Abstract
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.
Highlights
From the Departments of IIZnternul Medicine, §Molecular Genetics, and TBiochemistry, University of Texas Health Science Center at Dallas, SouthwesternMedical School, Dallas, Texas 75235
The transport of long chain fatty acids into mitochondria electrophoretic analysis of sodium dodecyl sulfate-solubilizedmembranesrevealed thatinmitochondria from both tissues all of the inhibitors interacted with a single protein
To address some of these questions we have examined the inhibitory effects of malonyl-CoA and a variety of irreversible inhibitors on carnitine palmitoyltransferasIein mitochondria from rat liver and skeletal muscle
Summary
Mitochondria (approximately 0.3mg of protein) were preincuhated at room temperaturein 0.9ml of medium containing the indicatedconcentrations of inhibitors andallcomponents of the carnitine palmitoyltransferase assay (method A) except substrates. Such behavior was noted previously for TG-CoA. Since all are derivatives of glycidyl-CoA we assume ultrafiltration cell, and dialyzed overnight against 5 mM potassium phosphate, pH 7.5, containing 0.5% Tween 20T. Anotherpotentand essentially irreversible inhibitor of carnitine palmitoyltransferase I is 2-bromopalmitoyl-CoA, which in the presence of same buffer containing 0.5% Tween 20, and the enzyme was eluted carnitine is believed to block the active site of the enzyme using a gradient of 5-100 mM potassium phosphate (pH 7.0), 0.5% through the self-catalyzed formation of the S-carboxypalmi-. In theabsence of added carnitine, bromopalmitoyl-CoA was much less effective; what inhibition was seen was presumably due to leakage of carnitine from the mitochondrial matrix [15].Despite the profound suppression of carnitinepalmitoyltransferase I inintactmitochondria
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