Purification and characterization of rat skeletal muscle acetyl-CoA carboxylase.
An acetyl-CoA carboxylase has been purified from rat hindlimb muscle using ammonium sulfate fractionation and avidin-Sepharose affinity chromatography. SDS/PAGE of the isolated enzyme showed a major protein band at approximately 272 kDa and a minor band at 265 kDa. The liver acetyl-CoA carboxylase gave a major protein band at 265 kDa and a minor band at 280 kDa. Adipose tissue acetyl-CoA carboxylase migrated to the 265-kDa position on the gel. Western blots performed using streptavidin-alkaline-phosphatase suggest that the bands from the three tissues contain biotin. The present study has characterized the muscle and adipose tissue enzymes under steady-state kinetics and determined Michaelis constants for the substrates. The activation constant for citrate, an essential activator for both preparations, was 2.13 +/- 0.05 mM for the muscle enzyme and 3.02 +/- 0.12 mM for adipose tissue (P < 0.01). The Km values for the muscle acetyl-CoA carboxylase compared to the adipose tissue acetyl-CoA carboxylase were: ATP, 57.6 +/- 0.9 microM compared to 106.5 +/- 2.6 microM, P < 0.01; acetyl-CoA, 31.7 +/- 1.5 microM compared to 21.5 +/- 1.0 microM, P < 0.01; bicarbonate, 2.25 +/- 0.10 mM compared to 2.73 +/- 0.29 mM, P > 0.05. The muscle acetyl-CoA carboxylase was inhibited by malonyl-CoA (Ki = 10.6 +/- 1.0 microM) and palmitoyl-CoA (Ki = 2.2 +/- 0.3 microM). These properties are consistent with the hypothesis that regulation of acetyl-CoA carboxylase plays an important role in governing the rate of fatty acid oxidation in the skeletal muscle.
Trumble
G E
GE
Department of Chemistry, Brigham Young University, Provo, Utah 84602, USA.
Smith
M A
MA
Winder
W W
WW
D000103:Acetyl-CoA Carboxylase / Q000037:antagonists & inhibitors / Q000302:isolation & purification* / Q000378:metabolism; D000818:Animals; D015153:Blotting, Western; D002951:Citrates / Q000494:pharmacology; D019343:Citric Acid; D004591:Electrophoresis, Polyacrylamide Gel; D004789:Enzyme Activation; D007700:Kinetics; D008297:Male; D008316:Malonyl Coenzyme A / Q000494:pharmacology; D018482:Muscle, Skeletal / Q000201:enzymology*; D010171:Palmitoyl Coenzyme A / Q000494:pharmacology; D051381:Rats; D017207:Rats, Sprague-Dawley; D013379:Substrate Specificity
D002951:Citrates; D010171:Palmitoyl Coenzyme A; D019343:Citric Acid; D008316:Malonyl Coenzyme A; D000103:Acetyl-CoA Carboxylase