Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:6.4.1.2 (acetyl-CoA carboxylase)
 2,876 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

An inhibitor and stimulator of in vitro hepatic fatty acid synthesis are present in renal microsomes. In addition, a stimulator of fatty acid synthesis is present in renal lysosomes. Renal microsomal inhibition of hepatic fatty acid synthesis is not due to the depletion of cofactors in the system. This inhibitor appears to be located exclusively in the kidney medullary microsomes. It is destroyed by Pronase and heat treatment suggesting it may be a protein. Its effects on fatty acid synthesis may be attributed in part to ATPase activity as well as a direct effect on the hepatic fatty acid synthesizing system. A stimulator of hepatic fatty acid synthesis is present in the buffer insoluble fraction of an acetone powder preparation of renal microsomes. This stimulator is relatively heat labile and does not appear to be a phospholipid. The lysosomal stimulator of hepatic fatty acid synthesis is associated with the contents of renal lysosomes and not with the lysosomal membranes. It acts at the acetyl-CoA carboxylase step and its activity is not affected by fasting or aminonucleoside induced nephrosis.
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PMID:Effect of renal microsomes and renal lysosomes on in vitro hepatic fatty acid synthesis. 113 13

The formation of palmitoylcarnitine is catalyzed by carnitine palmitoyl-transferase (CPT-I) and this catalysis is the first committed step in beta-oxidation. The malonyl-CoA-inhibited isoform appears to be distinct from latent (CPT-II) activity, which is localized to the matrix side of the mitochondrial inner membrane. Sarcoplasmic reticulum from canine cardiac muscle was fractionated on a discontinuous sucrose density gradient into three major bands, all of which contained Ca(2+)-ATPase activity. Only the fraction that banded at a concentration of 38% surcrose was slightly contaminated by mitochondria. Peroxisomal uricase was low or absent in fractionated SR. All sarcoplasmic reticulum fractions contained malonyl-CoA-sensitive medium- (COT) and long-chain (CPT) carnitine acyltransferase activities. CPT activity decreased in sarcoplasmic reticulum when Triton X-100 was present. Carnitine acyltransferase activities were inactivated by preincubating the sarcoplasmic reticulum with the sulfhydryl reagent, 5,5'-dithiobis-(2-nitrobenzoic acid) (DTNB). In contrast, mitochondrial CPT-II activity was stable in the presence of DTNB and activated by Triton X-100. Western blots of mitochondria and sarcoplasmic reticulum fractions showed that the mitochondrial fractions reacted with antibody to mitochondrial CPT-II but not with SR protein when both were added at comparable specific activities. The data suggest that cardiac SR contains a unique malonyl-CoA-sensitive isoform of CPT, and that synthesis of acylcarnitine may occur in the microenvironment of Ca2+ transport, where the extent of production of acylcarnitine is controlled by cardiac acetyl-CoA carboxylase activity.
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PMID:Evidence for malonyl-CoA-sensitive carnitine acyl-CoA transferase activity in sarcoplasmic reticulum of canine heart. 162 48

In the ATP-dependent carboxylation of biotin that is catalyzed by most biotin-dependent carboxylases, a fundamental mechanistic question is whether the ATP activates bicarbonate (via the formation of carboxyphosphate as an intermediate) or whether the ATP activates biotin (via the formation of O-phosphobiotin). We have resorted to three mechanistic tests using the biotin carboxylase subunit of acetyl-CoA carboxylase from Escherichia coli: positional isotope exchange, intermediate trapping, and 18O tracer experiments on the ATPase activity. First, no catalysis of positional isotope exchange in adenosine 5'-[( alpha, beta-18O, beta, beta-18O2]triphosphate) was observed when either biotin or bicarbonate was absent, nor was any exchange seen in the presence of both N-1-methylbiotin and bicarbonate. Second, the putative carboxyphosphate intermediate could not be trapped as its trimethyl ester, under conditions of incubation and analysis where the authentic triester was shown to be adequately stable. In the third test, however, we showed that the ATPase activity of biotin carboxylase that is seen in the absence of biotin, an activity that is known to parallel the normal carboxylase reaction when biotin is present, occurs with the transfer of an 18O label directly from [18O]bicarbonate into the product Pi. This result suggests that the bicarbonate-dependent biotin-independent ATPase reaction catalyzed by biotin carboxylase goes via carboxyphosphate and that the carboxylation of biotin itself may proceed analogously.
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PMID:On the intermediacy of carboxyphosphate in biotin-dependent carboxylations. 297

