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At least 127 records · Page 7Linked to original sources

PubMed1993

Expression of bovine vitamin K-dependent carboxylase activity in baculovirus-infected insect cells.

A vitamin K-dependent carboxylase has recently been purified from bovine liver microsomes and candidate cDNA clones have been isolated. Definitive identification of the carboxylase remains circumstantial since expression of candidate carboxylase cDNAs in mammalian cells is confounded by the presence of endogenous carboxylase activity. To overcome this problem, a recombinant strain of baculovirus (Autographa california nuclear polyhedrosis virus, AcMNPV) encoding a putative carboxylase (vbCbx/AcMNPV) was used to infect Sf9 insect cells, which we demonstrate have no endogenous carboxylase activity. Infection with vbCbx/AcMNPV conferred vitamin K-dependent carboxylase activity to Sf9 insect cells. Carboxylase activity was demonstrated to peak 2-3 days after infection with vbCbx/AcMNPV. Metabolic radiolabeling with L-[35S]methionine revealed that the 90-kDa recombinant protein is the major protein synthesized at the time of peak activity after infection. An anti-peptide antibody directed against residues 86-99 reacted with bovine liver carboxylase on Western blot analysis and immunoprecipitated recombinant carboxylase from infected Sf9 microsomal protein preparations. Since Sf9 insect cells lack endogenous vitamin K-dependent carboxylase activity, expression of carboxylase activity in Sf9 insect cells with recombinant baculovirus demonstrates that the protein encoded by this cDNA is a vitamin K-dependent gamma-glutamyl carboxylase.

Animals↗

PubMed1980

Evidence for two genetic complementation groups in pyruvate carboxylase-deficient human fibroblast cell lines.

We have examined genetic complementation in pyruvate carboxylase deficiency by comparing the enzyme activity in polyethylene glycol-induced heterokaryons with that in unfused mixtures of fibroblasts from three affected children. Complementation, manifested as a three- to sevenfold increase in pyruvate carboxylase activity, was observed in fusions between a biotin-responsive multiple carboxylase (pyruvate carboxylase, propionyl CoA carboxylase, and beta-methylcrotonyl CoA carboxylase) deficient fibroblast line and two other lines deficient only in pyruvate carboxylase activity. Kinetic analysis of complementing pyruvate carboxylase deficient lines, measured by the rate of restoration of enzyme activity as a function of time, revealed that maximum restoration was achieved within 10-24 hr after fusion. This profile is similar to those oberved for fusions between the multiple carboxylase deficient line and two lines deficient in propionyl CoA carboxylase activity that are known to represent different gene mutations. Although the patients with pyruvate carboxylase deficiency had similar clinica findings, our studies indicate that pyruvate carboxylase deficiency is genetically heterogeneous, with at least two distinct, probably intergenic, complementation groups.

Biotin↗

PubMed1997

Triiodothyronine stimulates and glucagon inhibits transcription of the acetyl-CoA carboxylase gene in chick embryo hepatocytes: glucose and insulin amplify the effect of triiodothyronine.

The mechanisms by which triiodothyronine (T3), glucose, insulin, and glucagon regulate acetyl-CoA carboxylase expression in primary cultures of chick embryo hepatocytes have been investigated. Incubating hepatocytes with T3 in the absence of glucose caused a fourfold increase in acetyl-CoA carboxylase activity. Addition of glucose (20 mM) enhanced the T3-induced increase in acetyl-CoA carboxylase activity by threefold but had no effect on enzyme activity in the absence of T3. The effects of T3 and glucose on acetyl-CoA carboxylase activity were accompanied by similar changes in acetyl-CoA carboxylase mRNA levels, indicating that regulation occurred at a pretranslational step. Xylitol mimicked the effect of glucose on acetyl-CoA carboxylase mRNA abundance, suggesting that an intermediate(s) of the nonoxidative branch of the pentose phosphate pathway may be involved in mediating this response. Insulin accelerated the accumulation of acetyl-CoA carboxylase mRNA abundance caused by T3 and glucose but had no effect on steady-state levels of acetyl-CoA carboxylase mRNA in the absence or presence of T3. Glucagon caused a 65% decrease in the accumulation of acetyl-CoA carboxylase mRNA in hepatocytes incubated with T3 and glucose. The effects of T3, glucose, insulin, and glucagon on the abundance of acetyl-CoA carboxylase mRNA were accounted for by changes in the transcription rate of the acetyl-CoA carboxylase gene. These data support the hypothesis that T3, glucose, insulin, and glucagon play a role in mediating the effects of nutritional manipulation on transcription of acetyl-CoA carboxylase in liver.

