scholarly journals Roles of the N- and C-terminal domains of carnitine palmitoyltransferase I isoforms in malonyl-CoA sensitivity of the enzymes: insights from expression of chimaeric proteins and mutation of conserved histidine residues

1998 ◽  
Vol 335 (3) ◽  
pp. 513-519 ◽  
Author(s):  
S. Todd SWANSON ◽  
Daniel W. FOSTER ◽  
J. Denis McGARRY ◽  
Nicholas F. BROWN
Keyword(s):  
Carnitine Palmitoyltransferase ◽  
Mitochondrial Outer Membrane ◽  
Irreversible Inhibitor ◽  
Muscle Enzymes ◽  
Histidine Residues ◽  
Terminal Domain ◽  
Malonyl Coa ◽  
Carnitine Palmitoyltransferase I ◽  
Cpt I ◽  
Terminal Domains

The mitochondrial outer membrane enzyme carnitine palmitoyltransferase I (CPT I) plays a major role in the regulation of fatty acid entry into the mitochondrial matrix for β-oxidation by virtue of its inhibition by malonyl-CoA. Two isoforms of CPT I, the liver type (L) and muscle type (M), have been identified, the latter being 100 times more sensitive to malonyl-CoA and having a much higher Km for the substrate carnitine. Here we have examined the roles of different regions of the CPT I molecules in their response to malonyl-CoA, etomoxir (an irreversible inhibitor) and carnitine. To this end, we analysed the properties of engineered rat CPT I constructs in which (a) the N-terminal domain of L-CPT I was deleted, (b) the N-terminal domains of L- and M-CPT I were switched, or (c) each of three conserved histidine residues located towards the N-terminus in L-CPT I was mutated. Several novel points emerged: (1) whereas the N-terminal domain is critical for a normal malonyl-CoA response, it does not itself account for the widely disparate sensitivities of the liver and muscle enzymes to the inhibitor; (2) His-5 and/or His-140 probably play a direct role in the malonyl-CoA response, but His-133 does not; (3) the truncated, chimaeric and point- mutant variants of the enzyme all bound the covalent, active-site- directed ligand, etomoxir; and (4) only the most radical alteration of L-CPT I, i.e. deletion of the N-terminal 82 residues, affected the response to carnitine. We conclude that the N-terminal domain of CPT I plays an essential, but permissive, role in the inhibition of the enzyme by malonyl-CoA. By contrast, the larger C-terminal region dictates the degree of sensitivity to malonyl-CoA, as well as the response to carnitine; it is also sufficient for etomoxir binding. Additionally, further weight is added to the notion that one or more histidine residues may be involved in the CPT I–malonyl-CoA interaction.

scholarly journals Topology of carnitine palmitoyltransferase I in the mitochondrial outer membrane

1997 ◽  
Vol 323 (3) ◽  
pp. 711-718 ◽  
Author(s):  
Fiona FRASER ◽  
Clark G. CORSTORPHINE ◽  
Victor A. ZAMMIT
Keyword(s):  
Outer Membrane ◽  
Carnitine Palmitoyltransferase ◽  
Transmembrane Domains ◽  
Proteinase K ◽  
Mitochondrial Outer Membrane ◽  
Malonyl Coa ◽  
Cytosolic Side ◽  
Carnitine Palmitoyltransferase I ◽  
Cpt I ◽  
Peptide Antibodies

