liver mitochondria (whereas in P. pastoris mitochondria the enzyme behaved anomalously), confirming that the purified preparation is a suitable model for studying the functional properties of the enzyme. Third, wide variation in the response to the inhibitor, malonyl-CoA, was observed depending only on the enzyme's membrane environment and independent of interaction with other proteins. In particular, the fluidity of the membrane had a direct influence on this parameter. These observations may help to explain the mechanism of the physiological changes in the properties of L-CPT I that occur in vivo and are consistent with the current topographical model of the enzyme.

Magnetic nanostructured lipid carrier for dual triggered curcumin delivery: Preparation, characterization and toxicity evaluation on isolated rat liver mitochondria

2021 ◽  
pp. 088532822110346
Author(s):  
Mohammad Yoozbashi ◽  
Hamid Rashidzadeh ◽  
Mehraneh Kermanian ◽  
Somayeh Sadighian ◽  
Mir-Jamal Hosseini ◽  
...  
Keyword(s):  
In Vitro Study ◽  
Liver Mitochondria ◽  
Rat Liver Mitochondria ◽  
Nanostructured Lipid Carrier ◽  
Mitochondrial Toxicity ◽  
X Ray Diffraction ◽  
Temperature Dependent ◽  
Reducing Ability ◽  
Transmission Electron

In this research, magnetic nanostructured lipid carriers (Mag-NLCs) were synthesized for curcumin (CUR) delivery. NLCs are drug-delivery systems prepared by mixing solid and liquid (oil) lipids. For preparation of NLCs, cetylpalmitate was selected as solid lipid and fish oil as liquid lipid. CUR-Mag-NLCs were prepared using high-pressure homogenization technique and were characterized by methods including X-ray diffraction (XRD), transmission electron microscopy (TEM), vibrating sample magnetometer (VSM), and dynamic light scattering (DLS). The CUR-Mag-NLCs were developed as a particle with a size of 140 ± 3.6 nm, a polydispersity index of 0.196, and a zeta potential of −22.6 mV. VSM analysis showed that the CUR-Mag-NLCs have excellent magnetic properties. Release rate of the drug was higher at 42 °C than 37 °C, indicating that release of the synthesized nanoparticles is temperature-dependent. Evaluation of mitochondrial toxicity was done using the isolated rats liver mitochondria including glutathione (GSH), malondialdehyde (MDA), and the ferric- reducing ability of plasma (FRAP) assays to study biosafety of the CUR-Mag-NLCs. Results of In vitro study on the isolated mitochondria revealed that both CUR-Mag-NLCs and curcumin have no specific mitochondrial toxicity.

Sign in / Sign up

Export Citation Format

Share Document