Acetyl-CoA Carboxylase↗

PubMed1985

Activation of acetyl-CoA carboxylase. Purification and properties of a Mn2+-dependent phosphatase.

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.

Acetyl-CoA Carboxylase↗

PubMed2004

Kinetic characterization of mutations found in propionic acidemia and methylcrotonylglycinuria: evidence for cooperativity in biotin carboxylase.

Acetyl-CoA carboxylase catalyzes the committed step in fatty acid synthesis in all plants, animals, and bacteria. The Escherichia coli form is a multifunctional enzyme consisting of three separate proteins: biotin carboxylase, carboxyltransferase, and the biotin carboxyl carrier protein. The biotin carboxylase component, which catalyzes the ATP-dependent carboxylation of biotin using bicarbonate as the carboxylate source, has a homologous functionally identical subunit in the mammalian biotin-dependent enzymes propionyl-CoA carboxylase and 3-methylcrotonyl-CoA carboxylase. In humans, mutations in either of these enzymes result in the metabolic deficiency propionic acidemia or methylcrotonylglycinuria. The lack of a system for structure-function studies of these two biotin-dependent carboxylases has prevented a detailed analysis of the disease-causing mutations. However, structural data are available for E. coli biotin carboxylase as is a system for its overexpression and purification. Thus, we have constructed three site-directed mutants of biotin carboxylase that are homologous to three missense mutations found in propionic acidemia or methylcrotonylglycinuria patients. The mutants M169K, R338Q, and R338S of E. coli biotin carboxylase were selected for study to mimic the disease-causing mutations M204K and R374Q of propionyl-CoA carboxylase and R385S of 3-methylcrotonyl-CoA carboxylase. These three mutants were subjected to a rigorous kinetic analysis to determine the function of the residues in the catalytic mechanism of biotin carboxylase as well as to establish a molecular basis for the two diseases. The results of the kinetic studies have revealed the first evidence for negative cooperativity with respect to bicarbonate and suggest that Arg-338 serves to orient the carboxyphosphate intermediate for optimal carboxylation of biotin.

Amino Acid Metabolism, Inborn Errors↗

PubMed1994

Molecular cloning of two different cDNAs for maize acetyl CoA carboxylase.

Acetyl CoA carboxylase (EC 6.4.1.2) in plants is a chloroplast-localized, biotin-containing enzyme that catalyses the carboxylation of acetyl CoA to malonyl CoA, the first committed step of the fatty acid biosynthesis pathway. Acetyl CoA carboxylase is the target site for the monocotyledon-specific aryloxyphenoxypropionate and cyclohexanedione groups of herbicides. We have purified a herbicide-sensitive acetyl CoA carboxylase from maize leaves to homogeneity (specific activity 7 mumol min-1 mg-1), separating it during the purification from a minor herbicide-resistant acetyl CoA carboxylase. The purified enzyme is a dimer of 230 kDa subunits. Antibodies raised to the purified acetyl CoA carboxylase detected three cross-reacting clones in a maize leaf cDNA expression library, each having an insert of 4-4.5 kb. Restriction analysis and sequencing showed that the cDNAs were derived from two different transcripts. Comparison of the deduced amino acid sequences with those of chicken and yeast acetyl CoA carboxylases confirmed that both types encoded acetyl CoA carboxylase, corresponding to the C-terminal half of the enzyme. The overall identity of the maize and chicken sequences was 37% (58% similarity) but for some shorter regions was much higher. Analysis of six other acetyl CoA carboxylase clones recovered from the maize cDNA library showed four belonged to one type and two to the other. The nucleotide sequence similarity between the two types of cDNA was approximately 95% in the coding region but considerably less in the 3'-untranslated region. Northern blot analysis of maize RNA showed a single band of 8.2-8.5 kb for acetyl CoA carboxylase mRNA. Southern blot hybridisations indicated that there are probably no more than two genes in maize for acetyl CoA carboxylase. The possible significance of two different cDNAs for acetyl CoA carboxylase is discussed.

