Reversible Phosphorylation and Inactivation of Acetyl-CoA Carboxylase from Lactating Rat Mammary Gland by Cyclic AMP-Dependent Protein Kinase

Superose 6 chromatography was used to separate rapidly the polymeric and dimeric forms of acetyl-CoA carboxylase. With preparations of acetyl-CoA carboxylase purified by Sepharose-avidin chromatography, it is shown that citrate promotes polymerization and that the extent of polymerization is diminished, but not eliminated, after phosphorylation by cyclic-AMP-dependent protein kinase. After exposure of rat epididymal adipose tissue to insulin, evidence was obtained for a marked increase in polymerization. The polymeric form, which was active in the absence of citrate, exhibited increased phosphorylation, particularly on a tryptic peptide designated the I-peptide in an earlier study [Brownsey & Denton (1982) Biochem. J. 202, 77-86]. In contrast, in tissue exposed to the beta-agonist isoprenaline, most of the phosphorylated acetyl-CoA carboxylase appeared to be in the dimeric form if chromatography was carried out in the absence of citrate, whereas in the presence of citrate the degree of polymerization was diminished.

Download Full-text

Chronic Control of Cyclic AMP-Dependent Protein Kinase of Rat Mammary Gland

Hormone and Metabolic Research ◽

10.1055/s-2007-1003358 ◽

1992 ◽

Vol 24 (09) ◽

pp. 450-451

Author(s):

R. Vernon ◽

S. Lindsay-Watt

Keyword(s):

Mammary Gland ◽

Protein Kinase ◽

Cyclic Amp ◽

Dependent Protein Kinase ◽

Dependent Protein ◽

Rat Mammary Gland

Download Full-text

Regulation of acetyl-CoA carboxylase in rat mammary gland. Effects of incubation with Ca2+, Mg2+ and ATP on enzyme activity in tissue extracts

Biochemical Journal ◽

10.1042/bj2000639 ◽

1981 ◽

Vol 200 (3) ◽

pp. 639-644 ◽

Cited By ~ 9

Author(s):

E M McNeillie ◽

R A Clegg ◽

V A Zammit

Keyword(s):

Mammary Gland ◽

Protein Kinase ◽

Cyclic Amp ◽

Kinase Inhibitor ◽

Enzyme Activation ◽

Acetyl Coa Carboxylase ◽

Acetyl Coa ◽

Carboxylase Activity ◽

Rat Mammary Gland ◽

Subsequent Activation

1. The effect of preincubation of extracts of lactating rat mammary gland with ATP, Mg2+ and micromolar concentrations of Ca2+ on the activity of acetyl-CoA carboxylase was studied. 2. Both Mg2+ and Ca2+ activated the enzyme. Activation with Mg2+ (5 mM) was larger than that with Ca2+ (calculated free Ca2+ concentration = 20-50 microM), but the activity decreased after reaching a peak. The activation obtained with Ca2+ was stable for up to 180 min. 3. Incubation with Ca2+ and Mg2+ together resulted in an activation that was slightly higher than that with Mg2+ only and was stable (compare the results for Ca2+ alone). 4. Preincubation in the absence of Mg2+, but not in the absence of Ca2+, resulted in the impairment of subsequent activation with either Mg2+ (when preincubation was with Ca2+ alone) or Mg2+ plus Ca2+. 5. KF (50 mM) prevented the activation of acetyl-CoA carboxylase by Ca2+ and Mg2+. 6. MgATP2- reversed (Mg2+ + Ca2+)-mediated activation and decreased the activity of acetyl-CoA carboxylase to about 10% of initial activity. Inhibition by ATP was unaffected by addition of cyclic AMP or cyclic AMP-dependent protein kinase inhibitor. 7. 32P was incorporated into acetyl-CoA carboxylase when incubations were carried out in the presence of [gamma-32P]ATP. Subsequent removal of ATP from the incubation medium resulted in rapid loss of 32P from acetyl-CoA carboxylase. 8. It is suggested that extracts of rat mammary gland contain endogenous protein kinase and phosphatase activities that modulate acetyl-CoA carboxylase activity through reversible phosphorylation and dephosphorylation. The phosphatase activity is sensitive to both Mg2+ and micromolar concentrations of Ca2+, whereas the kinase does not appear to be cyclic AMP-dependent.

Download Full-text

Evidence that glucagon-mediated inhibition of acetyl-CoA carboxylase in isolated adipocytes involves increased phosphorylation of the enzyme by cyclic AMP-dependent protein kinase

Biochemical Journal ◽

10.1042/bj2260139 ◽

1985 ◽

Vol 226 (1) ◽

pp. 139-145 ◽

Cited By ~ 20

Author(s):

R Holland ◽

D G Hardie ◽

R A Clegg ◽

V A Zammit

Keyword(s):

Protein Kinase ◽

Cyclic Amp ◽

Kinetic Parameters ◽

Acetyl Coa Carboxylase ◽

Response Curves ◽

Dependent Protein Kinase ◽

Acetyl Coa ◽

Dependent Protein ◽

Isolated Adipocytes

The kinetic parameters and phosphorylation state of acetyl-CoA carboxylase were analysed after purification of the enzyme by avidin-Sepharose chromatography from extracts of isolated adipocytes treated with glucagon or adrenaline. The results provide evidence that the mechanism of inhibition of acetyl-CoA carboxylase in adipocytes treated with glucagon [Zammit & Corstorphine (1982) Biochem. J. 208, 783-788] involves increased phosphorylation of the enzyme. Hormone treatment had effects on the kinetic parameters of the enzyme similar to those of phosphorylation of the enzyme in vitro by cyclic AMP-dependent protein kinase. Glucagon treatment of adipocytes led to increased phosphorylation of acetyl-CoA carboxylase in the same chymotryptic peptide as that containing the major site phosphorylated on the enzyme by purified cyclic AMP-dependent protein kinase in vitro [Munday & Hardie (1984) Eur. J. Biochem. 141, 617-627]. The dose-response curves for inhibition of enzyme activity and increased phosphorylation of the enzyme were very similar, with half-maximal effects occurring at concentrations of glucagon (0.5-1 nM) which are close to the physiological range. In general, the patterns of increased 32P-labelling of chymotryptic peptides induced by glucagon or adrenaline were similar, although there were quantitative differences between the effects of the two hormones on individual peptides. The results are discussed in terms of the possible roles of cyclic AMP-dependent and -independent protein kinases in the regulation of acetyl-CoA carboxylase activity and of lipogenesis in white adipose tissue.

Download Full-text