The topology of carnitine palmitoyltransferase I (CPT I) in the outer membrane of rat liver mitochondria was studied using several approaches. 1. The accessibility of the active site and malonyl-CoA-binding site of the enzyme from the cytosolic aspect of the membrane was investigated using preparations of octanoyl-CoA and malonyl-CoA immobilized on to agarose beads to render them impermeant through the outer membrane. Both immobilized ligands were fully able to interact effectively with CPT I. 2. The effects of proteinase K and trypsin on the activity and malonyl-CoA sensitivity of CPT I were studied using preparations of mitochondria that were either intact or had their outer membranes ruptured by hypo-osmotic swelling (OMRM). Proteinase K had a marked but similar effect on CPT I activity irrespective of whether only the cytosolic or both sides of the membrane were exposed to it. However, it affected sensitivity more rapidly in OMRM. By contrast, trypsin only reduced CPT I activity when incubated with OMRM. The sensitivity of the residual CPT I activity was unaffected by trypsin. 3. The proteolytic fragments generated by these treatments were studied by Western blotting using three anti-peptide antibodies raised against linear epitopes of CPT I. These showed that a proteinase K-sensitive site close to the N-terminus was accessible from the cytosolic side of the membrane. No trypsin-sensitive sites were accessible in intact mitochondria. In OMRM, both proteinase K and trypsin acted from the inter-membrane space side of the membrane. 4. The ability of intact mitochondria and OMRM to bind to each of the three anti-peptide antibodies was used to study the accessibility of the respective epitopes on the cytosolic and inter-membrane space sides of the membrane. 5. The results of all these approaches indicate that CPT I adopts a bitopic topology within the mitochondrial outer membrane; it has two transmembrane domains, and both the N- and C-termini are exposed on the cytosolic side of the membrane, whereas the linker region between the transmembrane domains protrudes into the intermembrane space.

Cold acclimation increases carnitine palmitoyltransferase I activity in oxidative muscle of striped bass

1994 ◽  
Vol 266 (2) ◽  
pp. R405-R412 ◽  
Author(s):  
K. J. Rodnick ◽  
B. D. Sidell
Keyword(s):  
Cold Acclimation ◽  
Striped Bass ◽  
Fatty Acyl ◽  
Thermal Acclimation ◽  
Carnitine Palmitoyltransferase ◽  
Malonyl Coa ◽  
Red Muscle ◽  
Carnitine Palmitoyltransferase I ◽  
Cpt I ◽  
Long Chain

The effect of thermal acclimation on the activity of carnitine palmitoyltransferase I (CPT I), the rate-limiting enzyme for beta-oxidation of long-chain fatty acids, was determined in oxidative red muscle of striped bass (Morone saxatilis) acclimated at 5 or 25 degrees C. As observed in mammalian tissues, malonyl-CoA potently inhibited CPT I activity of mitochondria. Inhibition by malonyl-CoA required inclusions of both bovine serum albumin (BSA) and palmitoyl-CoA in the reaction media. Because BSA binds long-chain fatty acyl-CoAs, this observation suggests that free fatty acyl-CoAs may disrupt mitochondrial membranes and affect the CPT I protein. Cold acclimation increased citrate synthase activity 1.6-fold and total CPT activity 2-fold in homogenates of red muscle; free carnitine increased 62%, and specific activity of CPT I in mitochondria increased 2-fold. No differences were observed between cold- and warm-acclimated fish in substrate-binding properties of CPT I at an assay temperature of 15 degrees C, as judged by the Michaelis constant (Km) for carnitine (0.11 +/- 0.02 vs. 0.13 +/- 0.02 mM) or inhibition of CPT I, as determined by the half-maximal inhibition concentration (IC50) for malonyl-CoA (0.14 +/- 0.05 vs. 0.09 +/- 0.03 microM). Thermal sensitivity of CPT I (Q10 = 2.91 +/- 0.12 vs. 3.02 +/- 0.20) and preference of CPT I for different long-chain fatty acyl-CoA substrates (16:1-CoA = 16:0-CoA > 18:1-CoA) were not altered by thermal acclimation.(ABSTRACT TRUNCATED AT 250 WORDS)

Carnitine palmitoyltransferase I control of acetogenesis, the major pathway of fatty acid β-oxidation in liver of neonatal swine

2010 ◽  
Vol 298 (5) ◽  
pp. R1435-R1443 ◽  
Author(s):  
Xi Lin ◽  
Kwanseob Shim ◽  
Jack Odle
Keyword(s):  
Fatty Acid ◽  
Ketone Bodies ◽  
Carnitine Palmitoyltransferase ◽  
Acid Oxidation ◽  
Major Pathway ◽  
Malonyl Coa ◽  
Suckling Piglets ◽  
Carnitine Palmitoyltransferase I ◽  
Newborn Pigs ◽  
Cpt I

