Carnitine palmitoyltransferase

Carnitine palmitoyltransferases I and II catalyze the transfer of long-chain acyl coenzyme A into mitochondria. The I isozyme is located on the cytosol side of the inner membrane and catalyzes the formation acylcarnitine from acyl-CoA and carnitine. After acylcarnitine crosses the inner membrane, it is converted back to acyl-CoA by the action of the II isozyme. This assay measures the activity of carnitine palmitoyltransferase I in intact mitochondria. 

The enzyme was assayed by quantitating the coenzyme A released. The assays are run in the presence and absence of L-carnitine to correct for enzymatic and nonenzymatic hydrolysis of palmitoyl-CoA. The separation was made at 40°C on a LiChrosorb RP-18 column (4.0 mm x 250 mm, 5 /urn). The mobile phase was prepared from solvent A [220 mM NaH2P04 and 0.05% (v/v) /3-thiodiglycol] and solvent B [125 mM NaH2P04, 43% (v/v) methanol, 0.9% (v/v) chloroform, and 0.05% (v/v) /3-thiodiglycol]. The composition of the mobile phase was 12% solvent B at time 0, 15% B at 8 minutes, 65% B at 28.5 minutes, and 100% B at 29 to 31 minutes. The mobile phase was returned to starting conditions within 1 minute and maintained there for 28 minutes before injecting the next sample. The column effluent was monitored at 254 nm. 

The enzyme assay contained in a final volume of 1.0 mL, 75 mM KC1,50 mM mannitol, 25 mM Hepes (pH 7.0), 0.2 mM EGTA, 2 mM KCN, 5 mM dithiothreitol, 1.75 mg fatty-acid-free bovine serum albumin, 30 to 120 yM palmitoyl-CoA, and 0.5 mM L-camitine. After 2 minutes of preincubation at 25°C, the reaction was initiated by adding 100 fiL (0.1-0.3 mg protein) of mitochondrial suspension. The reactions were terminated after 5 minutes by the addition of 50 yL of 60% (v/v) perchloric acid. The supemates were adjusted to pH 2 to 3 by adding 2 M potassium phosphate, cooled on ice, and 

The source of enzyme was rat liver mitochondria suspended in 0.25 M sucrose containing 2 mM Hepes (pH 7.4) and 1 mM EGTA. 

Fatty acid w-hydroxylation is involved in the.metabolism of prostaglandins and leukotrienes, and is the first step of dicarboxylic acid formation in the cell. In this assay, lauric acid is hydroxylated to form 12-hydroxylauric acid, which is then fluorescence-labeled on the carboxyl group with 3-bromomethyl-7-methoxy-l,4-benzoxazin-2-one. 

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Long-chain acyl-CoA esters are then converted to acylcamitine esters by readily reversible reactions with L-camitine catalyzed by carnitine palmitoyltransferase I (CPT I). 

Esser, V., Britton, C.H., Weiss, B.C., Foster, D.W. McGarry, J.D. (1993). Cloning, sequencing and expression of a cDNA encoding rat liver carnitine palmitoyltransferase 1. Direct evidence that a single polypeptide is involved in inhibitor interaction and catalytic function. J. Biol. Chem. 268, 5817-5822. 

Murthy, M.S.R. Pande, S.V. (1987). Malonyl-CoA binding site and the overt carnitine palmitoyltransferase activity reside on the opposite sides of the outer mitochondrial membrane. Proc. Nat. Acad. Sci. USA 84,378-382. 

Increased fatty acid oxidation is a characteristic of starvation and of diabetes meUims, leading to ketone body production by the Ever (ketosis). Ketone bodies are acidic and when produced in excess over long periods, as in diabetes, cause ketoacidosis, which is ultimately fatal. Because gluconeogenesis is dependent upon fatty acid oxidation, any impairment in fatty acid oxidation leads to hypoglycemia. This occurs in various states of carnitine deficiency or deficiency of essential enzymes in fatty acid oxidation, eg, carnitine palmitoyltransferase, or inhibition of fatty acid oxidation by poisons, eg, hypoglycin. 

Carnitine (p-hydroxy-y-trimethylammonium butyrate), (CHjljN"—CH2—CH(OH)—CH2—COO , is widely distributed and is particularly abundant in muscle. Long-chain acyl-CoA (or FFA) will not penetrate the inner membrane of mitochondria. However, carnitine palmitoyltransferase-I, present in the outer mitochondrial membrane, converts long-chain acyl-CoA to acylcarnitine, which is able to penetrate the inner membrane and gain access to the P-oxidation system of enzymes (Figure 22-1). Carnitine-acylcar-nitine translocase acts as an inner membrane exchange transporter. Acylcarnitine is transported in, coupled with the transport out of one molecule of carnitine. The acylcarnitine then reacts with CoA, cat-... 

Figure 22-9. Regulation of ketogenesis. -(Dshow three crucial steps in the pathway of metabolism of free fatty adds (FFA) that determine the magnitude of ketogenesis. (CPT-I, carnitine palmitoyltransferase-l.)...

