Quantitation by immunoblotting of the in vivo induction and subcellular distribution of hepatic acetyl-CoA carboxylase.
The in vivo induction of rat liver acetyl-CoA carboxylase (ACC) the rate-limiting enzyme of fatty acid biosynthesis, has been examined by immunoblotting, avidin blotting, and enzyme isolation. Three high-molecular-weight immunoreactive bands (Mr 220,000-260,000) were recognized in liver extracts by an anti-carboxylase polyclonal antiserum. Two bands, A and B, comigrated on sodium dodecyl sulfate polyacrylamide gels with purified acetyl-CoA carboxylase, were avidin binding, and were dramatically induced following high carbohydrate refeeding. Only band A was recognized on immunoblots using a monoclonal antibody directed against acetyl-CoA carboxylase, suggesting that band B is a proteolytic fragment in which the epitope recognized by the monoclonal antibody is absent. Following refeeding, approximately 57% of acetyl-CoA carboxylase mass (band A + band B) was present in the high-speed supernatant fraction, while 34 and 9% were in the high-speed (microsomal) and low-speed pellet fractions, respectively. Refeeding caused a large increase in total acetyl-CoA carboxylase mass, the magnitude of which differed in the various fractions. In the low-speed supernatant, a 20-fold increase in ACC mass was observed, while a 12-fold increase was seen in the high-speed supernatant. The fold increase in the high-speed pellet was even greater (greater than 27-fold). Acetyl-CoA carboxylase purified by avidin-Sepharose chromatography from fasted/refed rats had an approximate 4-fold higher Vmax and a significantly lower Ka for citrate than enzyme purified from fasted animals. The results of this study indicate that the induction of hepatic ACC that occurs during high carbohydrate refeeding of the fasted rat predominantly involves increases in enzyme content in both cytosol and microsomes, but is also accompanied by an increase in enzyme specific activity.
D000103:Acetyl-CoA Carboxylase / Q000008:administration & dosage / Q000096:biosynthesis* / Q000276:immunology; D000284:Administration, Oral; D000818:Animals; D000911:Antibodies, Monoclonal; D003101:Collodion; D004591:Electrophoresis, Polyacrylamide Gel; D004790:Enzyme Induction; D007118:Immunoassay; D008025:Ligases / Q000096:biosynthesis*; D008099:Liver / Q000166:cytology / Q000201:enzymology*; D008297:Male; D010209:Paper; D010766:Phosphorylation; D051381:Rats; D011919:Rats, Inbred Strains; D013347:Subcellular Fractions / Q000201:enzymology
D000911:Antibodies, Monoclonal; D003101:Collodion; D008025:Ligases; D000103:Acetyl-CoA Carboxylase