scholarly journals Kinetic studies on two isoforms of acetyl-CoA carboxylase from maize leaves

1996 ◽  
Vol 318 (3) ◽  
pp. 997-1006 ◽  
Author(s):  
Derek HERBERT ◽  
Lindsey J PRICE ◽  
Claude ALBAN ◽  
Laure DEHAYE ◽  
Dominique JOB ◽  
...  
Keyword(s):  
Kinetic Studies ◽  
Product Inhibition ◽  
Hill Equation ◽  
Acetyl Coa Carboxylase ◽  
Acetyl Coa ◽  
Maize Leaves ◽  
Ic50 Values ◽  
A Minor ◽  
The Hill

The steady-state kinetics of two multifunctional isoforms of acetyl-CoA carboxylase (ACCase) from maize leaves (a major isoform, ACCase1 and a minor isoform, ACCase2) have been investigated with respect to reaction mechanism, inhibition by two graminicides of the aryloxyphenoxypropionate class (quizalofop and fluazifop) and some cellular metabolites. Substrate interaction and product inhibition patterns indicated that ADP and Pi products from the first partial reaction were not released before acetyl-CoA bound to the enzymes. Product inhibition patterns did not match exactly those predicted for an ordered Ter Ter or a random Ter Ter mechanism, but were close to those postulated for an ordered mechanism. ACCase2 was about 1/2000 as sensitive as ACCase1 to quizalofop but only about 1/150 as sensitive to fluazifop. Fitting inhibition data to the Hill equation indicated that binding of quizalofop or fluazifop to ACCase1 was non-cooperative, as shown by the Hill constant (napp) values of 0.86 and 1.16 for quizalofop and fluazifop respectively. Apparent inhibition constant values (K´ from the Hill equation) for ACCase1 were 0.054 µM for quizalofop and 21.8 µM for fluazifop. On the other hand, binding of quizalofop or fluazifop to ACCase2 exhibited positive co-operativity, as shown by the napp values of 1.85 and 1.59 for quizalofop and fluazifop respectively. K´ values for ACCase2 were 1.7 mM for quizalofop and 140 mM for fluazifop. Kinetic parameters for the co-operative binding of quizalofop to maize ACCase2 were close to those of another multifunctional ACCase of limited sensitivity to graminicide, ACC220 from pea. Inhibition of ACCase1 by quizalofop was mixed-type with respect to acetyl-CoA or ATP, but the concentration of acetyl-CoA had the greater effect on the level of inhibition. Neither ACCase1 nor ACCase2 was appreciably sensitive to CoA esters of palmitic acid (16:0) or oleic acid (18:1). Approximate IC50 values were 10 µM (ACCase2) and 50 µM (ACCase1) for both CoA esters. Citrate concentrations up to 1 mM had no effect on ACCase1 activity. Above this concentration, citrate was inhibitory. ACCase2 activity was slightly stimulated by citrate over a broad concentration range (0.25–10 mM). The significance of possible effects of acyl-CoAs or citrate in vivo is discussed.

scholarly journals Polymer-protomer transition of acetyl-CoA carboxylase occurs in vivo and varies with nutritional conditions.

1980 ◽  
Vol 255 (21) ◽  
pp. 10033-10035
Author(s):  
B.A. Ashcraft ◽  
W.S. Fillers ◽  
S.L. Augustine ◽  
S.D. Clarke
Keyword(s):  
Acetyl Coa Carboxylase ◽  
Acetyl Coa ◽  
Nutritional Conditions

scholarly journals Regulation of acetyl-CoA carboxylase in rat mammary gland. Effects of starvation and of insulin and prolactin deficiency on the fraction of the enzyme in the active form in vivo

1982 ◽  
Vol 204 (1) ◽  
pp. 273-280 ◽  
Author(s):  
Elizabeth M. McNeillie ◽  
Victor A. Zammit
Keyword(s):  
Mammary Gland ◽  
Enzyme Activities ◽  
Enzyme Concentration ◽  
Acetyl Coa Carboxylase ◽  
Initial Activity ◽  
Acetyl Coa ◽  
Activity Ratios ◽  
Rat Mammary Gland ◽  
Rate Limiting