Acetyl-CoA Carboxylase↗

PubMed1980

Yeast mutants defective in acetyl-coenzyme A carboxylase and biotin: apocarboxylase ligase.

Among more than 7000 mutants of Saccharomyces cerevisiae, requiring saturated fatty acids, 61 acetyl-CoA-carboxylase-deficient strains have been identified. According to their mutual complementation characteristics these mutants have been assigned to two different genes, acc1 and acc2. Both acetyl-CoA carboxylase genes are unlinked to each other and to the fatty acids synthetase genes fas1 and fas2. The acetyl-CoA carboxylases of several acc1 and acc2 mutants have been purified and assayed for their overall and component enzyme activities. Besides overall acetyl-CoA carboxylation, which was lost in all cases, both component enzymes, biotin carboxylase and transcarboxylase, were simultaneously affected in most mutants, though often to a different relative extent. Similarly, the comparison of biochemical and genetic complementation data revealed no basis for a clear distinction between specific biotin carboxylase and transcarboxylase mutants. These results suggest that acc1 is a cluster gene coding for a multifunctional protein harboring both acetyl-CoA carboxylase component enzyme activities on the same polypeptide chain. The acetyl-CoA carboxylase isolated from acc2 mutants was free of biotin. Correspondingly, biotin:apoacetyl-CoA-carboxylase ligase activity was missing in acc2 mutants. Therefore, it is concluced that the primary defect in acc2 mutants is in the biotin:apocarboxylase ligase. In agreement with this conclusion, the acc2 acetyl-CoA carboxylase can be activated, in the presence of biotin and ATP, by ligase preparations from wild-type or acc1 mutant cells. By the use of these mutants, evidence was obtained that in vivo the biotinylation of both acetyl-CoA carboxylase and pyruvate carboxylase is catalyzed by the same ligase.

Acetyl-CoA Carboxylase↗

PubMed1986

Maize phosphoenolpyruvate carboxylase. Cloning and characterization of mRNAs encoding isozymic forms.

The isozymic forms of maize phosphoenolpyruvate carboxylase (P-enolpyruvate carboxylase) involved in photosynthetic CO2 fixation were shown by protein gel blot analysis to consist of 100-kDa subunits. The nonautotrophic isoform found in roots is comprised of 96-kDa subunits and is about 50-100-fold less prevalent. Further analysis of P-enolpyruvate carboxylase isoforms made use of cloned cDNA probes. Two cDNA clones were isolated from a library constructed from maize leaf poly(A) RNA. The largest clone was complementary to about 25% of P-enolpyruvate carboxylase mRNA, which is 3.4 kilobases in length. The quantity of P-enolpyruvate carboxylase mRNA in green, mature leaf tissue was estimated to be 0.20% of poly(A) RNA, whereas P-enolpyruvate carboxylase mRNA in roots was about 100-fold less prevalent. We used thermal denaturation of a P-enolpyruvate carboxylase cDNA probe hybridized to RNA gel blots to estimate the degree of sequence difference between mRNAs encoding different P-enolpyruvate carboxylase isoforms. There appear to be at least two prevalent P-enolpyruvate carboxylase mRNAs in green leaves which are significantly different in sequence, as are P-enolpyruvate carboxylase mRNAs in roots and shoots. The hybridization pattern of maize genomic DNA Southern blots indicates that P-enolpyruvate carboxylase is encoded by a small gene family.

Carboxy-Lyases↗

PubMed1993

Interactions between pyruvate carboxylase and other mitochondrial enzymes.

