scholarly journals Effects of dl-2-bromopalmitoyl-CoA and bromoacetyl-CoA in rat liver and heart mitochondria. Inhibition of carnitine palmitoyltransferase and displacement of [14C]malonyl-CoA from mitochondrial binding sites

1985 ◽  
Vol 230 (1) ◽  
pp. 169-179 ◽  
Author(s):  
M R Edwards ◽  
M I Bird ◽  
E D Saggerson
Keyword(s):  
Binding Site ◽  
Rat Liver ◽  
Binding Sites ◽  
Liver Mitochondria ◽  
Carnitine Palmitoyltransferase ◽  
Heart Mitochondria ◽  
Nitrobenzoic Acid ◽  
Malonyl Coa ◽  
Tissue Differences ◽  
The Relationship

The overt form of carnitine palmitoyltransferase (CPT1) in rat liver and heart mitochondria was inhibited by DL-2-bromopalmitoyl-CoA and bromoacetyl-CoA. S-Methanesulphonyl-CoA inhibited liver CPT1. The inhibitory potency of DL-2-bromopalmitoyl-CoA was 17 times greater with liver than with heart CPT1. Inhibition of CPT1 by DL-2-bromopalmitoyl-CoA was unaffected by 5,5′-dithiobis-(2-nitrobenzoic acid) or (in liver) by starvation. In experiments in which DL-2-bromopalmitoyl-CoA displaced [14C]malonyl-CoA bound to liver mitochondria, the KD (competing) was 25 times the IC50 for inhibition of CPT1 providing evidence that the malonyl-CoA-binding site is unlikely to be the same as the acyl-CoA substrate site. Bromoacetyl-CoA inhibition of CPT1 was more potent in heart than in liver mitochondria and was diminished by 5,5′-dithiobis-(2-nitrobenzoic acid) or (in liver) by starvation. Bromoacetyl-CoA displaced bound [14C]malonyl-CoA from heart and liver mitochondria. In heart mitochondria this displacement was competitive with malonyl-CoA and was considerably facilitated by L-carnitine. In liver mitochondria this synergism between carnitine and bromoacetyl-CoA was not observed. It is suggested that bromoacetyl-CoA interacts with the malonyl-CoA-binding site of CPT1. L-Carnitine also facilitated the displacement by DL-2-bromopalmitoyl-CoA of [14C]malonyl-CoA from heart, but not from liver, mitochondria. DL-2-Bromopalmitoyl-CoA and bromoacetyl-CoA also inhibited overt carnitine octanoyl-transferase in liver and heart mitochondria. These findings are discussed in relation to inter-tissue differences in (a) the response of CPT1 activity to various inhibitors and (b) the relationship between high-affinity malonyl-CoA-binding sites and those sites for binding of L-carnitine and acyl-CoA substrates.

scholarly journals Binding of malonyl-CoA to isolated mitochondria. Evidence for high- and low-affinity sites in liver and heart and relationship to inhibition of carnitine palmitoyltransferase activity

1984 ◽  
Vol 222 (3) ◽  
pp. 639-647 ◽  
Author(s):  
M I Bird ◽  
E D Saggerson
Keyword(s):  
Binding Sites ◽  
Maximal Activity ◽  
Liver Mitochondria ◽  
Carnitine Palmitoyltransferase ◽  
Male Rats ◽  
Heart Mitochondria ◽  
Binding Characteristics ◽  
Functional Sites ◽  
High Affinity ◽  
Malonyl Coa

