Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:2.3.1.21 (CPT)
4,580 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Dissection of the mitochondrial carnitine palmitoyltransferase (CPT) enzyme system in terms of its structure/function relationships has proved to be a formidable task. Although no one formulation has gained universal agreement we believe that the weight of evidence supports a model with the following features: a) in any given tissue CPT I and CPT II are distinct proteins; b) CPT I, unlike CPT II, is detergent labile; c) within a species CPT II is expressed body wide, whereas CPT I exists as tissue specific isoforms; d) malonyl-CoA and other CPT I inhibitors probably interact at the catalytic center of the enzyme, not with a regulatory subunit. The amino acid sequences of rat and human CPT II (deduced from cDNA clones) show them to be similar proteins (greater than 80% identity) but encoded by mRNAs of significantly different sizes. Efforts to clone and sequence the cDNA for rat liver CPT I are presently underway.
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PMID:New insights into the mitochondrial carnitine palmitoyltransferase enzyme system. 203 61

Carnitine palmitoyltransferase II of rat heart mitochondria was purified to homogeneity by a rapid method exploiting the hydrophobic nature of the protein. The method involves solubilization of mitochondrial membrane proteins by detergents and subsequent fractionation by hydrophobic affinity chromatography. Sepharose, cross-linked via a primary amino group of 1,omega-diaminoalkane, 4-aminobutyric acid, 6-aminocaproic acid, or 6-aminohexanol, was found to reversibly bind carnitine palmitoyltransferase under nondenaturing conditions. A homologous series of n-alkyl-agarose resins with n = 2 to 8 and phenyl-Sepharose were also found to reversibly bind the enzyme. Alkyl-Superose, phenyl-Superose, and Superose 12 chromatographies were also very useful in fractionating the enzyme. Successive chromatography on three or four hydrophobic columns yielded a highly pure enzyme preparation. The purified preparation appeared as one major protein band on polyacrylamide electrophoresis gels in the presence of sodium dodecyl sulfate (M(r) 68,000). The isolated enzyme had significant activity (sp act = 15.0 mumol/min/mg protein when 80 microM palmitoyl-CoA and 20 mM carnitine were used as substrates). Antibodies against this protein recognized (in immunoblots) one major protein band in crude preparations of rat heart mitochondria (M(r) 68,000), indistinguishable from purified carnitine palmitoyltransferase II. L-Palmitoylcarnitine (0.1 mM) and coenzyme A (0.1 mM), products of the enzyme-catalyzed reaction, inhibited carnitine palmitoyltransferase activity 66 and 71%, respectively. D-Palmitoylcarnitine (0.1 mM), however, did not inhibit the activity. Malonyl-CoA, a specific inhibitor of membrane-bound carnitine palmitoyltransferase I, did not show significant inhibition.
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PMID:Purification of carnitine palmitoyltransferase from heart mitochondria by hydrophobic affinity chromatography. 213 38

In animal cells long chain fatty acids are transferred into the mitochondria for oxidation as acylcarnitines. Carnitine palmitoyltransferase I in the outer membrane, and carnitine translocase plus carnitine palmitoyltransferase II in the inner membrane catalyse the transfer. Carnitine palmitoyltransferase I is inhibited by malonyl-CoA, an intermediate in fatty acid synthesis. In the liver of fasted, diabetic, or thyreotoxic animals this enzyme shows increased activity and less inhibition by malonyl-CoA. Peroxisomes also contain carnitine acyltransferases and a beta-oxidation enzyme system. This system is particularly active in the shortening of very long chain fatty acids. The carnitine acyltransferases of the peroxisomes presumably are active in the transfer of the shortened acyl-CoAs and the acetyl-CoA to the mitochondria for complete oxidation. The carnitine acyltransferases of the mitochondria can catalyse the formation of propionylcarnitine and branched chain acylcarnitines from branched chain amino acids, and methylthiopropionylcarnitine from methionine. Their formation may represent a "security valve" preventing acyl-CoA accumulation in the mitochondria. The liver, which normally releases carnitine for other tissues, releases the branched chain acylcarnitines even more easily. This may be important for the development of secondary carnitine deficiency in some inborn errors of metabolism which are accompanied by the accumulation of acyl-CoAs in the tissue.
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PMID:The role of carnitine in intracellular metabolism. 219 93