Ketogenesis is regulated at three cmcial steps (1) control of free fatty acid mobihzation from adipose tissue (2) the activity of carnitine palmitoyltransferase-1 in hver, which determines the proportion of the fatty acid flux that is oxidized rather than esteri-fied and (3) partition of acetyl-CoA between the pathway of ketogenesis and the citric acid cycle. 

GMBS or sulfo-GMBS have been used for studying carnitine palmitoyltransferase-1 in its formation of a complex within the outer mitochondrial membrane (Faye et al., 2007), for investigating protein organization of the postsynaptic density (Liu et al., 2006), and in studying the structure and dynamics of rhodopsin (Jacobsen et al., 2006). 

Faye, A., Esnous, C., Price, N.T., Onfray, M.A., Girard, J., and Prip-Buus, C. (2007) Rat liver carnitine palmitoyltransferase 1 forms an oligomeric complex within the outer mitochondrial membrane.. Biol. Cbem. 10.1074/jbc.M705418200. 

Carnitine palmitoyltransferase deficiency is an autosomal recessive myopathy caused by a genetic defect of the mitochondrial enzyme CPT (Fig. 42-2). The disease is prevalent in men (male female ratio, 5.5 1) and appears to be the most common cause of recurrent myoglobinuria in adults [4]. 

Mitochondria contain all the enzymes necessary for oxidation of fatty acids but, before this can take place, the fatty acids have to be transported into the mitochondria. Transport requires the formation of an ester of the fatty acid with a compound, carnitine, in a reaction catalysed by the enzyme carnitine palmitoyltransferase ... 

It is the fatty acyl-camitine that is transported across the inner mitochondrial membrane from the cytosol to the matrix so that two different enzymes are reqnired for the transport. The first enzyme, carnitine palmitoyltransferase-1, is located on the outer surface of this membrane and the second enzyme, carnitine pahnitoyltransferase-II, is located on the inner side of this membrane (Figure 7.11). Carnitine may have this role since it is smaller than CoASH and has no net charge. 

The activity of carnitine palmitoyltransferase-I plays an important role in the regulation of fatty acid oxidation malonyl-CoA is an allosteric exhibitor of the enzyme. Malonyl-CoA is a key intermediate in fatty acid synthesis, which ensures that fatty acid oxidation is decreased when synthesis is taking place. Nonetheless, malonyl-CoA has a major role in the control of fatty acid oxidation in all tissues in which fatty acid oxidation occurs, even if no synthesis takes place. 

Figure 7.15 Inhibition of acetyl-CoA carboxylase by cyclic AMP dependent protein kinase and AMP dependent protein kinase the dual effect of glucagon. Phosphorylation of acetyl-CoA carboxylase by either or both enzymes inactivates the enzyme which leads to a decrease in concentration of malonyl-CoA, and hence an increase in activity of carnitine palmitoyltransferase-I and hence an increase in fatty acid oxidation. Insulin decreases the cyclic AMP concentration maintaining an active carboxylase and a high level of malonyl-CoA to inhibit fatty acid oxidation.

There is a marked increase in the activity of the key enzymes that convert fatty acids into ketone bodies carnitine palmitoyltransferase and HMG-CoA synthase. 

The carnitine palmitoyltransferase is insensitive to the inhibitor malonyl-Co A, so that it cannot be inhibited by this allosteric effector (see above). 

Defects in several proteins involved in fatty acid oxidation are known. These are carnitine palmitoyltransferases, any of the three acyl-CoA dehydrogenases, or the protein that... 

Malonyl-CoA is also involved in the regulation of fatty acid oxidation, via inhibition of carnitine palmitoyltransferase. In non-lipogenic tissues, the only role of the carboxylase is provision of malonyl-CoA for regulation of the rate of fatty acid oxidation. 

CARNITINE ACETYLTRANSFERASE CARNITINE OCTANOYLTRANSFERASE CARNITINE PALMITOYLTRANSFERASE... 

Figure 8-3. The carnitine shuttle. A long-chain fatty acyl CoA (LCFA CoA) can diffuse across the outer mitochondrial membrane but must be carried across the inner membrane as acyl-carnitine. The active sites of CPT-I and CPT-II are oriented toward the interiors of their respective membranes. CPT, carnitine palmitoyltransferase.

In 1955, Fritz determined that carnitine plays an essential role in fatty acid -oxidation (FAO), and in 1973 the first two clinically relevant disorders affecting this pathway were described primary carnitine deficiency by Engel and Angelini, and carnitine palmitoyltransferase (CPT) type II (CPT-II) deficiency by DiMauro and DiMauro [6, 7]. To date, more than 20 different enzyme deficiency states affecting fatty acid transport and mitochondrial / -oxidaLion have been described [8] and additional enzymes involved in this pathway are still being discovered [9, 10]. 

Bonnefont JP, Djouadi F, Prip-Buus C, Gobin S, Munnich A, Bastin J (2004) Carnitine palmitoyltransferases 1 and 2 biochemical, molecular and medical aspects. Mol Aspects Med 25 495-520... 

Inhibitor Long-chain fatty acyl CoA (inhibits acetyl CoA carboxylase) Malonyl CoA (inhibits carnitine palmitoyltransferase)... 

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