The ‘initial’ (I), endogenous phosphatase-activated (A) and citrate-activated (C) activities of acetyl-CoA carboxylase were measured in mammary-gland extracts of pregnant and lactating rats. There was a 10-fold increase in the A and C enzyme activities in the transition from early to peak lactation [cf. data of Mackall & Lane (1977) Biochem. J.162, 635–642], but there was no significant increase in the ratio of the initial activity to the A and C activities of the enzyme. Starvation (24h) or short-term (3h) streptozotocin-induced diabetes both resulted in a 40% decrease in I/A and I/C activity ratios. In starvation this was accompanied by a decrease in the absolute values of the A and C activities such that the initial activity in mammary glands of starved animals was 45% that in glands from fed animals. Insulin treatment of starved or diabetic animals 60min before killing increased the I activity without affecting the A or C enzyme activities. Removal of the pups for 24h from animals in peak lactation (weaning) resulted in a marked but similar decrease in all three activities such that, although the initial activity was only 10% of that in suckled animals, the I/A and I/C activity ratios remained high and unaltered. Inhibition of prolactin secretion by injection of 2-bromo-α-ergocryptine gave qualitatively similar results to those during weaning. Simultaneous administration of ovine prolactin completely prevented the effects of bromoergocryptine. It is suggested that the initial activity of acetyl-CoA carboxylase in rat mammary gland is regulated by at least two parallel mechanisms: (i) an acute regulation of the proportion of the enzyme in the active state and (ii) a longer-term modulation of enzyme concentration in the gland. Insulin appeared to mediate its acute effects through mechanism (i), whereas prolactin had longer-term effects on enzyme concentration in the gland. A comparison of initial enzyme activities (I) obtained in the present study with rates of lipogenesis measured in vivo [Agius & Williamson (1980) Biochem. J.192, 361–364; Munday & Williamson (1981) Biochem. J.196, 831–837] gave good agreement between the two sets of data for all conditions studied except for 24h-starved and streptozotocin-diabetic animals. It is suggested that acetyl-CoA carboxylase activity is rate-limiting for lipogenesis in the mammary gland in normal, fed, suckled or weaned animals but that in starved and short-term diabetic animals changes in the activity of the enzyme by covalent modification alone may not be sufficient to maintain the enzyme in its rate-limiting role.

scholarly journals P5375A default in acetyl-CoA carboxylase phosphorylation increases thrombus growth in vitro and in vivo, and in a collagen-dependent manner

2017 ◽  
Vol 38 (suppl_1) ◽  
Author(s):  
S. Lepropre ◽  
S. Kautbally ◽  
L. Bertrand ◽  
G.R. Steinberg ◽  
B.E. Kemp ◽  
...  
Keyword(s):  
Dependent Manner ◽  
Acetyl Coa Carboxylase ◽  
Acetyl Coa

Abstract 3560: Use of Hyperpolarized Magnetic Resonance for Non-Invasive Observation of Metabolic Regulation

Circulation ◽  
2008 ◽  
Vol 118 (suppl_18) ◽  
Author(s):  
Marie A Schroeder ◽  
Lisa C Heather ◽  
Helen J Atherton ◽  
Kieran Clarke ◽  
George K Radda ◽  
...  
Keyword(s):  
Magnetic Resonance ◽  
Metabolic Regulation ◽  
Krebs Cycle ◽  
Enzyme Regulation ◽  
Product Inhibition ◽  
Acetyl Coa ◽  
Fed State ◽  
Fasted Rats ◽  
Pyruvate Ratio