To examine the regulation of hepatic acetogenesis in neonatal swine, carnitine palmitoyltransferase I (CPT I) activity was measured in the presence of varying palmitoyl-CoA (substrate) and malonyl-CoA (inhibitor) concentrations, and [1-14C]-palmitate oxidation was simultaneously measured. Accumulation rates of 14C-labeled acetate, ketone bodies, and citric acid cycle intermediates within the acid-soluble products were determined using radio-HPLC. Measurements were conducted in mitochondria isolated from newborn, 24-h (fed or fasted), and 5-mo-old pigs. Acetate rather than ketone bodies was the predominant radiolabeled product, and its production increased twofold with increasing fatty acid oxidation during the first 24-h suckling period. The rate of acetogenesis was directly proportional to CPT I activity. The high activity of CPT I in 24-h-suckling piglets was not attributable to an increase in CPT I gene expression, but rather to a large decrease in the sensitivity of CPT I to malonyl-CoA inhibition, which offset a developmental decrease in affinity of CPT I for palmitoyl-CoA. Specifically, the IC50 for malonyl-CoA inhibition and Km value for palmitoyl-CoA measured in 24-h-suckling pigs were 1.8- and 2.7-fold higher than measured in newborn pigs. The addition of anaplerotic carbon from malate (10 mM) significantly reduced 14C accumulation in acetate ( P < 0.003); moreover, the reduction was much greater in newborn (80%) than in 24-h-fed (72%) and 5-mo-old pigs (55%). The results demonstrate that acetate is the primary product of hepatic mitochondrial β-oxidation in Sus scrofa and that regulation during early development is mediated primarily via kinetic modulation of CPT I.

scholarly journals Evidence that the sensitivity of carnitine palmitoyltransferase I to inhibition by malonyl-CoA is an important site of regulation of hepatic fatty acid oxidation in the fetal and newborn rabbit. Perinatal development and effects of pancreatic hormones in cultured rabbit hepatocytes

1990 ◽  
Vol 269 (2) ◽  
pp. 409-415 ◽  
Author(s):  
C Prip-Buus ◽  
J P Pegorier ◽  
P H Duee ◽  
C Kohl ◽  
J Girard
Keyword(s):  
Fatty Acid ◽  
Cyclic Amp ◽  
Fatty Acid Oxidation ◽  
Carnitine Palmitoyltransferase ◽  
Acid Oxidation ◽  
Fetal Hepatocytes ◽  
Malonyl Coa ◽  
Carnitine Palmitoyltransferase I ◽  
Cpt I ◽  
Hepatic Fatty Acid Oxidation

The temporal changes in oleate oxidation, lipogenesis, malonyl-CoA concentration and sensitivity of carnitine palmitoyltransferase I (CPT 1) to malonyl-CoA inhibition were studied in isolated rabbit hepatocytes and mitochondria as a function of time after birth of the animal or time in culture after exposure to glucagon, cyclic AMP or insulin. (1) Oleate oxidation was very low during the first 6 h after birth, whereas lipogenesis rate and malonyl-CoA concentration decreased rapidly during this period to reach levels as low as those found in 24-h-old newborns that show active oleate oxidation. (2) The changes in the activity of CPT I and the IC50 (concn. causing 50% inhibition) for malonyl-CoA paralleled those of oleate oxidation. (3) In cultured fetal hepatocytes, the addition of glucagon or cyclic AMP reproduced the changes that occur spontaneously after birth. A 12 h exposure to glucagon or cyclic AMP was sufficient to inhibit lipogenesis totally and to cause a decrease in malonyl-CoA concentration, but a 24 h exposure was required to induce oleate oxidation. (4) The induction of oleate oxidation by glucagon or cyclic AMP is triggered by the fall in the malonyl-CoA sensitivity of CPT I. (5) In cultured hepatocytes from 24 h-old newborns, the addition of insulin inhibits no more than 30% of the high oleate oxidation, whereas it stimulates lipogenesis and increases malonyl-CoA concentration by 4-fold more than in fetal cells (no oleate oxidation). This poor effect of insulin on oleate oxidation seems to be due to the inability of the hormone to increase the sensitivity of CPT I sufficiently. Altogether, these results suggest that the malonyl-CoA sensitivity of CPT I is the major site of regulation during the induction of fatty acid oxidation in the fetal rabbit liver.

