scholarly journals Coenzyme A is a potent inhibitor of acetyl-CoA carboxylase from rat epididymal fat-pads

1992 ◽  
Vol 283 (1) ◽  
pp. 35-38 ◽  
Author(s):  
S K Moule ◽  
N J Edgell ◽  
A C Borthwick ◽  
R M Denton
Keyword(s):  
Potent Inhibitor ◽  
Ion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Acetyl Coa Carboxylase ◽  
Inhibitory Effects ◽  
Fat Pad ◽  
Maximal Inhibition ◽  
Acetyl Coa ◽  
Epididymal Fat ◽  
Fat Pads

Rat epididymal fat-pad extracts have previously been shown to contain an insulin-stimulated acetyl-CoA carboxylase kinase, which is co-eluted from Mono Q ion-exchange chromatography with a potent inhibitor of acetyl-CoA carboxylase [Borthwick, Edgell & Denton (1990) Biochem. J. 270, 795-801]. A variety of tests, including reactivity with thiol reagents, identify this inhibitor as CoA. Inhibition requires the presence of MgATP, but is independent of any phosphorylation of the enzyme. The effect is complete in about 5 min and is associated with depolymerization of acetyl-CoA carboxylase. Half-maximal inhibition is observed at about 40 nM-CoA. The inhibitory effects of CoA can be partially reversed by incubation with citrate and more fully overcome by treatment of the enzyme with the insulin-stimulated acetyl-CoA carboxylase kinase.

scholarly journals Effects of protein phosphatase inhibitors on the regulation of insulin-sensitive enzymes within rat epididymal fat-pads and cells

1991 ◽  
Vol 276 (3) ◽  
pp. 649-654 ◽  
Author(s):  
G A Rutter ◽  
A C Borthwick ◽  
R M Denton
Keyword(s):  
Okadaic Acid ◽  
Pyruvate Dehydrogenase ◽  
Protein Phosphatase ◽  
Acid Treatment ◽  
Acetyl Coa Carboxylase ◽  
Acetyl Coa ◽  
Phosphatase Inhibitors ◽  
Epididymal Fat ◽  
Protein Phosphatase Inhibitors ◽  
Fat Pads

1. The effects of the protein phosphatase inhibitors okadaic acid and microcystin LR on the regulation by insulin of pyruvate dehydrogenase and acetyl-CoA carboxylase have been studied in rat epididymal fat-pads and isolated cells. These inhibitors both completely blocked the phosphatase activity (against phosphorylase a) present in extracts of epididymal fat-pads, with half-maximal effects in the nanomolar range. 2. Okadaic acid treatment of pads and cells lowered the activity of acetyl-CoA carboxylase assayed in tissue extracts, both before and after treatment of the extracts with the activator, citrate. Further, okadaic acid treatment abolished the 2-3-fold difference in activity observed between extracts from control and insulin-treated tissues, assayed without prior treatment with citrate. 3. Incubation of pads with [32P]Pi, sufficient to label the intracellular pool of ATP, demonstrated that okadaic acid increased the overall phosphorylation of acetyl-CoA carboxylase on a number of distinct sites, as judged by two-dimensional mapping of tryptic peptides. These included the ‘I-peptide’ [Brownsey & Denton (1982) Biochem. J. 202, 77-86], the phosphorylation of which may be associated with the stimulation of the activity of the enzyme by insulin, as well as inhibitory phosphorylation sites. 4. Incubation with 1 microM-okadaic acid had no effect on the basal level of active pyruvate dehydrogenase apparent after tissue extraction, but abolished the 2-3-fold increase in this parameter which was elicited by insulin in the absence of okadaic acid. However, okadaic acid treatment did not affect the persistent increase in active pyruvate dehydrogenase levels which was apparent in mitochondria subsequently isolated from insulin-treated pads and re-incubated with an oxidizable substrate. It is concluded that the effects of okadaic acid are exerted through changes in metabolite concentrations rather than some direct action on the signalling pathway whereby insulin stimulates pyruvate dehydrogenase. 5. Microcystin LR did not mimic the effects of okadaic acid on intact cells and pads described above.