Hyperpolarized magnetic resonance (HP MR) has enabled real time visualization of in vivo metabolism. In this study, we postulated that HP MR could also non-invasively provide a measure of metabolic regulation. We focused on regulation of pyruvate dehydrogenase (PDH), a highly controlled enzyme that catalyzes the oxidation of pyruvate to acetyl CoA and CO2/HCO3-. We compared PDH flux in conditions of normal and attenuated enzyme activity, and in the presence of normal and augmented Krebs cycle flux, to determine the contributions of PDH activity and end product inhibition to enzyme regulation. Six rats were examined in the fed and fasted states (to modulate PDH activity), with 40 μmol HP 13C1-pyruvate alone and 40 μmol HP pyruvate co-infused with 40 μmol malate (to manipulate Krebs cycle flux/acetyl CoA uptake). HP tracer was infused into the rats in an MR scanner and cardiac spectra were acquired every second for 1 min. Conversion of pyruvate to 13HCO3-was monitored and the 13HCO3-/pyruvate ratio was used as a marker of PDH flux. Infusion of malate increased PDH flux by 31% compared with pyruvate alone, indicating that removal of acetyl CoA by incorporation into the Krebs cycle increased PDH flux. PDH flux was 57% lower in fasted rats injected with pyruvate alone compared with fed rats, and did not change with malate co-infusion. Here, low PDH activity prevented additional enzyme flux. These results suggest that end product inhibition limits fed state PDH flux, whereas PDH activity regulates pyruvate oxidation in the fasted state. In conclusion, this study has provided evidence that HP MR may be useful to obtain details of metabolic regulation, rather than just reflecting metabolic state. Figure 1 Bicarbonate/pyruvate ratio in fed and fasted rats, following an injection of pyruate or pyruate plus malate. In fed rats, co-infusion of malate increased PDH flux by 31% compared with injection of pyruvate alone (*p=0.02). Fasting reduced PDH flux by 57% (**p=0.002) following injection of pyruvate alone. Co-infusion with malate did not affect PDH flux in fasted rats.

scholarly journals RNA-seq reveals novel mechanistic targets of withaferin A in prostate cancer cells

2020 ◽  
Vol 41 (6) ◽  
pp. 778-789 ◽  
Author(s):  
Su-Hyeong Kim ◽  
Eun-Ryeong Hahm ◽  
Krishna B Singh ◽  
Sruti Shiva ◽  
Jacob Stewart-Ornstein ◽  
...  
Keyword(s):  
Prostate Cancer ◽  
Fatty Acid ◽  
Fatty Acid Synthase ◽  
Acid Synthesis ◽  
Acetyl Coa Carboxylase ◽  
Rna Seq ◽  
Atp Citrate Lyase ◽  
Acetyl Coa ◽  
Citrate Lyase

Abstract Withaferin A (WA) is a promising phytochemical exhibiting in vitro and in vivo anticancer activities against prostate and other cancers, but the mechanism of its action is not fully understood. In this study, we performed RNA-seq analysis using 22Rv1 human prostate cancer cell line to identify mechanistic targets of WA. Kyoto Encyclopedia of Genes and Genomes pathway analysis of the differentially expressed genes showed most significant enrichment of genes associated with metabolism. These results were validated using LNCaP and 22Rv1 human prostate cancer cells and Hi-Myc transgenic mice as models. The intracellular levels of acetyl-CoA, total free fatty acids and neutral lipids were decreased significantly following WA treatment in both cells, which was accompanied by downregulation of mRNA (confirmed by quantitative reverse transcription-polymerase chain reaction) and protein levels of key fatty acid synthesis enzymes, including ATP citrate lyase, acetyl-CoA carboxylase 1, fatty acid synthase and carnitine palmitoyltransferase 1A. Ectopic expression of c-Myc, but not constitutively active Akt, conferred a marked protection against WA-mediated suppression of acetyl-CoA carboxylase 1 and fatty acid synthase protein expression, and clonogenic cell survival. WA was a superior inhibitor of cell proliferation and fatty acid synthesis in comparison with known modulators of fatty acid metabolism including cerulenin and etomoxir. Intraperitoneal WA administration to Hi-Myc transgenic mice (0.1 mg/mouse, three times/week for 5 weeks) also resulted in a significant decrease in circulating levels of total free fatty acids and phospholipids, and expression of ATP citrate lyase, acetyl-CoA carboxylase 1, fatty acid synthase and carnitine palmitoyltransferase 1A proteins in the prostate in vivo.

scholarly journals Localization and characterization of two structurally different forms of acetyl-CoA carboxylase in young pea leaves, of which one is sensitive to aryloxyphenoxypropionate herbicides

1994 ◽  
Vol 300 (2) ◽  
pp. 557-565 ◽  
Author(s):  
C Alban ◽  
P Baldet ◽  
R Douce
Keyword(s):  
Molecular Mass ◽  
Subunit Structure ◽  
Acetyl Coa Carboxylase ◽  
Leaf Extracts ◽  
Acetyl Coa ◽  
Native Molecular Mass ◽  
Molecular Masses ◽  
Minor Form ◽  
A Minor