Cytological evidence that the C-terminus of carnitine palmitoyltransferase I is on the cytosolic face of the mitochondrial outer membrane

1999 ◽  
Vol 341 (3) ◽  
pp. 777-784 ◽  
Author(s):  
Feike R. VAN DER LEIJ ◽  
Anita M. KRAM ◽  
Beatrijs BARTELDS ◽  
Han ROELOFSEN ◽  
Gioia B. SMID ◽  
...  
Keyword(s):  
Fusion Protein ◽  
Outer Membrane ◽  
Carnitine Palmitoyltransferase ◽  
Laser Scanning Microscopy ◽  
Mitochondrial Outer Membrane ◽  
Confocal Laser ◽  
Immunogold Electron Microscopy ◽  
C Terminus ◽  
Carnitine Palmitoyltransferase I ◽  
Cpt I

Carnitine palmitoyltransferase I (CPT I) is a key enzyme in the regulation of β-oxidation. The topology of this enzyme has been difficult to elucidate by biochemical methods. We studied the topology of a fusion protein of muscle-type CPT I (M-CPT I) and green fluorescent protein (GFP) by microscopical means. To validate the use of the fusion protein, designated CPT I-GFP, we checked whether the main characteristics of native CPT I were retained. CPT I-GFP was expressed in HeLa cells after stable transfection. Confocal laser scanning microscopy in living cells revealed an extranuclear punctate distribution of CPT I-GFP, which coincided with a mitochondrial fluorescent marker. Immunogold electron microscopy localized CPT I-GFP almost exclusively to the mitochondrial periphery and showed that the C-terminus of CPT I must be on the cytosolic face of the mitochondrial outer membrane. Western analysis showed a protein that was 6 kDa smaller than predicted, which is consistent with previous results for the native M-CPT I. Mitochondria from CPT I-GFP-expressing cells showed an increased CPT activity that was inhibited by malonyl-CoA and was lost on solubilization with Triton X-100. We conclude that CPT I-GFP adopts the same topology as native CPT I and that its C-terminus is located on the cytosolic face of the mitochondrial outer membrane. The evidence supports a recently proposed model for the domain structure of CPT I based on biochemical evidence.

scholarly journals Restoration of the properties of carnitine palmitoyltransferase I in liver mitochondria during re-feeding of starved rats

1986 ◽  
Vol 239 (2) ◽  
pp. 485-488 ◽  
Author(s):  
B D Grantham ◽  
V A Zammit
Keyword(s):  
Liver Mitochondria ◽  
Carnitine Palmitoyltransferase ◽  
Kinetic Properties ◽  
Malonyl Coa ◽  
Carnitine Palmitoyltransferase I ◽  
Cpt I

The recovery of the parameters of the kinetic properties of carnitine palmitoyltransferase (CPT) I in liver mitochondria of starved rats was studied after re-feeding animals for various periods of time. There were no significant changes either in the activity of the enzyme at high palmitoyl-CoA concentrations or in the affinity of the enzyme for palmitoyl-CoA, or in the sensitivity of CPT I to malonyl-CoA inhibition after 3 h or 6 h re-feeding. After 24 h re-feeding, both the affinity of the enzyme for palmitoyl-CoA and the activity of the enzyme were still not significantly different from those for the enzyme in mitochondria from 24 h-starved animals. By contrast, the sensitivity of CPT I to malonyl-CoA inhibition was largely, but not fully, restored to that observed in mitochondria from fed rats.

scholarly journals Sensitivity of inhibition of rat liver mitochondrial outer-membrane carnitine palmitoyltransferase by malonyl-CoA to chemical- and temperature-induced changes in membrane fluidity