Kinesin, an ATP-dependent microtubule motor, can be studied in vitro in motility assays where the kinesin is nonspecifically adsorbed to a surface. However, adsorption can inactivate kinesin and may alter its reaction kinetics. We therefore prepared a biotinated kinesin derivative, K612-BIO, and characterized its activity in solution and when bound to streptavidin-coated surfaces. K612-BIO consists of the N-terminal 612 amino acids of the Drosophila kinesin alpha subunit linked to the 87-amino acid C-terminal domain of the biotin carboxyl carrier protein subunit of Escherichia coli acetyl-CoA carboxylase. The C-terminal domain directs the efficient post-translational biotination of the protein. We expressed K612-BIO at high levels using the baculovirus expression vector system and purified it to near-homogeneity. The expressed protein is completely soluble, and > 90% is bound by streptavidin. K612-BIO steady-state ATPase kinetics (KM,ATP = 24 microM, K0.5, microtubule = 0.61 mg ml-1, Vmax = approximately 25 s-1 head-1, 25 degrees C) are similar to those reported for intact kinesin. ATPase kinetics are not affected by the addition of streptavidin. Enzyme bound to a surface coated with streptavidin drove microtubule gliding in the presence of 2 mM ATP at 750 +/- 130 nm s-1 (26 degrees C). Activity was abolished by pretreatment of the surface with biotin, indicating that the microtubule movements are due to specifically bound enzyme. Motility assays based on specific attachment of biotinated enzyme to streptavidin-coated surfaces will be useful for quantitative analysis of kinesin motility and may provide a way to detect activity in kinesin derivatives or kinesin-like proteins that have not yet been shown to move microtubules.
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PMID:Microtubule movement by a biotinated kinesin bound to streptavidin-coated surface. 813 86

Acetyl-CoA carboxylase catalyzes the first committed step in the biosynthesis of long-chain fatty acids. The Escherichia coli form of the enzyme consists of a biotin carboxylase protein, a biotin carboxyl carrier protein, and a carboxyltransferase protein. In this report a system for site-directed mutagenesis of the biotin carboxylase component is described. The wild-type copy of the enzyme, derived from the chromosomal gene, is separated from the mutant form of the enzyme which is coded on a plasmid. Separation of the two forms is accomplished using a histidine-tag attached to the amino terminus of the mutant form of the enzyme and nickel affinity chromatography. This system was used to mutate four active site residues, E211, E288, N290, and R292, to alanine followed by their characterization with respect to several different reactions catalyzed by biotin carboxylase. In comparison to wild-type biotin carboxylase, all four mutant enzymes gave very similar results in all the different assays, suggesting that the mutated residues have a common function. The mutations did not affect the bicarbonate-dependent ATPase reaction. In contrast, the mutations decreased the maximal velocity of the biotin-dependent ATPase reaction 1000-fold but did not affect the Km for biotin. The activity of the ATP synthesis reaction catalyzed by biotin carboxylase where carbamoyl phosphate reacts with ADP was decreased 100-fold by the mutations. The ATP synthesis reaction required biotin to stimulate the activity in the wild-type; however, biotin did not stimulate the activity of the mutant enzymes. The results showed that the mutations have abolished the ability of biotin to increase the activity of the enzyme. Thus, E211, E288, N290, and R292 were responsible, at least in part, for the substrate-induced synergism by biotin in biotin carboxylase.
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PMID:Mutations at four active site residues of biotin carboxylase abolish substrate-induced synergism by biotin. 1007 84