Properties of the carnitine palmitoyltransferase (EC 2.3.1.21) (CPT) enzyme system were compared in isolated mitochondria from a range of tissues in rodents, monkey, and man. Common features were as follows: (a) while membrane-bound, CPT I, but not CPT II, was inhibited reversibly by malonyl-coenzyme A (CoA) and irreversibly by CoA esters of certain oxirane carboxylic acids; (b) the detergent, Tween-20, readily solubilized CPT II in active form while leaving CPT I membrane associated and catalytically functional; (c) octyl glucoside and Triton X-100 released active CPT II but caused essentially complete loss of CPT I activity. Use of [3H]tetradecylglycidyl-CoA, a covalent ligand for CPT I, yielded estimates of the enzyme's monomeric molecular size: approximately 86 kDa in non-hepatic tissues and approximately 90-94 kDa in liver, depending upon species. A polyclonal antibody to purified rat liver CPT II recognized a single protein in each tissue; its apparent molecular mass was approximately 70 kDa in all rat tissues and approximately 68 kDa in all mouse tissues as well as monkey and human liver. On Northern blot analysis a rat liver CPT II cDNA probe detected a single approximately 2.5-kilobase mRNA in all rat and mouse tissues examined. The following points are emphasized. First, CPT I and II are different proteins. Second, within a species CPT II, but not CPT I, is probably conserved across tissue lines. Third, slight variations in size of both enzymes were found in different species, although, at least in the case of CPT II, significant amino acid identity exists among the various isoforms. Fourth, CPT I, unlike CPT II, requires membrane integrity for catalytic function. Finally, the strategic use of detergents provides a simple means of discriminating between the two enzyme activities.
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PMID:Inter-tissue and inter-species characteristics of the mitochondrial carnitine palmitoyltransferase enzyme system. 235 17

We report the isolation and characterization of a full-length cDNA encoding rat liver carnitine palmitoyltransferase II (CPT II). Beginning with the purified protein CNBr fragments were generated and sequenced. Corresponding oligonucleotides were used to screen a rat liver cDNA library constructed in the plasmid cloning vector, pcDV. The clone ultimately obtained consisted of a 62 nucleotide 5'-untranslated region, a single open reading frame of 1,974 bases predicting a protein of 658 amino acids (Mr = 74,119), and a 3'-untranslated segment of 260 nucleotides followed by the poly (A) tail. The identity of the cDNA was confirmed by the findings that (a) the open reading frame encoded all three peptides found in the original protein; (b) a fourth peptide synthesized from a portion of the deduced amino acid sequence and used to immunize a rabbit resulted in the generation of an antibody that recognized pure CPT II on a Western blot; (c) in vitro transcription and translation of the cDNA (ligated into pBlue-script KS (+] generated a protein that was specifically immunoprecipitated by anti-CPT II antibody and having a Mr slightly greater than that of mature CPT II; (d) transfection of COS cells with the cDNA subcloned into the expression vector, pCMV4, resulted in a 6-fold induction of mitochondrial CPT II catalytic activity. It seems likely that the de novo synthesized enzyme gains entry into the mitochondrion via a targeting peptide that is subsequently cleaved. The mature protein probably associates (relatively loosely) with the inner membrane through a limited number of membrane spanning domains. The predicted amino acid sequence of CPT II shows strong identity with those of two other acyltransferases, namely, rat liver peroxisomal carnitine octanoyltransferase and porcine choline acetyltransferase.
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PMID:Cloning, sequencing, and expression of a cDNA encoding rat liver mitochondrial carnitine palmitoyltransferase II. 235 18

After coronary occlusion and reflow, carbohydrate catabolism is enhanced, whereas fatty acid utilization is delayed. To test the hypothesis that "stunning" of fatty acid use by ischemic heart reflects reduced fatty acid transport into the mitochondria, two activities involved in the transport were examined: carnitine-acylcarnitine translocase and carnitine palmitoyltransferase II (CPT II). The maximal velocity for carnitine exchange of the translocase is reduced 55% in mitochondria isolated from ischemic canine heart (60-min left circumflex occlusion). Mitochondria from ischemic heart show 50% depletion in total matrix glutathione, a 200% increase in glutathione disulfide (GSSG), and an 80% decrease in the ratio of reduced glutathione (GSH) to GSSG, suggesting that the loss of translocase activity may be a consequence of protein sulfhydryl modifications. In support of this, treatment of these mitochondria with the sulfhydryl-reducing agents, GSH or dithiothreitol, restores carnitine exchange to control. Partial return of mitochondrial GSH and a decrease in GSSG are observed with a 20-min reperfusion of the ischemic myocardium. Continued depression in carnitine exchange with reperfusion suggests that other mechanisms may prevent restoration of activity. Import of palmitoylcarnitine on the translocase is coupled to palmitoyl-CoA production by CPT II. Mitochondria from ischemic heart with decreased coupling activity also have the lowest palmitoylcarnitine-supported respiratory rates, suggesting that in severely ischemic tissue the translocation-transesterification sequence may become rate limiting to fatty acid oxidation.
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PMID:Carnitine-acylcarnitine translocase in ischemia: evidence for sulfhydryl modification. 312 91

A 20-year-old man was shown to have a deficiency of carnitine palmitoyltransferase (CPT) II in skeletal muscle. The evidence was: (i) there was no significant oxidation of [9,10-3H]-palmitate or of [1-14C]palmitate in mitochondrial fractions from fresh skeletal muscle from the patient; (ii) all the CPT activity in a homogenate of fresh muscle from the patient was overt (CPT I) with no increase in activity after the inner membrane was disrupted; (iii) all the CPT activity in the patient's muscle was inhibited by malonyl-CoA; and (iv) an immunoreactive peptide of 67 kDa corresponding to CPT II, present in mitochondria from controls, was absent in those from the patient.
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PMID:A case of carnitine palmitoyltransferase II deficiency in human skeletal muscle. 319 28