scholarly journals Evidence for a protein regulator from rat liver which activates acetyl-CoA carboxylase

1993 ◽  
Vol 292 (1) ◽  
pp. 75-84 ◽  
Author(s):  
K A Quayle ◽  
R M Denton ◽  
R W Brownsey
Keyword(s):  
Ion Exchange ◽  
Rat Liver ◽  
High Speed ◽  
Deae Cellulose ◽  
Ion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Size Exclusion ◽  
Acetyl Coa Carboxylase ◽  
Acetyl Coa ◽  
Carboxylase Activity

1. A regulator of acetyl-CoA carboxylase has been identified in high-speed supernatant fractions from rat liver. The regulator was found to activate highly purified acetyl-CoA carboxylase 2-3-fold at physiological citrate concentrations (0.1-0.5 mM). The effects of the regulator on acetyl-CoA carboxylase activity were dose-dependent, and half-maximal activation occurred in 7-8 min at 30 degrees C. 2. The acetyl-CoA carboxylase regulator was non-dialysable and was inactivated by heating or by exposure to carboxypeptidase. The regulator was enriched from rat liver cytosol by first removing the endogenous acetyl-CoA carboxylase and then using a combination of purification steps, including (NH4)2SO4 precipitation, ion-exchange chromatography and size-exclusion chromatography. The regulator activity appeared to be a protein with a molecular mass of approx. 75 kDa, which could be eluted from mono-Q with approx. 0.35 M KCl as a single peak of activity. 3. Studies of the effects of the regulator on phosphorylation or subunit size of acetyl-CoA carboxylase indicated that the changes in enzyme activity are most unlikely to be explained by dephosphorylation or by proteolytic cleavage. 4. The regulator co-migrates with acetyl-CoA carboxylase through several purification steps, including ion-exchange chromatography and precipitation with (NH4)2SO4; however, the proteins may be separated by Sepharose-avidin chromatography, and the association between the proteins is also disrupted by addition of avidin in solution. Furthermore, the binding of the regulator itself to DEAE-cellulose is altered by the presence of acetyl-CoA carboxylase. Taken together, these observations suggest that the effects of the regulator on acetyl-CoA carboxylase may be explained by direct protein-protein interaction in vitro.

scholarly journals Inhibition of acetyl-CoA carboxylase activity in isolated rat adipocytes incubated with glucagon. Interactions with the effects of insulin, adrenaline and adenosine deaminase

1982 ◽  
Vol 208 (3) ◽  
pp. 783-788 ◽  
Author(s):  
Victor A. Zammit ◽  
Clark G. Corstorphine
Keyword(s):  
Adenosine Deaminase ◽  
Acetyl Coa Carboxylase ◽  
Initial Activity ◽  
Maximal Inhibition ◽  
Acetyl Coa ◽  
Carboxylase Activity ◽  
Control Activity ◽  
Fat Pads ◽  
Isolated Rat

1. Adipocytes isolated from epididymal fat-pads of fed rats were incubated with different concentrations of glucagon, insulin, adrenaline and adenosine deaminase, and the effects of these agents on the ‘initial’ activity of acetyl-CoA carboxylase in the cells were studied. 2. Glucagon (at concentrations between 0.1 and 10nm) inhibited acetyl-CoA carboxylase activity. Maximal inhibition was approx. 70% of the ‘control’ activity in the absence of added hormone, and the concentration of hormone required for half-maximal inhibition was 0.3–0.5nm-glucagon. 3. Incubation of cells with adenosine deaminase resulted in a similar inhibition of acetyl-CoA carboxylase activity. Preincubation of adipocytes with adenosine deaminase did not alter either the sensitivity of carboxylase activity to increasing concentrations of glucagon or the maximal extent of inhibition. 4. Adrenaline inhibited acetyl-CoA carboxylase to the same extent as glucagon. Preincubation of the cells with glucagon did not alter the sensitivity of enzyme activity to adrenaline or the degree of maximal inhibition. 5. Insulin activated the enzyme by 70–80% of ‘control’ activity. Preincubation of the cells with glucagon did not alter the concentration of insulin required to produce half the maximal stimulatory effect (about 12μunits of insulin/ml). The effects of insulin and glucagon appeared to be mediated completely independently, and were approximately quantitatively similar but opposite. These characteristics resulted in the mutual cancellation of the effects of the two hormones when they were both present at equally effective concentrations. 6. The implications of these findings with regard to current concepts about the mechanism of regulation of acetyl-CoA carboxylase and to the regulation of the enzyme in vivo are discussed.