[14C]Malonyl-CoA bound to intact mitochondria isolated from rat liver and heart in a manner consistent with the presence of two independent classes of binding sites in each tissue. The binding characteristics for mitochondria obtained from fed male rats were: for heart, KD(1) = 11-18nM, KD(2) = 30 microM, N1 = 7pmol/mg of protein, N2 = approx. 660pmol/mg of protein; for liver, KD(1) = 0.1 microM, KD(2) = 5.6 microM, N1 = 11pmol/mg of protein, N2 = 165pmol/mg of protein. In the presence of 40 microM-palmitoyl-CoA the characteristics of binding at the high-affinity sites were changed, so that for heart KD(1) = 0.26 microM, with no change in N1 and for liver KD(1) = approx. 2 microM, with N1 increased to approx. 40pmol/mg of protein. Differences between the two tissues in tightness of malonyl-CoA binding at the high-affinity sites explains the considerably greater sensitivity of heart CPT1 (overt form of carnitine palmitoyltransferase) to inhibition by malonyl-CoA [Saggerson & Carpenter, (1981) FEBS Lett. 129, 229-232; McGarry, Mills, Long & Foster (1983) Biochem. J. 214, 21-28]. Starvation (24h) did not change the characteristics of [14C]malonyl-CoA binding to liver mitochondria and did not alter the I50 (concentration giving 50% inhibition) for displacement of [14C]malonyl-CoA by palmitoyl-CoA. Therefore the decreased sensitivity of liver CPT1 to inhibition by malonyl-CoA in starvation [Saggerson & Carpenter (1981) FEBS Lett. 129, 225-228; Bremer (1981) Biochim. Biophys. Acta 665, 628-631] is not explained by differences in malonyl-CoA binding. Percentage occupancy of the high-affinity sites in heart mitochondria by malonyl-CoA correlated closely with percentage inhibition of CPT1 measured under similar conditions. This finding supports the proposal that the high-affinity binding sites are the functional sites mediating inhibition of CPT1 by malonyl-CoA. Similar experiments with liver mitochondria also suggested that the occupancy of high-affinity sites by malonyl-CoA regulates CPT1 activity. 5,5′-Dithiobis-(2-nitrobenzoic acid), which decreased the sensitivity of heart or liver CPT1 to inhibition by malonyl-CoA [Saggerson & Carpenter (1982) FEBS Lett. 137, 124-128], also decreased [14C]malonyl-CoA binding to the high-affinity sites of heart mitochondria. N1 values for [14C]malonyl-CoA binding to high-affinity sites in liver mitochondria were determined in various physiological states which encompassed a 7-fold range of CPT1 maximal activity (fed, starved, pregnant, hypothyroid, foetal). The N1 value did not change in these states.(ABSTRACT TRUNCATED AT 400 WORDS)

scholarly journals Interacting effects of l-carnitine and malonyl-CoA on rat liver carnitine palmitoyltransferase

1985 ◽  
Vol 230 (1) ◽  
pp. 161-167 ◽  
Author(s):  
M I Bird ◽  
E D Saggerson
Keyword(s):  
Binding Sites ◽  
Concentration Ratio ◽  
Liver Mitochondria ◽  
Carnitine Palmitoyltransferase ◽  
Heart Mitochondria ◽  
Albumin Concentration ◽  
Latent Form ◽  
Malonyl Coa ◽  
Carnitine Palmitoyltransferase Activity ◽  
Overt Form

Malonyl-CoA significantly increased the Km for L-carnitine of overt carnitine palmitoyltransferase in liver mitochondria from fed rats. This effect was observed when the molar palmitoyl-CoA/albumin concentration ratio was low (0.125-1.0), but not when it was higher (2.0). In the absence of malonyl-CoA, the Km for L-carnitine increased with increasing palmitoyl-CoA/albumin ratios. Malonyl-CoA did not increase the Km for L-carnitine in liver mitochondria from 24h-starved rats or in heart mitochondria from fed animals. The Km for L-carnitine of the latent form of carnitine palmitoyltransferase was 3-4 times that for the overt form of the enzyme. At low ratios of palmitoyl-CoA/albumin (0.5), the concentration of malonyl-CoA causing a 50% inhibition of overt carnitine palmitoyltransferase activity was decreased by 30% when assays with liver mitochondria from fed rats were performed at 100 microM-instead of 400 microM-carnitine. Such a decrease was not observed with liver mitochondria from starved animals. L-Carnitine displaced [14C]malonyl-CoA from liver mitochondrial binding sites. D-Carnitine was without effect. L-Carnitine did not displace [14C]malonyl-CoA from heart mitochondria. It is concluded that, under appropriate conditions, malonyl-CoA may decrease the effectiveness of L-carnitine as a substrate for the enzyme and that L-carnitine may decrease the effectiveness of malonyl-CoA to regulate the enzyme.

scholarly journals The effect of malonyl-CoA on overt and latent carnitine acyltransferase activities in rat liver and adipocyte mitochondria

1983 ◽  
Vol 210 (2) ◽  
pp. 591-597 ◽  
Author(s):  
E D Saggerson ◽  
C A Carpenter
Keyword(s):  
Rat Liver ◽  
Liver Mitochondria ◽  
Carnitine Palmitoyltransferase ◽  
Nitrobenzoic Acid ◽  
Carnitine Acyltransferase ◽  
Malonyl Coa ◽  
Before And After ◽  
Carnitine Palmitoyltransferase Activity

1. Carnitine palmitoyltransferase and carnitine octanoyltransferase activities were measured in mitochondria at various acyl-CoA concentrations before and after sonication, thus permitting assessment of both overt and latent activities. 2. Overt carnitine palmitoyltransferase in liver and adipocyte mitochondria and overt carnitine octanoyltransferase in liver mitochondria were inhibited by malonyl-CoA. None of the latent activities were affected by this metabolite. 3. 5,5′-Dithiobis-(2-nitrobenzoic acid) stimulated latent hepatic carnitine palmitoyltransferase at low [palmitoyl-CoA]. 4. Starvation (24 h) decreased overt carnitine palmitoyltransferase activity in adipocyte mitochondria, but did not alter the sensitivity of this activity to malonyl-CoA.

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 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 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 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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