Although pyruvate carboxylase associated with both mitochondrial aspartate aminotransferase and malate dehydrogenase, it had a higher affinity for the amino-transferase. Furthermore, the aminotransferase enhanced dissociation of malate dehydrogenase from pyruvate carboxylase. Glutamate dehydrogenase did not associate with pyruvate carboxylase alone, but it apparently associated with the pyruvate carboxylase-aminotransferase complex, and malate dehydrogenase associated with the resulting ternary complex. Citrate synthase and other proteins tested did not associate with pyruvate carboxylase. However, citrate synthase associated with the pyruvate carboxylase-malate dehydrogenase complex. Apparently as a consequence of these heteroenzyme interactions, the rate of the pyruvate carboxylase reaction was slightly greater when coupled with malate dehydrogenase or both malate dehydrogenase and citrate synthase than when coupled with citrate synthase alone. In addition, in the presence of both coupling enzymes, the rate of conversion of pyruvate to citrate was higher than predicted on the basis of the Michaelis-Menten relationship of the two coupling enzymes. Therefore, binding of malate dehydrogenase to pyruvate carboxylase enhances pyruvate carboxylase activity. Association of citrate synthase with the malate dehydrogenase-pyruvate carboxylase binary complex does not alter activation of pyruvate carboxylase but results in citrate synthase being more reactive than free citrate synthase with oxalacetate.

Animals↗

PubMed1983

Requirement of acetyl-coenzyme A carboxylase kinase for coenzyme A.

Phosphorylation and inactivation of acetyl-coenzyme A (CoA) carboxylase by acetyl-CoA carboxylase kinase in the presence of ATP and Mg2+ requires coenzyme A. Coenzyme A did not enhance the phosphorylation of alternative substrates of the carboxylase kinase such as protamine or histones. Analogs of coenzyme A were also effective in stimulating the inactivation of carboxylase. The KA of CoA for stimulated carboxylase inactivation was 25 microM. The presence of coenzyme A did not alter the Km of the carboxylase kinase for its substrates, ATP and acetyl-CoA carboxylase. Fluorescence binding studies showed that CoA binds to carboxylase but not to the kinase. The KD of CoA binding to carboxylase is 27 microM. These results indicate that coenzyme A, acting on acetyl-CoA carboxylase, may play an important role in the regulation of the covalent modification mechanism for acetyl-CoA carboxylase.

Allosteric Regulation↗

PubMed1995

Mutagenesis of vitamin K-dependent carboxylase demonstrates a carboxyl terminus-mediated interaction with vitamin K hydroquinone.

The gamma-glutamyl carboxylase and vitamin K epoxidase activities of a series of mutants of bovine vitamin K-dependent carboxylase with progressively larger COOH-terminal deletions have been analyzed. The recombinant wild-type (residues 1-758) and mutant protein carboxylases, Cbx 711, Cbx 676, and Cbx 572, representing residues 1-711, 1-676, and 1-572, respectively, were expressed in baculovirus-infected Sf9 cells. Wild-type carboxylase had a Km for the substrate Phe-Leu-Glu-Glu-Leu (FLEEL) of 0.87 mM; the carboxylation of FLEEL was stimulated 2.5-fold by proPT18, the propeptide of prothrombin. Its Km for vitamin K hydroquinone was 23 microM and the specific epoxidase activity of the carboxylase was 938 pmol vitamin KO/30 min/pmol of carboxylase. Cbx 711, which was also stimulated by proPT18, had a Km for FLEEL, a Km for vitamin K hydroquinone, and a specific epoxidase activity that was comparable to the wild-type carboxylase. In contrast Cbx 572 lacked both carboxylase and epoxidase activities. Although Cbx 676 had a normal carboxylase active site in terms of the Km for FLEEL and its stimulation by proPT18, the Km for vitamin K hydroquinone was 540 microM, and the specific epoxidase activity was 97 pmol KO/30 min/pmol of Cbx 676. The catalytic efficiencies of Cbx 676 for glutamate carboxylation and vitamin K epoxidation were decreased 15- and 400-fold, respectively, from wild-type enzyme reflecting the requirement for formation of an activated vitamin K species for carboxylation to occur. These data indicate that the truncation of COOH-terminal segments of the carboxylase had no effect on FLEEL or propeptide recognition, but in the case of Cbx 676, selectively affected the interaction with vitamin K hydroquinone and the generation of epoxidase activity. These data suggest that a vitamin K epoxidase activity domain may reside near the COOH terminus while the carboxylase active site domain resides toward the NH2 terminus.