The yeast Pyc1 isoform of pyruvate carboxylase has been further characterized and shown to differ from the Pyc2 isoform in its K(a) for K(+) activation. Pyc1 differs from chicken liver pyruvate carboxylase in the lack of effect of acetyl-CoA on ADP phosphorylation by carbamoyl phosphate, which may be a result of differences in the loci of action of the effector between the two enzymes. Solvent D(2)O isotope effects have been measured with Pyc1 on the full pyruvate carboxylation reaction, the ATPase reaction in the absence of pyruvate, and the carbamoyl phosphate-ADP phosphorylation reaction for the first time for pyruvate carboxylase. Proton inventories indicate that the measured isotope effects are due to a single proton transfer step in the reaction. The inverse isotope effects observed in all reactions suggest that the proton transfer step converts the enzyme from an inactive to an active form. Kinetic measurements on the C249A mutant enzyme suggest that C249 is involved in the binding and action of enzyme activators K(+) and acetyl-CoA. C249 is not involved in ATP binding as was observed for the corresponding residue in the biotin carboxylase subunit of Escherichia coli acetyl-CoA carboxylase, nor is it directly responsible for the measured inverse (D)(k(cat)/K(m)) isotope effects. The size of the inverse isotope effects indicates that they may result from formation of a low-barrier hydrogen bond. Modification of the wild type and C249A mutant with o-phthalaldehyde suggests that C249 is involved in isoindole formation but that the modification of this residue is not directly responsible for the accompanying major loss of enzyme activity.
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PMID:Kinetic characterization of yeast pyruvate carboxylase isozyme Pyc1 and the Pyc1 mutant, C249A. 1474 53

Previously we reported that a mutant of Corynebacterium glutamicum ATCC14067 with reduced H+-ATPase activity, F172-8, showed an approximately two times higher specific rate of glucose consumption than the parent, but no glutamic acid productivity under the standard biotin-limited culture conditions, where biotin concentration was set at 5.5 microg/l in the production medium (Sekine et al., Appl. Microbiol. Biotechnol., 57, 534-540 (2001)). In this study, various culture conditions were tested to check the glutamic acid productivity of strain F172-8. The mutant was found to produce glutamic acid under exhaustive biotin limitation, where the biotin concentration of the medium was set at 2.5 microg/l with much smaller inoculum size. When strain F172-8 was cultured under the same biotin-limited conditions using a jar fermentor, 53.7 g/l of glutamic acid was produced from 100 g/l glucose, while the parent produced 34.9 g/l of glutamic acid in a medium with 5.5 microg/l biotin. The glutamic acid yield of strain F172-8 also increased under Tween 40-triggered production conditions (1.2-fold higher than the parent strain). The amounts of biotin-binding enzymes were investigated by Western blot analysis. As compared to the parent, the amount of pyruvate carboxylase was lower in the mutant; however, the amount of acetyl-CoA carboxylase did not significantly change under the glutamic acid production conditions. To the best of our knowledge, this is the first report showing that the H+-ATPase-defective mutant of C. glutamicum is useful in glutamic acid production.
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PMID:Enhanced glutamic acid production by a H+-ATPase-defective mutant of Corynebacterium glutamicum. 1611 73

The assay of acetyl-CoA carboxylase (EC 6.4.1.2) does not follow ideal zero-order kinetics when assayed in a crude extract from wheat (Triticum aestivum L.) germ. Our results show that the lack of ideality is the consequence of contamination by ATPase and adenylate kinase. These enzyme activities generate significant amounts of ADP and AMP in the assay mixture, thus limiting the availability of ATP for the carboxylase reaction. Moreover, ADP and AMP are competitive inhibitors, with respect to ATP, of acetyl-CoA carboxylase. Similar relationships between adenylate nucleotides and acetyl-CoA carboxylase are found in isolated chloroplasts. There is no evidence that acetyl-CoA carboxylase activity in the extracts of the plant systems examined is altered by covalent modification, such as a phosphorylation-dephosphorylation cycle. A scheme is presented that illustrates the dependency of acetyl-CoA carboxylase and fatty acid synthesis on the energy demands of the chloroplasts in vivo.
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PMID:Regulation of Plant Acetyl-CoA Carboxylase by Adenylate Nucleotides. 1666 80

