Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: 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)

Fatty acid synthetase was purified 13-fold from lactating rabbit mammary glands by a procedure which involved chromatography on DEAE-cellulose, ammonium sulphate precipitation and gel filtration on Sepharose 4B. The preparation was completed within two days and over 100 mg of enzyme was isolated from 100--150 g of mammary tissue, which represented a yield of over 40%. The preparation was homogeneous by the criteria of polyacrylamide gel electrophoresis and ultracentrifugal analysis. The sedimentation constant, S20,w was 13.3 S, the absorption coefficient, A280nm1%, measured refractometrically was 10.0 +/- 0.1, and the amino acid composition was determined. The subunit molecular weight determined by gel electrophoresis in the presence of sodium dodecyl sulphate was 252,000 +/- 6,000, and the molecular weight of the native enzyme measured by sedimentation equilibrium was 515,000. These experiments indicate that at the concentrations which exist in mammary tissue (2--4 mg/ml) fatty acid synthetase is a dimer. The purified enzyme did however show a tendency to dissociate to a monomeric 9-9S species on storage for several days or following exposure to a low ionic strength buffer at pH 8.3. There was only a small quantity of alkali labile phosphate (0.2 molecules per subunit) bound covalently to the purified enzyme. Acetyl-CoA carboxylase was purified 300-fold in a 50% yield within 24 h by ammonium sulphate and polyethylene glycol precipitations [Hardie, D.G. and Cohen, P. (1978) FEBS Lett. 91, 1--7]. The preparation was in a state approaching homogeneity as judged by polyacrylamide gel electrophoresis, gel filtration on Sepharose 4B and ultracentrifugal analysis. The sedimentation constant, S20,w, was 50.5 S, the absorption index, A280nm1%, was 14.5 +/- 0.7, and the amino acid composition was determined. The subunit molecular weight of acetyl-CoA carboxylase determined by gel electrophoresis in the presence of sodium dodecyl sulphate was identical to that of fatty acid synthetase (252,000) as shown by electrophoresis of a mixture of the two proteins. The preparations also contained two minor components of molecular weight 235,000 and 225,000, which appear to be derived from the major species of mol. wt 252,000. A large emount of phosphate (3.2 molecules per subunit) was found to be bound covalently to the purified enzyme. The properties of fatty acid synthetase and acetyl-CoA carboxylase are compared to those obtained by other workers.
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PMID:Purification and physicochemical properties of fatty acid synthetase and acetyl-CoA carboxylase from lactating rabbit mammary gland. 3 36

Acetyl-CoA carboxylase is the pivotal enzyme in the de novo synthesis of fatty acids and is the only carboxylase with a biotin-containing subunit greater than 200,000 daltons. The biotin moiety is covalently linked to the active site and has a high affinity (Kd = 10(-15) M) for the protein avidin. This relationship has been used in previous studies to identify acetyl-CoA carboxylase isolated from mammalian species. However, acetyl-CoA carboxylase has not been isolated and characterized in a poikilothermic species such as the rainbow trout. The present study describes the isolation and identification of acetyl-CoA carboxylase in the cytosol of rainbow trout (Salmo gairdneri) liver. The enzyme was isolated using two distinct procedures--polyethylene glycol precipitation and avidin-Sepharose affinity chromatography. Identification of the isolated protein as acetyl-CoA carboxylase was made by the following: (1) sodium dodecyl sulfate-polyacrylamide gel electrophoresis; (2) avidin binding; (3) in vivo labeling with [14C]biotin; and (4) acetyl-CoA carboxylase-specific activity. The subunit molecular weight of the major protein was 230,000 daltons +/- 3.3%. This protein was shown to bind avidin (Mr = 16,600) prior to sodium dodecyl sulfate-polyacrylamide gel electrophoresis, indicating the presence of biotin. In addition, protein isolated from fish that had previously received intraperitoneal injections of [14C]biotin, showed the majority of radioactivity associated with the 230,000 dalton protein.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Isolation and identification of acetyl-CoA carboxylase from rainbow trout (Salmo gairdneri) liver. 256 57