Amino Acid Sequence↗

PubMed2005

Marginal maternal biotin deficiency in CD-1 mice reduces fetal mass of biotin-dependent carboxylases.

Marginal maternal biotin deficiency reduces hepatic activity of biotin-dependent carboxylases and causes high rates of fetal birth defects in mice. We tested the hypothesis that the decreased carboxylase activity observed in deficient dams and their offspring is mediated by decreased abundance of biotinylated carboxylases, decreased expression of their mRNAs, or both. During gestation, CD-1 mice were fed a diet that induced biotin deficiency or a biotin-sufficient diet. On gestational d 17, gravid uteri were removed, and each live fetus was examined grossly for defects. The expected high incidence of cleft palate (83%) in offspring was observed. In maternal and fetal liver, acetyl-CoA carboxylase, pyruvate carboxylase, propionyl-CoA carboxylase, and beta-methylcrotonyl-CoA carboxylase abundances were determined by Western blotting; the content of mRNAs for most of these enzymes and holocarboxylase synthetase was determined by real-time RT-PCR. Biotin deficiency significantly reduced the abundance of the carboxylases in maternal and fetal liver; neither the content of mRNAs for the carboxylases nor holocarboxylase synthetase changed. This study provides evidence that the decrease in carboxylase activities is attributable to a decrease in the abundance of biotinylated carboxylases; further, this effect is more severe in fetuses than dams.

Animals↗

PubMed1988

Early processing of prothrombin and factor X by the vitamin K-dependent carboxylase.

Binding interactions between the membrane-associated vitamin K-dependent carboxylase and its prothrombin and factor X substrates have been investigated in liver microsomes. Both substrates are firmly attached to microsomal membrane fragments which also harbor the carboxylase. In vitro 14CO2 gamma-carboxylation of these substrates, triggered by reduced vitamin K1H2, resulted in release of 14C-labeled prothrombin precursors from the membrane fragments, but no release of 14C-labeled factor X precursors could be demonstrated, which suggested a difference in early processing of these substrates by the carboxylase. Warfarin treatment of rats resulted in a 3-fold increase in the membrane concentration of factor X antigens and a 20-fold increase in 14C gamma-carboxylation of the membrane pool of factor X carboxylase substrates. There was a dose-response relationship between the amount of drug administered to the rats and 14C labeling of the membrane pool of factor X carboxylase substrates. On the other hand, the membrane concentration of prothrombin antigens did not increase in response to the drug, and 14CO2 gamma-carboxylation of the membrane pool of prothrombin carboxylase substrates was the same in warfarin and saline-treated rats. The results demonstrate significant differences in the interaction between the carboxylase and its prothrombin and factor X substrates. It appears that the different interactions result from binding of the prothrombin and the factor X precursors to separate microsomal membrane proteins that are involved in the gamma-carboxylation reaction. Warfarin appears to induce the factor X precursor-specific but not the prothrombin precursor-specific binding proteins, which suggests a new mechanism for the action of warfarin. These binding proteins may be under different genetic control. Treatment of the prothrombin and the factor X carboxylase substrates with endonuclease H showed that the rat prothrombin and the human factor X carboxylase substrates are high mannose glycoproteins. The human prothrombin and the rat factor X carboxylase substrates did not, on the other hand, change their migration in sodium dodecyl sulfate-polyacrylamide gel electrophoresis gels after endonuclease H treatment. The data demonstrate differences in the glycoprotein nature of the rat and the human carboxylase substrates.