The cardiac glycoside ouabain initiates a cascade of signaling events through Na+,K+-ATPase, leading to an increase in cell growth and proliferation in different cell types. We explored the effects of ouabain on glucose metabolism in skeletal muscle and clarified the mechanisms of ouabain signal transduction. In rat soleus muscle 200 microM ouabain decreased basal glucose uptake without effect on insulin-stimulated glucose uptake. Ouabain increased glycogen synthesis additively to insulin and this effect was abolished in the presence of a MEK1/2 inhibitor (PD98059) or a c-Src inhibitor (PP2). Ouabain exposure reduced glucose oxidation, and this effect was reversed in the presence of PP2. Incubation with ouabain did not affect intramuscular ATP and its metabolites; however acetyl-CoA carboxylase phosphorylation was reduced, with no effect on AMPK phosphorylation. Insulin-stimulated Akt phosphorylation was not affected by ouabain. Ouabain reduced basal and insulin-stimulated phosphorylation of PKC alpha/beta and delta isoforms, whereas phosphorylation of PKCzeta was unchanged. Ouabain exposure increased interaction of 1- and 2-subunits of Na-pump with c-Src, as assessed by co-immunoprecipitation with c-Src. Phosphorylation of ERK1/2, GSK 3 / and p90rsk activity was increased in response to ouabain, and these effects were prevented in the presence of PD98059 and PP2. In conclusion, the cardiac glycoside ouabain stimulates glycogen synthesis additively to insulin in rat skeletal muscle. This effect is mediated by activation of c-Src-, ERK1/2- p90rsk- and GSK3-dependent signaling pathway.
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PMID:Metabolic and signaling events mediated by cardiotonic steroid ouabain in rat skeletal muscle. 1753 36

The effects of diabetes on heart function may be initiated or compounded by the exaggerated reliance of the diabetic heart on fatty acids and ketones as metabolic fuels. beta-Blocking agents such as metoprolol have been proposed to inhibit fatty acid oxidation. We hypothesized that metoprolol would improve cardiac function by inhibiting fatty acid oxidation and promoting a compensatory increase in glucose utilization. We measured ex vivo cardiac function and substrate utilization after chronic metoprolol treatment and acute metoprolol perfusion. Chronic metoprolol treatment attenuated the development of cardiac dysfunction in streptozotocin (STZ)-diabetic rats. After chronic treatment with metoprolol, palmitate oxidation was increased in control hearts but decreased in diabetic hearts without affecting myocardial energetics. Acute treatment with metoprolol during heart perfusions led to reduced rates of palmitate oxidation, stimulation of glucose oxidation, and increased tissue ATP levels. Metoprolol lowered malonyl-CoA levels in control hearts only, but no changes in acetyl-CoA carboxylase phosphorylation or AMP-activated protein kinase activity were observed. Both acute metoprolol perfusion and chronic in vivo metoprolol treatment led to decreased maximum activity and decreased sensitivity of carnitine palmitoyltransferase I to malonyl-CoA. Metoprolol also increased sarco(endo)plasmic reticulum Ca(2+)-ATPase expression and prevented the reexpression of atrial natriuretic peptide in diabetic hearts. These data demonstrate that metoprolol ameliorates diabetic cardiomyopathy and inhibits fatty acid oxidation in streptozotocin-induced diabetes. Since malonyl-CoA levels are not increased, the reduction in total carnitine palmitoyltransferase I activity is the most likely factor to explain the decrease in fatty acid oxidation. The metabolism changes occur in parallel with changes in gene expression.
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PMID:Metoprolol improves cardiac function and modulates cardiac metabolism in the streptozotocin-diabetic rat. 1820 48


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