Acetyl-CoA carboxylase was isolated from rat liver by polyethylene glycol precipitation and avidin affinity chromatography. Sodium dodecyl sulfate electrophoresis of the enzyme gives one protein band (Mr 250,000). Phosphate analysis of the carboxylase showed the presence of 8.3 mol of phosphate/mol of subunit (Mr 250,000). The purified carboxylase has low activity in the absence of citrate (specific activity = 0.3 units/mg). However, addition of 10 mM citrate activates the carboxylase 10-fold, with half-maximal activation observed at 2 mM citrate, well above the physiological citrate level. Using this carboxylase as a substrate, we have isolated from rat liver a protein that activates the enzyme about 10-fold. This protein has been purified to near homogeneity (Mr 90,000). Incubation of this protein with 32P-labeled acetyl-CoA carboxylase results in a time-dependent activation of carboxylase with concomitant release of 32Pi, indicating that this protein is a phosphoprotein phosphatase. Both activation and dephosphorylation are dependent on Mn2+, but not citrate. This phosphatase does not hydrolyze p-nitrophenyl phosphate but does show high affinity for acetyl-CoA carboxylase (Km = 0.2 microM) as compared to its action on phosphorylase a (Km = 5.5 microM) and phosphohistone (Km = 20 microM). Activated acetyl-CoA carboxylase was isolated after dephosphorylation by the phosphatase. Such preparations contain about 5 mol of phosphate/mol of subunit and have specific activities of 2.6-3.0 units/mg in the absence of citrate. These activities are comparable to those of the phosphorylated carboxylase in the presence of 10 mM citrate. Thus, dephosphorylation by the Mn2+-dependent phosphatase renders the carboxylase citrate-independent, as compared to the phosphorylated form, which is citrate-dependent. To our knowledge this is the first report of a preparation of animal acetyl-CoA carboxylase that has substantial catalytic activity independent of citrate.
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PMID:Activation of acetyl-CoA carboxylase. Purification and properties of a Mn2+-dependent phosphatase. 286 Jan 6

Acetyl-CoA carboxylase from rat epididymal fat tissue is activated by incubation at 30 C in the absence of citrate or metal ions. This activation is accompanied by a corresponding loss of 32P from the labeled enzyme, and it is not blocked by the heat-stable phosphorylase phosphatase inhibitor proteins from rabbit muscle. We have succeeded in separating an activity which activates and dephosphorylates acetyl-CoA carboxylase from the carboxylase using polyethylene glycol-6000. These results suggest that the temperature-dependent activation of acetyl-CoA carboxylase in crude or partially purified preparations results from dephosphorylation of the carboxylase by bound phosphatase.
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PMID:Heat activation of rat epididymal fat tissue acetyl-coa carboxylase is due to dephosphorylation by its endogenous phosphatase. 611 34

Rat liver acetyl-CoA carboxylase has been purified to homogeneity by a new method involving polyethylene glycol precipitation, and DEAE and Sepharose 4B chromatography. The final product displays a single band on SDS polyacrylamide gel electrophoresis of estimated molecular weight 240,000. This material contains 5.5 +/- 0.3 moles of alkali-labile phosphate per subunit and has a specific activity of 1.2 +/- 0.2 units per mg protein. As compared to previous purification procedures for the liver enzyme, this product has a higher phosphate content, lower specific activity, and an absence of major proteolysis. Trypsin digestion of 32P-labeled acetyl-CoA carboxylase from hepatocytes reveals that the 32P-labeled phosphorylation sites are extremely labile to proteolytic digestion. Potential modification of isolated liver acetyl-CoA carboxylase by proteolysis and/or dephosphorylation must be ascertained prior to in vitro enzymatic studies.
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PMID:A new method for the isolation of rat liver acetyl-CoA carboxylase. 611 63