1990 ◽  
Vol 272 (2) ◽  
pp. 421-425 ◽  
Author(s):  
M P Kolodziej ◽  
V A Zammit
Keyword(s):  
Outer Membrane ◽  
Membrane Fluidity ◽  
Rat Liver ◽  
Isoamyl Alcohol ◽  
Membrane Lipid ◽  
Carnitine Palmitoyltransferase ◽  
Mitochondrial Outer Membrane ◽  
Rat Liver Mitochondria ◽  
Malonyl Coa ◽  
Cpt I

We have tested the possibility that alterations in the fluidity of the outer membrane of rat liver mitochondria could result in changes in the sensitivity of overt carnitine palmitoyltransferase (CPT I) to malonyl-CoA [Zammit (1986) Biochem. Soc. Trans. 14. 676-679]. The sensitivity of CPT I to malonyl-CoA inhibition was measured by using highly purified mitochondrial outer membranes prepared from fed or 48 h-starved rats in the presence and absence of agents that increase membrane fluidity by perturbing membrane lipid order [benzyl alcohol, isoamyl alcohol (3-methylbutan-l-ol) and 2-(2-methoxyethoxy)ethyl-8-(cis-2-n-octylpropyl)octanoate (A2C)]. All these agents resulted in marked decreases in the ability of malonyl-CoA to inhibit CPT I. This effect was accompanied by a modest increase in the absolute activity of CPT I in the absence of malonyl-CoA when the short-chain alcohols were used, but not when A2C was used, suggesting that the effect of increased membrane fluidity to decrease the malonyl-CoA sensitivity of CPT I may occur independently from other actions that may affect more directly the active site of the enzyme. In confirmation of the potential importance of fluidity changes, we showed that a marked increase in sensitivity of CPT I to malonyl-CoA could be produced when assays were performed at lower temperatures than those normally employed. These observations are discussed in the context of the slowness of the changes in CPT I sensitivity to malonyl-CoA inhibition that are induced by physiological perturbations.

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.

scholarly journals Cholate extracts of mitochondrial outer membranes increase inhibition by malonyl-CoA of carnitine palmitoyltransferase-I by a mechanism involving phospholipids

1994 ◽  
Vol 299 (3) ◽  
pp. 761-767 ◽  
Author(s):  
R L Mynatt ◽  
J J Greenhaw ◽  
G A Cook
Keyword(s):  
Regulatory Protein ◽  
Sodium Cholate ◽  
Carnitine Palmitoyltransferase ◽  
Proteinase K ◽  
Membrane Composition ◽  
Outer Membranes ◽  
Malonyl Coa ◽  
Increased Sensitivity ◽  
Carnitine Palmitoyltransferase I ◽  
Cpt I

It has been reported that sodium cholate can separate the catalytic component of carnitine palmitoyltransferase-I (CPT-I) from a putative malonyl-CoA-binding regulatory protein capable of conferring sensitivity to malonyl-CoA on CPT-II. We found that cholate preferentially extracted a contaminating malonyl-CoA-sensitive CPT from mitochondrial inner membranes. When cholate extracts of outer membranes were incubated either with cholate extracts of inner membranes or with osmotically swollen mitochondria, inhibition of CPT by malonyl-CoA was increased. Treatment of intact mitochondria with subtilisin abolished the increased inhibition by malonyl-CoA, suggesting that the outer-membrane CPT-I was responsible for the increased inhibition. Incubation of cholate extracts with proteinase K did not prevent the increased inhibition. Fractionation of the cholate extract indicated the presence of phospholipids. Addition of cardiolipin or phosphatidylglycerol to osmotically swollen mitochondria increased sensitivity of CPT to malonyl-CoA, but several other phospholipids did not. When cardiolipin was added to intact mitochondria from either starved or fed rats, there were large increases in inhibition by malonyl-CoA; sensitivity in mitochondria from starved rats increased to that normally observed with mitochondria from fed rats. These results suggest that phospholipids are responsible for the increased inhibition of CPT by malonyl-CoA with added cholate extracts and that changes in membrane composition may be involved in the physiological regulation of CPT-I.

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