scholarly journals Rat epididymal adipose tissue acetyl-CoA carboxylase

1970 ◽  
Vol 48 (8) ◽  
pp. 915-921 ◽  
Author(s):  
P. R. Desjardins ◽  
K. Dakshinamurti
Keyword(s):  
Adipose Tissue ◽  
Epididymal Adipose Tissue ◽  
Acetyl Coa Carboxylase ◽  
Acetyl Coa ◽  
Ph Optimum ◽  
Epididymal Fat ◽  
Ph Dependent ◽  
Rat Adipose Tissue ◽  
Cold Inactivation ◽  
Fat Pads

The properties of a partially purified acetyl-CoA carboxylase (acetyl-CoA:CO2 ligase (ADP), EC 6.4.1.2) from rat epididymal fat pads have been studied. The properties of the rat adipose tissue enzyme are similar to those of the liver in regard to the pH optimum and affinity for substrates and inhibitors. The rat adipose tissue carboxylase shows a pH-dependent, reversible cold inactivation.

The inhibitory effects of some adenosine nucleolipids on the lipolysis in rat epididymal fat pads in vitro

1979 ◽  
Vol 44 (5) ◽  
pp. 1651-1656 ◽  
Author(s):  
Sixtus Hynie ◽  
Jiří Smrt
Keyword(s):  
Fatty Acids ◽  
Cyclic Amp ◽  
Dibutyryl Cyclic Amp ◽  
Inhibitory Effects ◽  
Epididymal Fat ◽  
Fat Pads

3'-Oleolyl-2,3-dihydroxypropyl-AMP, 3'-stearoyl-2,3-dihydroxypropyl-AMP, octadecyl-AMP and palmitamidoethyl-AMP inhibited in comparison with adenosine or fatty acids much stronger the lipolysis in rat epididymal fat pads in vitro stimulated by isoproterenol, theophylline and dibutyryl cyclic AMP. The inhibition of the effects of the two latter drugs suggest that the described effect is caused not only by the inhibition of the cyclic AMP production but also by the inhibition of its effect on the following steps in process of lipolysis.

scholarly journals Expression, biotinylation and purification of a biotin-domain peptide from the biotin carboxy carrier protein of Escherichia coli acetyl-CoA carboxylase

1994 ◽  
Vol 302 (3) ◽  
pp. 881-887 ◽  
Author(s):  
A Chapman-Smith ◽  
D L Turner ◽  
J E Cronan ◽  
T W Morris ◽  
J C Wallace
Keyword(s):  
Escherichia Coli ◽  
Exchange Chromatography ◽  
Carrier Protein ◽  
Acetyl Coa Carboxylase ◽  
Acetyl Coa ◽  
E Coli ◽  
Biotin Ligase ◽  
Domain Peptide

A protein segment consisting of the C-terminal 87 residues of the biotin carboxy carrier protein from Escherichia coli acetyl-CoA carboxylase was overexpressed in E. coli. The expressed biotin-domain peptide can be fully biotinylated by coexpression with a plasmid that overproduces E. coli biotin ligase. The extent of biotinylation was limited in vivo, but could be taken to completion in cell lysates on addition of ATP and biotin. We used the coexpression of biotin ligase and acceptor protein to label the biotin-domain peptide in vitro with [3H]biotin, which greatly facilitated development of a purification procedure. The apo (unbiotinylated) form of the protein was prepared by induction of biotin-domain expression in a strain lacking the biotin-ligase-overproduction plasmid. The apo domain could be separated from the biotinylated protein by ion-exchange chromatography or non-denaturing PAGE, and was converted into the biotinylated form of the peptide on addition of purified biotin ligase. The identify of the purified biotin-domain peptide was confirmed by N-terminal sequence analysis, amino acid analysis and m.s. The domain was readily produced and purified in sufficient quantities for n.m.r. structural analysis.