Young pea leaves contain two structurally different forms of acetyl-CoA carboxylase (EC 6.4.1.2; ACCase). A minor form, which accounted for about 20% of the total ACCase activity in the whole leaf, was detected in the epidermal tissue. This enzyme was soluble and was purified to homogeneity from young pea leaf extracts. It consisted of a dimer of two identical biotinyl subunits of molecular mass 220 kDa. In this respect, this multifunctional enzyme was comparable with that described in other plants and in other eukaryotes. A predominant form was present in both the epidermal and mesophyll tissues. In mesophyll protoplasts, ACCase was detected exclusively in the soluble phase of chloroplasts. This enzyme was partially purified from pea chloroplasts and consisted of a freely dissociating complex, the activity of which may be restored by combination of its separated constituents. The partially purified enzyme was composed of several subunits of molecular masses ranging from 32 to 79 kDa, for a native molecular mass > 600 kDa. One of these subunits, of molecular mass 38 kDa, was biotinylated. This complex subunit structure was comparable with that of microorganisms and was referred to as a ‘prokaryotic’ form of ACCase. Biochemical parameters were determined for both ACCase forms. Finally, both pea leaf ACCases exhibited different sensitivities towards the grass ACCase herbicide, diclofop. This compound had no effect on the ‘prokaryotic’ form of ACCase, while the ‘eukaryotic’ form was strongly inhibited.

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 Insulin activation of lipogenesis in isolated mammary acini from lactating rats fed on a high-fat diet. Evidence that acetyl-CoA carboxylase is a site of action

1987 ◽  
Vol 242 (3) ◽  
pp. 905-911 ◽  
Author(s):  
M R Munday ◽  
D H Williamson
Keyword(s):  
Glucose Uptake ◽  
High Fat Diet ◽  
Acetyl Coa Carboxylase ◽  
High Fat ◽  
Acetyl Coa ◽  
Carboxylase Activity ◽  
Glucose Incorporation ◽  
Site Of Action ◽  
A Site

Feeding lactating rats on high-fat cheese crackers in addition to laboratory chow increased the dietary intake of fat from 2 to 20% of the total weight of food eaten and decreased mammary-gland lipogenesis in vivo by approx. 50%. This lipogenic inhibition was also observed in isolated mammary acini, where it was accompanied by decreased glucose uptake. These inhibitions were completely reversed by incubation with insulin. Insulin had no effect on the rate of glucose transport into acini, nor on pyruvate dehydrogenase activity as estimated by the accumulation of pyruvate and lactate, suggesting that these are not the sites of lipogenic inhibition. Insulin stimulated the incorporation of [1-14C]acetate into lipid in acini from high-fat-fed rats. In the presence of alpha-cyanohydroxycinnamate, a potent inhibitor of mitochondrial pyruvate transport, and with glucose as the sole substrate, neither [1-14C]glucose incorporation into lipid nor glucose uptake were stimulated by insulin. Insulin did stimulate the incorporation of [1-14C]acetate into lipid in the presence of alpha-cyanohydroxycinnamate, and this was accompanied by an increase in glucose uptake by the acini. This indicated that increased glucose uptake was secondary to the stimulation of lipogenesis by insulin, which therefore must occur via activation of a step in the pathway distal to mitochondrial pyruvate transport. Insulin stimulated acetyl-CoA carboxylase activity measured in crude extracts of acini from high-fat-fed rats, restoring it to values close to those of chow-fed controls. The effects of insulin on acetyl-CoA carboxylase activity and lipogenesis were not antagonized by adrenaline or dibutyryl cyclic AMP.

Characteristics of two forms of acetyl-CoA carboxylase from maize leaves

1994 ◽  
Vol 22 (3) ◽  
pp. 261S-261S ◽  
Author(s):  
DEREK HERBERT ◽  
CLAUDE ALBAN ◽  
DAVID J. COLE ◽  
KENNETH E. PALLETT ◽  
JOHN L. HARWOOD
Keyword(s):  
Acetyl Coa Carboxylase ◽  
Acetyl Coa ◽  
Maize Leaves
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