1. Estimates of the functional sizes of the molecular species responsible for the overt (I) and latent (II) activities of carnitine palmitoyltransferase (CPT) in 48 h-starved rat liver mitochondria were obtained from radiation inactivation experiments. 2. The decay in the activity of total CPT and that of CPT II only (after inhibition of CPT I) was measured in mitochondrial samples exposed to different doses of high-energy ionizing radiation. 3. The decay curves obtained by plotting residual activity of total CPT as a logarithm function of irradiation dose suggested the contribution of more than one target towards total CPT activity. 4. By contrast, in mitochondria in which CPT I activity was approximately 95% inhibited, the activity of CPT decayed in a simple mono-exponential manner. Target-size analysis yielded an approximate Mr of 69,700 for this component (CPT II). 5. This information, as well as that on the relative non-irradiated activities of CPT I and CPT II, was used in graphical and statistical methods to obtain the parameters of the decay curve for CPT I. These analyses yielded an approximate Mr of 96,700 for CPT I.
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PMID:Evidence for distinct functional molecular sizes of carnitine palmitoyltransferases I and II in rat liver mitochondria. 335 31

The effects of various inhibitors of carnitine palmitoyltransferase I were examined in mitochondria from rat liver and skeletal muscle. Three types of inhibitors were used: malonyl-CoA (reversible), tetradecylglycidyl-CoA and three of its analogues (irreversible), and 2-bromopalmitoyl-CoA (essentially irreversible when added with carnitine). Competitive binding studies between labeled and unlabeled ligands together with electrophoretic analysis of sodium dodecyl sulfate-solubilized membranes revealed that in mitochondria from both tissues all of the inhibitors interacted with a single protein. While the binding capacity for inhibitors was similar in liver and muscle (6-8 pmol/mg of mitochondrial protein) the proteins involved were of different monomeric size (Mr 94,000 and 86,000, respectively). Treatment of mitochondria with the detergent, octyl glucoside, yielded a soluble form of carnitine palmitoyltransferase and residual membranes that were devoid of enzyme activity. The solubilized enzyme displayed the same activity regardless of whether carnitine palmitoyltransferase I of the original mitochondria had first been exposed to an irreversible inhibitor or destroyed by chymotrypsin. It eluted as a single activity peak through four purification steps. The final product from both liver and muscle migrated as single band on sodium dodecyl sulfate-polyacrylamide electrophoresis with Mr of approximately 80,000. The data are consistent with the following model. The inhibitor binding protein is carnitine palmitoyltransferase I itself (as opposed to a regulatory subunit). The hepatic monomer is larger than the muscle enzyme. Each inhibitor interacts via its thioester group at the palmitoyl-CoA binding site of the enzyme but also at a second locus that is probably different for each agent and dictated by the chemical substituent on carbon 2. Disruption of the mitochondrial inner membrane by octyl glucoside causes inactivation of carnitine palmitoyltransferase I while releasing carnitine palmitoyltransferase II in active form. The latter is readily purified, is a smaller protein than carnitine palmitoyltransferase I, and has the same molecular weight in liver and muscle. It is insensitive to inhibitors where on or off the mitochondrial membrane.
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PMID:Characterization of the mitochondrial carnitine palmitoyltransferase enzyme system. I. Use of inhibitors. 359 41

Exposure of rat liver mitochondrial membranes to octyl glucoside, Triton X-100, or Tween 20 solubilized an active and tetradecylglycidyl-CoA (TG-CoA)-insensitive carnitine palmitoyltransferase (presumed to be carnitine palmitoyltransferase II). The residual membranes after octyl glucoside or Triton X-100 treatment were devoid of all transferase activity. By contrast, Tween 20-extracted membranes were still rich in transferase; this was completely blocked by TG-CoA and thus was presumed to be carnitine palmitoyltransferase I. The residual carnitine palmitoyltransferase activity disappeared from the membranes upon subsequent addition of octyl glucoside or Triton X-100 and could not be recovered in the supernatant fraction. Antibody raised against purified rat liver transferase II (Mr 80,000) recognized only this protein in immunoblots from untreated liver mitochondrial membranes containing both transferases I and II. Tween 20-extracted membranes, which contained only transferase I, did not react with the antibody. Purified transferase II from skeletal muscle (also of Mr 80,000) was readily recognized by the antiserum, suggesting antigenic similarity with the liver enzyme. These and other studies on the effects of detergents on the mitochondrial [3H]TG-CoA binding protein provide further support for the model of carnitine palmitoyltransferase proposed in the preceding paper. They suggest that: 1) carnitine palmitoyltransferases I and II in rat liver are immunologically distinct proteins; 2) transferase I is more firmly anchored into its membrane environment than transferase II; 3) association of carnitine palmitoyltransferase I with a membrane component(s) is necessary for catalytic activity. While carnitine palmitoyltransferase I is a different protein in liver and muscle, it seems likely that both tissues share the same transferase II.
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PMID:Characterization of the mitochondrial carnitine palmitoyltransferase enzyme system. II. Use of detergents and antibodies. 359 42


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