Animals↗

PubMed1985

Simultaneous preparation of the three biotin-containing mitochondrial carboxylases from rat liver.

Affinity chromatography on avidin-Sepharose column was used to bind the biotin-containing carboxylases from rat liver. With a biotin gradient (0-0.3 mM), peaks of activity of pyruvate, propionly CoA and beta-methylcrotonyl CoA carboxylases co-eluted. Subsequent separation of the three carboxylases was attained using DEAE-Sepharose chromatography. Sodium dodecyl sulphate-polyacrylamide gel electrophoresis showed each of the enzymes to be pure, with pyruvate carboxylase giving a single subunit band (Mr 130 000), propionyl-CoA carboxylase giving two bands (Mr 73 000 and 56 500) and beta-methylcrotonyl-CoA carboxylase giving two bands (Mr 75 000 and 60 000). The specific activity of propionyl-CoA carboxylase (15.8 munits/mg) and beta-methylcrotonyl-CoA carboxylase (24.2 munits/mg) were comparable with reported activities for these purified enzymes, while that of pyruvate carboxylase (1.25 munits/mg) was low. This is a suitable method for the simultaneous preparation of purified carboxylases for the specific purpose of raising antisera to these enzymes.

Animals↗

PubMed1999

Evolution of pyruvate carboxylase and other biotin containing enzymes in developing rat liver and kidney.

The evolution of pyruvate carboxylase has been studied in rat liver and kidney during perinatal development. The pyruvate carboxylase activity, amount of enzyme and mRNA levels have been assayed from 2 days before delivery to weaning. In liver, there is a peak of activity and amount of enzyme 24 h before delivery and 2 peaks, at 12 h and 6 days, after parturition. The transcription of the enzyme gene followed a similar pattern, with mRNA peaks preceding those of activity and amount of enzyme. However, in kidney, pyruvate carboxylase activity, amount and mRNA remain low until weaning. These results confirm the limited role of renal gluconeogenesis during the perinatal development. Since all carboxylases contain biotin as prosthetic group, the biotinylation of pyruvate carboxylase during the perinatal period was investigated by western-blot using streptavidin-biotin peroxidase. In the mitochondrial samples from liver and kidney, all the pyruvate carboxylase detected was fully biotinylated, indicating an early development of the holocarboxylase synthetase activity in the perinatal period. This Western-blot technique also allowed us the detection of other biotin-enzymes based on their molecular weight. In liver, during the perinatal development propionyl-coA and 3-methyl-crotonyl-coA carboxylases followed a pattern of induction similar to pyruvate carboxylase. In kidney, the expression of mitochondrial carboxylases was lower compared to liver and propionyl-coA carboxylase was not detected during the studied period.

Animals↗

PubMed1975

Mode of action of the macrolide-type antibiotic, chlorothricin. Effect of the antibiotic on the catalytic activity and some structural parameters of pyruvate carboxylases purified from rat and chicken liver.

The macrolide-type antibiotic chlorothricin inhibits pyruvate carboxylases purified from rat liver, chicken liver and Azotobacter vinelandii. Under standard assay conditions the concentration of chlorothricin required for half-maximal inhibition of oxalacetate synthesis is 0.26 mM (rat liver), 0.12 mM (chicken liver), and 0.5 mM (Azobacter vinelandii). Inhibition by chlorothricin appears non-competitive in character when measured as a function of the concentration of the substrates of the pyruvate carboxylase reaction as well as of CoASAc and Mg2+. This pattern of inhibition suggests that this antibiotic interacts at unique sites on chicken and rat liver pyruvate carboxylase which are distinct from both the catalytic and activator sites. Interaction of chlorothricin with the two vertebrate liver pyruvate carboxylases differs from the effect exerted by this antibiotic on pyruvate carboxylase purified from Azotobacter vinelandii. A sigmoidal relationship between initial velocity and inhibitor concentration is observed for the vertebrate enzymes under most conditions whereas a hyperbolic profile characterizes the concentration dependence of inhibition of the Azotobacter vinelandii enzyme by chlorothricin. In the case of rat liver pyruvate carboxylase chlorothricin does not alter the extent of cooperativity in the relationship between initial rate and CoASAc concentration. However, a small but significant increase of the Hill coefficient from a value of 2.7 in the absence of antibiotic to that of 3.3 in the presence of 0.5 mM chlorothricin is observed for chicken liver pyruvate carboxylase. Chlorothricin decreases the rate of inactivation observed when rat liver pyruvate carboxylase is incubated with trinitrobenzenesulfonate and when chicken liver pyruvate carboxylase is incubated at 2 degrees C. The maximal decrease in inactivation observed in the presence of saturating concentrations of antibiotic is 50% for cold inactivation of the chicken liver enzyme and 60% for inactivation of the enzyme from rat liver by trinitrobenzenesulfonate. In both cases a sigmoidal relationship is observed between inactivation rate and chlorothricin concentration. These data as well as the initial rate studies suggest that multiple interacting sites for this antibiotic are present on the vertebrate liver pyruvate carboxylases. The occupancy of these sites appears to cause significant distortion of both the catalytic and the activator sites.

