Acetyl-coenzyme-A carboxylase from rat liver. Subunit structure and proteolytic modification

Area of Science:

• Biochemistry
• Enzymology
• Molecular Biology

Background:

• Acetyl-CoA carboxylase (ACC) is a key enzyme in fatty acid synthesis.
• Understanding ACC's subunit structure is crucial for metabolic regulation.
• Mammalian ACC subunit structure has been debated, with varying reports.

Purpose of the Study:

• To elucidate the native subunit structure of rat liver acetyl-CoA carboxylase.
• To investigate the role of biotinylation in ACC subunit assembly.
• To determine if smaller subunits arise from proteolytic degradation.

Main Methods:

• Polyacrylamide gel electrophoresis (PAGE) in dodecylsulfate.
• [14C]biotin labeling of the enzyme.
• Proteolytic digestion studies with trypsin, chymotrypsin, and rat liver proteases.
• Immunoprecipitation from crude rat liver extracts.

Main Results:

• Purified rat liver ACC preparations showed heterogeneous subunit patterns (230 kDa, 124 kDa, 118 kDa).
• [14C]biotin was associated with the 230 kDa and 124 kDa polypeptides, but not the 118 kDa one.
• Proteolytic treatment of the 230 kDa subunit generated smaller polypeptides.
• Immunoprecipitation consistently yielded only the 230 kDa subunit, with 1 mol biotin per 237 kDa.

Conclusions:

• Rat liver ACC exists as a single, heavy subunit (230 kDa) containing one biotin molecule.
• Observed smaller subunits are likely artifacts of proteolytic modification during purification.
• Mammalian ACC exhibits a highly integrated subunit structure, distinct from prokaryotic and plant forms.