A protein kinase which phosphorylates and inactivates acetyl-CoA carboxylase has been purified to apparent homogeneity from rat liver. The kinase was found to exist in two forms: bound to carboxylase in a complex or in a free form that is in different stages of aggregation over a wide range of molecular weights. The purification of the kinase involved first partial purification of acetyl-CoA carboxylase through polyethylene glycol precipitation and DEAE-cellulose chromatography. The kinase was then separated from acetyl-CoA carboxylase by Sepharose 2B chromatography. The molecular weight of the kinase subunit was 170,000 as determined by sodium dodecyl sulfate-gel electrophoresis. The incorporation of 1 mol of phosphate/mole of carboxylase subunit caused complete inactivation of the carboxylase. Acetyl-CoA carboxylase, inactivated by the kinase, can be dephosphorylated and reactivated when incubated with phosphorylase phosphatase. The Km values of the kinase for acetyl-CoA carboxylase and ATP are 90 nM and 20 microM, respectively. The kinase was found to be cyclic AMP-independent, but activated by CoA. The protein kinase can phosphorylate acetyl-CoA carboxylase, protamine, and histones, but could not act on hydroxymethylglutaryl-CoA reductase or phosphorylase b.
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PMID:Purification and properties of a kinase which phosphorylates and inactivates acetyl-CoA carboxylase. 612 Jan 70

Maize leaf acetyl-CoA carboxylase was purified from whole tissue homogenates by precipitation with polyethylene glycol and ammonium sulfate, and gel filtration. Recoveries were approximately 5% with 100-fold increases in specific activity. The molecular weight of the native enzyme is estimated at 500,000 from the elution volume of a calibrated Ultrogel AcA 22 column. Electrophoresis in polyacrylamide gel containing 1% sodium dodecyl sulfate revealed a single subunit of Mr 60,000-61,000. Investigation of the kinetic properties of the purified enzyme indicates that Mg X ATP is the active substrate, with free ATP inhibiting and Mg2+ activating the enzyme. Km's for acetyl-CoA and HCO3- are about 0.1 and 2 mM, respectively. ADP inhibition is competitive with respect to ATP, but uncompetitive with respect to acetyl-CoA. The observed responses of purified acetyl-CoA carboxylase to changes in pH, and in concentrations of Mg2+, ATP, and ADP, and the reported changes in the chloroplastic concentrations of these effectors during light-dark transitions of chloroplasts are consistent with increased acetyl-CoA carboxylase activity upon illumination of chloroplasts.
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PMID:Purification and characterization of maize leaf acetyl-coenzyme A carboxylase. 614 67

Three cyclic AMP-independent acetyl-CoA carboxylase kinases (A, B1 and B2) have been isolated from lactating rat mammary gland, using phosphocellulose chromatography, high performance gel filtration, and affinity chromatography on casein-Sepharose and phosvitin-Sepharose. These protein kinases have been identified with previously described kinases by the following criteria. Kinase A phosphorylates the same sites on rabbit mammary acetyl-CoA carboxylase as acetyl-CoA carboxylase kinase 2, which was originally described as a contaminant of rabbit mammary acetyl-CoA carboxylase purified by the poly(ethylene glycol)procedure. Kinase A will henceforth be referred to as acetyl-CoA carboxylase kinase-2. Kinase B1 has been identified with casein kinase II by its heparin sensitivity, elution behaviour on phosphocellulose, molecular mass, substrate specificity and subunit composition. Kinase B2 has been identified with casein kinase I by its elution behaviour on phosphocellulose, molecular mass, substrate specificity and subunit composition. The three kinases phosphorylate distinct sites on acetyl-CoA carboxylase. Phosphorylation by either casein kinase I or II does not affect enzyme activity. However, acetyl-CoA carboxylase kinase 2 inactivates acetyl-CoA carboxylase reversibly, in an identical manner to cyclic-AMP-dependent protein kinase, and phosphorylates sites located on identical peptides. Acetyl-CoA carboxylase kinase-2 can, however, be distinguished from the free catalytic subunit of cyclic-AMP-dependent protein kinase by its molecular mass, its substrate specificity, its elution behaviour on phosphocellulose, and its complete lack of sensitivity to the protein inhibitor of cyclic-AMP-dependent protein kinase. We also present evidence that phosphorylation of acetyl-CoA carboxylase by cyclic-AMP-dependent protein kinase occurs directly and not via a bicyclic cascade system as proposed by other laboratories.
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PMID:Isolation of three cyclic-AMP-independent acetyl-CoA carboxylase kinases from lactating rat mammary gland and characterization of their effects on enzyme activity. 614 23