Adenosine Triphosphate↗

PubMed1984

Isolation of three cyclic-AMP-independent acetyl-CoA carboxylase kinases from lactating rat mammary gland and characterization of their effects on enzyme activity.

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.

Acetyl-CoA Carboxylase↗

PubMed1990

Acute hormonal control of acetyl-CoA carboxylase. The roles of insulin, glucagon, and epinephrine.

Acetyl-CoA carboxylase, purified from rapidly freeze-clamped livers of rats maintained on a normal laboratory diet and given 0-5 units of insulin shortly before death, gives a major protein band (Mr 265,000) on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The carboxylase from untreated rats has relatively low activity (0.8 unit/mg protein when assayed in the absence of citrate) and high phosphate content (8.5 mol of Pi/mol of subunit), while the enzyme from livers of rats that received 5 units of insulin has higher activity (2.0 units/mg protein) and lower phosphate content (7.0 mol of Pi/mol of subunit). Addition of citrate activates both preparations with half-maximal activation (K0.5) at 1.0 and 0.6 mM citrate, respectively. The enzyme from rats that did not receive insulin is mainly in the octameric state (Mr approximately 2 x 10(6)), while that from rats that received insulin is mainly in the polymeric state (Mr approximately 10 x 10(6)). Thus, short-term administration of insulin results in activation of acetyl-CoA carboxylase, lowering of its citrate requirement, and dephosphorylation and polymerization of the protein. The insulin-induced changes in the carboxylase are probably due to dephosphorylation of the protein since similar changes are observed when the enzyme from rats that did not receive insulin is dephosphorylated by the Mn2(+)-dependent [acetyl-CoA carboxylase]-phosphatase 2. The effect of glucagon or epinephrine administration on acetyl-CoA carboxylase was also investigated. The carboxylase from fasted/refed rats has a relatively high specific activity (3.4 units/mg protein in the absence of citrate), lower phosphate content (4.9 mol of Pi/mol of subunit), and is present mainly in the polymeric state (Mr approximately 10 x 10(6)). Addition of citrate activates the enzyme with K0.5 = 0.2 mM citrate. Glucagon or epinephrine injection of fasted/refed rats yielded carboxylase with lower specific activity (1.4 or 1.9 units/mg, respectively, in the absence of citrate), higher phosphate content (6.4 or 6.7 mol of Pi/mol of subunit, respectively), and mainly in the octameric state (Mr approximately 2 x 10(6)). Treatment of these preparations with [acetyl-CoA carboxylase]-phosphatase 2 reactivated the enzyme (specific activity approximately 8 units/mg protein in the absence of citrate) and polymerized the protein (Mr approximately 10 x 10(6]. These observations indicate that insulin and glucagon, by altering the phosphorylation state of the acetyl-CoA carboxylase, play antagonistic roles in the acetyl-control of its activity and therefore in the regulation of fatty acid synthesis.

Acetyl-CoA Carboxylase↗