Isolation and characterization of a novel acetyl-CoA carboxylase kinase from rat liver.
Acetyl-CoA carboxylase is regulated allosterically by citrate and covalently by a phosphorylation/dephosphorylation mechanism. We have isolated and purified from rat livers a novel kinase that phosphorylates and inactivates the carboxylase. This kinase is bound to the carboxylase and can be eluted in salt-rich solution. The native kinase exists as high molecular weight aggregates of a subunit that has a molecular weight of 40,000. The phosphorylation sites of the carboxylase were determined after tryptic and cyanogen bromide digestions of 32P-labeled carboxylase and separation of the peptides by various chromatographic procedures. Amino acid analyses of the phosphopeptides showed that the Ser77 and Ser1200 residues were the sites of phosphorylation. Treating the phosphorylated carboxylase with the Mn(2+)-dependent acetyl-CoA carboxylase phosphatase 2 removed the phosphate and reactivated the carboxylase. These results suggest that both this kinase and the acetyl-CoA carboxylase phosphatase 2 act at the same site(s) in the acetyl-CoA carboxylase molecule. Citrate dramatically inhibits the kinase-mediated phosphorylation of the carboxylase, suggesting that the allosteric modification and activation by citrate render the phosphorylation sites inaccessible to the kinase and therefore maintain high carboxylase activity. This observation indicates that there is a close interplay between the citrate effect on and phosphorylation of the carboxylase in regulating its activity.
Mohamed
A H
AH
Verna and Marrs McLean Department of Biochemistry, Baylor College of Medicine, Houston, Texas 77030.
Huang
W Y
WY
Huang
W
W
Venkatachalam
K V
KV
Wakil
S J
SJ
D000103:Acetyl-CoA Carboxylase / Q000302:isolation & purification / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D002846:Chromatography, Affinity; D002852:Chromatography, Ion Exchange; D004591:Electrophoresis, Polyacrylamide Gel; D005260:Female; D007700:Kinetics; D008099:Liver / Q000201:enzymology*; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010449:Peptide Mapping; D010748:Phosphopeptides / Q000737:chemistry / Q000302:isolation & purification; D010768:Phosphoserine / Q000032:analysis; D011494:Protein Kinases / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism*; D051381:Rats; D017207:Rats, Sprague-Dawley
D010748:Phosphopeptides; D010768:Phosphoserine; D011494:Protein Kinases; C033288:(acetyl-CoA carboxylase) kinase; D000103:Acetyl-CoA Carboxylase