scholarly journals Hormonal regulation of adipose-tissue acetyl-coenzyme A carboxylase by changes in the polymeric state of the enzyme. The role of long-chain fatty acyl-coenzyme A thioesters and citrate

1974 ◽  
Vol 142 (2) ◽  
pp. 365-377 ◽  
Author(s):  
Andrew P. Halestrap ◽  
Richard M. Denton
Keyword(s):  
Enzyme Activity ◽  
Coenzyme A ◽  
Total Activity ◽  
Fatty Acyl ◽  
Acetyl Coa Carboxylase ◽  
Initial Activity ◽  
Acetyl Coa ◽  
Coa Thioesters ◽  
Fat Pads

1. Acetyl-CoA carboxylase activity was measured in extracts of rat epididymal fat-pads either on preparation of the extracts (initial activity) or after incubation of the extracts with citrate (total activity). In the presence of glucose or fructose, brief exposure of pads to insulin increased the initial activity of acetyl-CoA carboxylase; no increase occurred in the absence of substrate. Adrenaline in the presence of glucose and insulin decreased the initial activity. None of these treatments led to a substantial change in the total activity of acetyl-CoA carboxylase. A large decrease in the initial activity of acetyl-CoA carboxylase also occurred with fat-pads obtained from rats that had been starved for 36h although the total activity was little changed by this treatment. 2. Conditions of high-speed centrifugation were found which appear to permit the separation of the polymeric and protomeric forms of the enzyme in fat-pad extracts. After the exposure of the fat-pads to insulin (in the presence of glucose), the proportion of the enzyme in the polymeric form was increased, whereas exposure to adrenaline (in the presence of glucose and insulin) led to a decrease in enzyme activity. 3. These changes are consistent with a role of citrate (as activator) or fatty acyl-CoA thioesters (as inhibitors) in the regulation of the enzyme by insulin and adrenaline; no evidence that the effects of these hormones involve phosphorylation or dephosphorylation of the enzyme could be found. 4. Changes in the whole tissue concentration of citrate and fatty acyl-CoA thioesters were compared with changes in the initial activity of acetyl-CoA carboxylase under a variety of conditions of incubation. No correlation between the citrate concentration and the initial enzyme activity was evident under any condition studied. Except in fat-pads which were exposed to insulin there was little inverse correlation between the concentration in the tissue of fatty acyl-CoA thioesters and the initial activity of acetyl-CoA carboxylase. 5. It is suggested that changes in the concentration of free fatty acyl-CoA thioesters (which may not be reflected in whole tissue concentrations of these metabolites) may be important in the regulation of the activity of acetyl-CoA carboxylase. The possibility is discussed that the concentration of free fatty acyl-CoA thioesters may be controlled by binding to a specific protein with properties similar to albumin.

Properties of a phosphoprotein phosphatase from rat epididymal fat pads: deactivation of hormone-sensitive triglyceride lipase and activation of glycogen synthase in adipose tissue

1980 ◽  
Vol 58 (3) ◽  
pp. 243-250 ◽  
Author(s):  
David L. Severson ◽  
Shellie Sloan
Keyword(s):  
Adipose Tissue ◽  
Phosphatase Activity ◽  
Glycogen Synthase ◽  
Adenine Nucleotides ◽  
Fat Pad ◽  
Phosphoprotein Phosphatase ◽  
Triglyceride Lipase ◽  
Epididymal Fat ◽  
Hormone Sensitive ◽  
Fat Pads

