PURIFICATION AND CHARACTERIZATION OF RAT SKELETAL-MUSCLE ACETYL-COA CARBOXYLASE

An acetyl-CoA carboxylase has been purified from rat hindlimb muscle u sing ammonium sulfate fractionation and avidin-Sepharose affinity chro matography. SDS/PAGE of the isolated enzyme showed a major protein ban d at approximately 272 kDa and a minor band at 265 kDa. The liver acet yl-CoA carboxylase gave a major protein band at 265 kDa and a minor ba nd at 280 kDa. Adipose tissue acetyl-CoA carboxylase migrated to the 2 65-kDa position on the gel. Western blots performed using streptavidin -alkaline-phosphatase suggest that the bands from the three tissues co ntain biotin. The present study has characterized the muscle and adipo se 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 th e muscle enzyme and 3.02+/-0.12 mM for adipose tissue (P<0.01). The K- m values for the muscle acetyl-CoA carboxylase compared to the adipose tissue acetyl-CoA carboxylase were: ATP, 57.6+/-0.9 mu M compared to 106.5+/-2.6 mu M, P<0.01; acetyl-CoA, 31.7+/-1.5 mu M compared to 21.5 +/-1.0 mu M, 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 malo nyl-CoA (K-i = 10.6+/-1.0 mu M) and palmitoyl-CoA (K-i = 2.2+/-0.3 mu M). These properties are consistent with the hypothesis that regulatio n of acetyl-CoA carboxylase plays an important role in governing the r ate of fatty acid oxidation in the skeletal muscle.