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KMID : 0371919900030010043
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Journal of Wonju College of Medicine
1990 Volume.3 No. 1 p.43 ~ p.56
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Abstract
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On the contrary to the report that acetyl CoA carboxylase is a single polypeptide of MW 220,000 ~260,000 that contains 2~6 moles of phosphate and 1 mole of biotin, the enzyme has been suggested to have MWs of 220,000~260,000 composed of 2 subunits with MWs of 123,000 and 108,000. It has been known that acetyl CoA carboxylase activity is stimulated by citrate or isocitrate temporarily and also regulated by nutritional and hormonal state for a long term in vivo. It has been reported that acetyl CoA carboxylase activity was stimulated ty trypsin treatment or by the prolonged storage in the refrigerator as well as citrate or is?citrate in vitro. In the present study, acetyl CoA carboxylase was purified from the liver of rats refed with a fat free high carbohydrate diet after the 3 days fasting, through polyethylene glycol, ammonium sulfate precipitation, Sepharose 2B gel filtration and DEAE-Sephacel column chromatography, and the MWs of subunits and the change in the enzyme activity were measured.
Acetyl CoA carboxylase purified 1,522 folds from the liver of rats fasted for 3 days then by refed with a fat free high carbohydrate diet and had a specific activity of 3.88 Units/mg protein. SDS-polyacrylamide gel electrophoresis of this enzyme showed three protein bands corresponding MWs of 220,000, 120,000, and 110,000. Specific activity of acetyl CoA carboxylase obtained after the DEAE- Sephacel column chromatography was 30 Units/mg protein and the enzyme was purified 12,000 folds.
Upon SDS-polyacrylamide gel electrophoresis, only two protein bands corresponding MWs of 120,000 and 110,000 were appeared with the disappearance of a protein band having MW of 220,000, indicating that proteolytic hydrolysis of the enzyme into two subunits during purification. The reason for the dramatic increase of the enzyme activity by proteolysis is not known.
Ouchterlony double immunodiffusion between rabbit antiserum against acetyl CoA carboxylase prepared through Sepharose 2B gel filtration step and 3% polyethylene glycol extract of the 20,000 xg supernatant of liver homogenate was carried out, 15㎖ of antiserum cross-reacted with liver homogenate and formed a sharp hexagonal precipitin. Addition of IgG prepared from the antiserum by protein A Sepharose column chromatography to the 5% polyethylene glycol precipitate of liver homogenate inhibited the acetyl CoA carboxylase activity in parallel to the IgG concentration indicating the presence of antibody against acetyl CoA carboxylase in the IgG.
Treatment of 0.625, 1.25 and 1.875 ㎍ of trypsin to 1 ㎎ of partially purified acetyl CoA carboxylase resulted in the increase of the enzyme activity in the order of 375,282 and 178% respectively. The inhibition of acetyl CoA carboxylase by trypsin (1.5 ㎍/㎎ protein) was dependent on the incubation time indicating that the extensive hydrolysis of the enzyme by trypsin decreased the enzyme activity.
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