Protein phosphatase-2B was purified from extracts of rabbit skeletal muscle by a procedure that involved fractionation with ammonium sulphate, chromatography on DEAE-Sepharose, fractionation with poly(ethylene glycol), gel filtration on Sephadex G-200 (Mr = 98000 +/- 4000), chromatography on Affi-Gel Blue and affinity chromatography on calmodulin-Sepharose. The enzyme was purified 3500-fold in seven days with an overall yield of 0.5%. The alpha-subunit of phosphorylase kinase, protein phosphatase inhibitor-1 and the myosin P-light chain from rabbit skeletal muscle were dephosphorylated by protein phosphatase-2B with similar kinetic constants. The alpha-subunit of phosphorylase kinase was dephosphorylated at least 100-fold more rapidly than the beta-subunit, while glycogen phosphorylase, glycogen synthase, histones H1 and H2B, ATP-citrate lyase, acetyl-CoA carboxylase, L-pyruvate kinase and protein synthesis initiation factor eIF-2 were not dephosphorylated at significant rates. Protein phosphatase-2B became activated 10-fold by calmodulin (A0.5 = 6 nM) after chromatography on DEAE-Sepharose and this degree of activation was maintained throughout the remainder of the purification. Calmodulin increased the Vmax of the reaction without altering the Km for inhibitor-1. The activity of protein phosphatase-2B was completely dependent on Ca2+ in the presence or absence of calmodulin. Half-maximal activation was observed at 1.0 microM Ca2+ in the absence, and at 0.5 microM Ca2+ in the presence, of 0.03 microM calmodulin. Protein phosphatase-2B was inhibited completely by trifluoperazine; half-maximal inhibition occurred at 45 microM in the absence and 35 microM in the presence of 0.03 microM calmodulin. The metabolic role of protein phosphatase-2B in vivo is discussed in the light of the observation that this enzyme is probably identical to a major calmodulin-binding protein of neural tissue termed calcineurin or CaM-BP80 [Stewart, A. A., Ingebritsen, T. S., Manalan, A., Klee, C. B., and Cohen, P. (1982) FEBS Lett. 137, 80-84].
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PMID:The protein phosphatases involved in cellular regulation. 5. Purification and properties of a Ca2+/calmodulin-dependent protein phosphatase (2B) from rabbit skeletal muscle. 630 28

Biotin-protein ligase is an enzyme that catalyzes the ATP-dependent biotinylation of a specific lysine residue in acetyl-CoA carboxylase. The biotin-protein ligase from Pyrococcus horikoshii OT3 has been cloned, overexpressed and purified. Crystallization was performed by the microbatch method or the vapour-diffusion method using PEG 2000 as a precipitant at 295 K. X-ray diffraction data have been collected to 1.6 A resolution from a native crystal and to 1.55 A resolution from a selenomethionine-derivative crystal for multiple anomalous dispersion phasing using synchrotron radiation at 100 K. The native crystal belongs to the monoclinic space group P2(1), with unit-cell parameters a = 38.601, b = 78.264, c = 70.147 A, beta = 101.48 degrees. Assuming a homodimer per asymmetric unit gives a VM value of 2.14 A3 Da(-1) and a solvent content of 42.5%. Cocrystals with biotin, ADP and biotinyl-5'-AMP were prepared and diffraction data sets were collected to 1.6, 1.6 and 1.45 A resolution, respectively.
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PMID:Purification, crystallization and preliminary crystallographic analysis of the biotin-protein ligase from Pyrococcus horikoshii OT3. 1651 Sep 91


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