A phosphoprotein phosphatase has been partially purified from rat epididymal fat pads by a procedure utilizing ammonium sulfate and ethanol precipitations and chromatography on DEAE-Sephadex A-50. The phosphatase was eluted from Sephadex G-75 columns with an apparent molecular weight of 28 000. The phosphoprotein phosphatase catalyzed the reversible deactivation of protein kinase activated chicken adipose tissue hormone-sensitive triglyceride lipase. Phosphatase activity measured with activated triglyceride lipase as substrate was completely dependent upon the presence of metal ions (Mg2+, Ca2+, or Mn2+) and was inhibited by inorganic phosphate and adenine nucleotides. The fat pad phosphatase increased the rate of activation of glycogen synthase in rat adipose tissue infranatant fractions from fed and 24-h-fasted rats but had little or no effect on synthase activity in infranatant fractions from rats fasted for 48 h. Fasting had no effect on rat fat pad phosphatase activity measured with triglyceride lipase as substrate, but phosphatase activity was decreased in preparations from diabetic rats.

scholarly journals Demonstration of the phosphorylation of acetyl-coenzyme A carboxylase within intact rat epididymal fat-cells

1977 ◽  
Vol 168 (3) ◽  
pp. 441-445 ◽  
Author(s):  
R W Brownsey ◽  
W A Hughes ◽  
R M Denton
Keyword(s):  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Fat Cells ◽  
Acetyl Coa Carboxylase ◽  
Acetyl Coa ◽  
Acetyl Coenzyme A ◽  
Phosphorylated Proteins ◽  
Epididymal Fat ◽  
Intracellular Proteins ◽  
Acetyl Coenzyme A Carboxylase

Intact rat epididymal fat-cells were incubated with 32Pi and the intracellular proteins separated by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. One of the phosphorylated proteins has the same RF value as [14C]biotin-labelled acetyl-CoA carboxylase purified from fat-cells and is specifically precipitated after incubation with antiserum raised against acetyl-CoA carboxylase. No significant changes in the extent of phosphorylation of acetyl-CoA carboxylase were detected after exposure of the cells to insulin.

scholarly journals A unified molecular mechanism for the regulation of acetyl-CoA carboxylase by phosphorylation

2016 ◽  
Vol 2 (1) ◽  
Author(s):  
Jia Wei ◽  
Yixiao Zhang ◽  
Tai-Yuan Yu ◽  
Kianoush Sadre-Bazzaz ◽  
Michael J Rudolph ◽  
...  
Keyword(s):  
Drug Discovery ◽  
Molecular Mechanism ◽  
Conformational Changes ◽  
Potent Inhibitor ◽  
Acetyl Coa Carboxylase ◽  
Single Chain ◽  
Electron Microscopic ◽  
Acetyl Coa ◽  
Microscopic Studies ◽  
And Function

Abstract Acetyl-CoA carboxylases (ACCs) are crucial metabolic enzymes and attractive targets for drug discovery. Eukaryotic acetyl-CoA carboxylases are 250 kDa single-chain, multi-domain enzymes and function as dimers and higher oligomers. Their catalytic activity is tightly regulated by phosphorylation and other means. Here we show that yeast ACC is directly phosphorylated by the protein kinase SNF1 at residue Ser1157, which potently inhibits the enzyme. Crystal structure of three ACC central domains (AC3–AC5) shows that the phosphorylated Ser1157 is recognized by Arg1173, Arg1260, Tyr1113 and Ser1159. The R1173A/R1260A double mutant is insensitive to SNF1, confirming that this binding site is crucial for regulation. Electron microscopic studies reveal dramatic conformational changes in the holoenzyme upon phosphorylation, likely owing to the dissociation of the biotin carboxylase domain dimer. The observations support a unified molecular mechanism for the regulation of ACC by phosphorylation as well as by the natural product soraphen A, a potent inhibitor of eukaryotic ACC. These molecular insights enhance our understanding of acetyl-CoA carboxylase regulation and provide a basis for drug discovery.

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