Inhibition of carnitine palmitoyltransferase of the hepatic mitochondrial outer membrane (CPT-I) by 2-bromopalmitoyl-CoA

1993 ◽  
Vol 21 (4) ◽  
pp. 503S-503S
Author(s):  
KHOSROW KASHFI ◽  
GEORGE A. COOK
Keyword(s):  
Outer Membrane ◽  
Carnitine Palmitoyltransferase ◽  
Mitochondrial Outer Membrane ◽  
Cpt I

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 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 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.

scholarly journals Evidence against direct involvement of phosphorylation in the activation of carnitine palmitoyltransferase by okadaic acid in rat hepatocytes

1994 ◽  
Vol 300 (3) ◽  
pp. 693-699 ◽  
Author(s):  
M Guzman ◽  
M P Kolodziej ◽  
A Caldwell ◽  
C G Corstorphine ◽  
V A Zammit
Keyword(s):  
Outer Membrane ◽  
Okadaic Acid ◽  
Outer Membrane Proteins ◽  
Rat Hepatocytes ◽  
Carnitine Palmitoyltransferase ◽  
Mitochondrial Outer Membrane ◽  
Fluoride Ions ◽  
Malonyl Coa ◽  
The Difference ◽  
Cpt I

The mechanism of activation of mitochondrial overt carnitine palmitoyltransferase (CPT I) by treatment of hepatocytes with okadaic acid (OA) was investigated. Activation was observed when cells were permeabilized with digitonin, but not when a total membrane fraction was obtained by sonication. Both cell disruption methods preserved the activation of phosphorylase observed in OA-treated hepatocytes. Activation of CPT I was also observed in crude homogenates of OA-treated hepatocytes, but it was lost upon subsequent isolation of mitochondria from such homogenates. In all experiments, any activation observed did not depend on the presence or absence of fluoride ions in the permeabilization/homogenization media. When hepatocytes were permeabilized in the absence of fluoride and further incubated with exogenous phosphatases 1 and 2A, the OA-induced activation of CPT was not reversed, whereas the activation of glycogen phosphorylase in the same cells was rapidly reversed. Treatment of hepatocytes with OA, followed by permeabilization and incubation before assay of CPT I, demonstrated that OA had no short-term effect on the sensitivity of CPT I to malonyl-CoA, although the difference in sensitivity between cells isolated from fed and starved rats was fully preserved. Incubation of isolated mitochondria or purified mitochondrial outer membranes with cyclic AMP-dependent or AMP-activated protein kinases, under phosphorylating conditions, did not affect the activity of CPT I or its sensitivity to malonyl-CoA inhibition. Under the same conditions, the use of [32P]ATP resulted in the labelling of several outer-membrane proteins but, unlike [3H]etomoxir-labelled CPT I, none of them was specifically removed from membrane extracts by a specific polyclonal antibody to the enzyme. We conclude that the increase in overt CPT activity observed in permeabilized hepatocytes is not due to direct phosphorylation of CPT I, but may involve interactions between the mitochondrial outer membrane and other membranous or soluble cytosolic components of the cell.

scholarly journals Proteinase treatment of intact hepatic mitochondria has differential effects on inhibition of carnitine palmitoyltransferase by different inhibitors

1992 ◽  
Vol 282 (3) ◽  
pp. 909-914 ◽  
Author(s):  
K Kashfi ◽  
G A Cook
Keyword(s):  
Outer Membrane ◽  
Adenylate Kinase ◽  
Citrate Synthase ◽  
Physiological State ◽  
Carnitine Palmitoyltransferase ◽  
Mitochondrial Outer Membrane ◽  
Intermembrane Space ◽  
Mitochondrial Inner Membrane ◽  
Malonyl Coa ◽  
No Release

Proteolysis of intact mitochondria by Nagarse (subtilisin BPN') and papain resulted in limited loss of activity of the outer-membrane carnitine palmitoyltransferase, but much greater loss of sensitivity to inhibition by malonyl-CoA. In contrast with a previous report [Murthy & Pande (1987) Proc. Natl. Acad. Sci. U.S.A. 84, 378-382], we found that trypsin had no effect on malonyl-CoA sensitivity. Even when 80% of activity was destroyed by trypsin, there was no difference in the malonyl-CoA sensitivity of the enzyme remaining. Trypsin caused release of the intermembrane-space enzyme adenylate kinase, indicating loss of integrity of the mitochondrial outer membrane, whereas Nagarse and papain caused no release of that enzyme. Citrate synthase was not released by any of the three proteinases, indicating no damage to the mitochondrial inner membrane. When we examined the effects of proteolysis on the inhibition of carnitine palmitoyltransferase by a wide variety of inhibitors having different mechanisms of inhibition, we found differential proteolytic effects that were specific for those inhibitors (malonyl-CoA and hydroxyphenylglyoxylate) that have their inhibitory potencies diminished by changes in physiological state. Both of those inhibitors protected carnitine palmitoyltransferase from the effects of proteolysis, but did not inhibit the proteinases directly. Inhibition by two other inhibitors (DL-2-bromopalmitoyl-CoA and N-benzyladriamycin 14-valerate) was not altered by proteinase treatment, even when most of the enzyme activity had been destroyed. Inhibition by glyburide, which is minimally affected by physiological state, was affected only to a slight extent at the highest concentration of trypsin tested. Proteolysis by Nagarse appeared to produce loss of co-operativity in malonyl-CoA inhibition. The effects of proteolysis are discussed and compared with changes in Ki occurring with changing physiological states.

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