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.

scholarly journals Studies on the activation in vitro of carnitine palmitoyltransferase I in liver mitochondria from normal, diabetic and glucagon-treated rats

1987 ◽  
Vol 243 (1) ◽  
pp. 261-265 ◽  
Author(s):  
B D Grantham ◽  
V A Zammit
Keyword(s):  
Kinetic Parameters ◽  
Rat Liver ◽  
Liver Mitochondria ◽  
Carnitine Palmitoyltransferase ◽  
Rat Liver Mitochondria ◽  
Kinetic Characteristics ◽  
Carnitine Palmitoyltransferase I ◽  
Carnitine Palmitoyltransferase Activity ◽  
Catalytic Capacity

The activation of overt carnitine palmitoyltransferase activity that occurs when rat liver mitochondria are incubated at near-physiological temperatures and ionic strengths was studied for mitochondria obtained from animals in different physiological states. In all instances, it was found to be due exclusively to an increase in the catalytic capacity of the enzyme and not to an increase in affinity of the enzyme for palmitoyl-CoA. The enzyme in mitochondria from fed animals always showed a larger degree of activation than that in mitochondria from starved animals. This was the case even for mitochondria (e.g. from fed diabetic animals) in which the kinetic characteristics of carnitine palmitoyltransferase were more similar to those for the enzyme in mitochondria from starved rats. Glucagon treatment of rats before isolation of the mitochondria did not affect the characteristics either of the kinetic parameters of overt carnitine palmitoyltransferase or of its activation in vitro.

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.

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.

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 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 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 Demonstration of N- and C-terminal domain intramolecular interactions in rat liver carnitine palmitoyltransferase 1 that determine its degree of malonyl-CoA sensitivity

2005 ◽  
Vol 387 (1) ◽  
pp. 67-76 ◽  
Author(s):  
Audrey FAYE ◽  
Karen BORTHWICK ◽  
Catherine ESNOUS ◽  
Nigel T. PRICE ◽  
Stéphanie GOBIN ◽  
...  
Keyword(s):  
Rat Liver ◽  
Liver Mitochondria ◽  
Carnitine Palmitoyltransferase ◽  
Intramolecular Interactions ◽  
Rat Liver Mitochondria ◽  
Cross Linking ◽  
Terminal Domain ◽  
Malonyl Coa ◽  
Carnitine Palmitoyltransferase 1 ◽  
Terminal Domains

We have previously proposed that changes in malonyl-CoA sensitivity of rat L-CPT1 (liver carnitine palmitoyltransferase 1) might occur through modulation of interactions between its cytosolic N- and C-terminal domains. By using a cross-linking strategy based on the trypsin-resistant folded state of L-CPT1, we have now shown the existence of such N–C (N- and C-terminal domain) intramolecular interactions both in wild-type L-CPT1 expressed in Saccharomyces cerevisiae and in the native L-CPT1 in fed rat liver mitochondria. These N–C intramolecular interactions were found to be either totally (48-h starvation) or partially abolished (streptozotocin-induced diabetes) in mitochondria isolated from animals in which the enzyme displays decreased malonyl-CoA sensitivity. Moreover, increasing the outer membrane fluidity of fed rat liver mitochondria with benzyl alcohol in vitro, which induced malonyl-CoA desensitization, attenuated the N–C interactions. This indicates that the changes in malonyl-CoA sens-itivity of L-CPT1 observed in mitochondria from starved and diabetic rats, previously shown to be associated with altered membrane composition in vivo, are partly due to the disruption of N–C interactions. Finally, we show that mutations in the regulatory regions of the N-terminal domain affect the ability of the N terminus to interact physically with the C-terminal domain, irrespective of whether they increased [S24A (Ser24→Ala)/Q30A] or abrogated (E3A) malonyl-CoA sensitivity. Moreover, we have identified the region immediately N-terminal to transmembrane domain 1 (residues 40–47) as being involved in the chemical N–C cross-linking. These observations provide the first demonstration by a physico-chemical method that L-CPT1 adopts different conformational states that differ in their degree of proximity between the cytosolic N-terminal and the C-terminal domains, and that this determines its degree of malonyl-CoA sensitivity depending on the physiological state.

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