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PubMed1989

Formation of malonyl coenzyme A in rat heart. Identification and purification of an isozyme of A carboxylase from rat heart.

Acetyl-CoA carboxylase is thought to be absent in the heart since the latter is highly catabolic and nonlipogenic. It has been suggested that the high level of malonyl-CoA that is found in the heart is derived from mitochondrial propionyl-CoA carboxylase, which also uses acetyl-CoA. In the present study, acetyl-CoA carboxylase was identified and purified from homogenates of rat heart. The isolated enzyme had little activity in the absence of citrate (specific activity, less than 0.1 units/mg); however, citrate stimulated its activity (specific activity, 1.8 units/mg in the presence of 10 mM citrate). Avidin inhibited greater than 95% of activity, and addition of biotin reversed this inhibition. Further, malonyl-CoA (1 mM) and palmitoyl-CoA (100 microM) inhibited greater than 90% of carboxylase activity. Similar to acetyl-CoA carboxylase of lipogenic tissues, the heart enzyme could be activated greater than 6-fold by preincubation with liver (acetyl-CoA carboxylase)-phosphatase 2. The activation was accompanied by a decrease in the K0.5 for citrate to 0.68 mM. These observations suggest that the activity in preparations from heart is due to authentic acetyl-CoA carboxylase. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the preparation from heart showed the presence of one major protein band (Mr 280,000) and a minor band (Mr 265,000) while that from liver gave a major protein band (Mr 265,000). A Western blot probed with avidin-peroxidase suggested that both the 280- and 265-kDa species contained biotin. Antibodies to liver acetyl-CoA carboxylase, which inhibited greater than 95% of liver carboxylase activity, inhibited only 35% of heart enzyme activity. In an immunoblot (using antibodies to liver enzyme) the 265-kDa species, and not the major 280-kDa species, in the heart preparation was specifically stained. These observations suggest the presence of two isoenzymes of acetyl-CoA carboxylase that are immunologically distinct, the 265-kDa species being predominant in the liver and the 280-kDa species being predominant in the heart.

Acetyl-CoA Carboxylase↗

PubMed2001

Tethered processivity of the vitamin K-dependent carboxylase: factor IX is efficiently modified in a mechanism which distinguishes Gla's from Glu's and which accounts for comprehensive carboxylation in vivo.

The vitamin K-dependent (VKD) carboxylase binds VKD proteins via their propeptide and converts Glu's to gamma-carboxylated Glu's, or Gla's, in the Gla domain. Multiple carboxylation is required for activity, which could be achieved if the carboxylase is processive. In the only previous study to test for this capability, an indirect assay was used which suggested processivity; however, the efficiency was poor and raised questions regarding how full carboxylation is accomplished. To unequivocally determine if the carboxylase is processive and if it can account for comprehensive carboxylation in vivo, as well as to elucidate the enzyme mechanism, we developed a direct test for processivity. The in vitro carboxylation of a complex containing carboxylase and full-length factor IX (fIX) was challenged with an excess amount of a distinguishable fIX variant. Remarkably, carboxylation of fIX in the complex was completely unaffected by the challenge protein, and comprehensive carboxylation was achieved, showing conclusively that the carboxylase is processive and highly efficient. These studies also showed that carboxylation of individual fIX/carboxylase complexes was nonsynchronous and implicated a driving force for the reaction which requires the carboxylase to distinguish Glu's from Gla's. We found that the Gla domain is tightly associated with the carboxylase during carboxylation, blocking the access of a small peptide substrate (EEL). The studies describe the first analysis of preformed complexes, and the rate for full-length, native fIX in the complex was equivalent to that of the substrate EEL. Thus, intramolecular movement within the Gla domain to reposition new Glu's for catalysis is as rapid as diffusion-limited positioning of a small substrate, and the Gla domain is not sterically constrained by the rest of the fIX molecule during carboxylation. The rate of carboxylation of fIX in the preformed complex was 24-fold higher than for fIX modified by free carboxylase, which supports carboxylase processivity and which indicates that binding and/or release is the rate-limiting step in protein carboxylation. These data indicate a model of tethered processivity, in which the VKD proteins remain bound to the carboxylase throughout the reaction via their propeptide, while the Gla domain undergoes intramolecular movement to reposition new Glu's for catalysis to ultimately achieve comprehensive carboxylation.

Animals↗

PubMed2001

Function of Escherichia coli biotin carboxylase requires catalytic activity of both subunits of the homodimer.

Biotin carboxylase catalyzes the ATP-dependent carboxylation of biotin and is one component of the multienzyme complex acetyl-CoA carboxylase that catalyzes the first committed step in fatty acid synthesis. The Escherichia coli biotin carboxylase is readily isolated from the other components of the acetyl-CoA carboxylase complex such that enzymatic activity is retained. The three-dimensional structure of biotin carboxylase, determined by x-ray crystallography, demonstrated that the enzyme is a homodimer consisting of two active sites in which each subunit contains a complete active site. To understand how each subunit contributes to the overall function of biotin carboxylase, we made hybrid molecules in which one subunit had a wild-type active site, and the other subunit contained an active site mutation known to significantly affect the activity of the enzyme. One of the two genes encoded a poly-histidine tag at its N terminus, whereas the other gene had an N-terminal FLAG epitope tag. The two genes were assembled into a mini-operon that was induced to give high level expression of both enzymes. "Hybrid" dimers composed of one subunit with a wild-type active site and a second subunit having a mutant active site were obtained by sequential chromatographic steps on columns of immobilized nickel chelate and anti-FLAG affinity matrices. In vitro kinetic studies of biotin carboxylase dimers in which both subunits were wild type revealed that the presence of the N-terminal tags did not alter the activity of the enzyme. However, kinetic assays of hybrid dimer biotin carboxylase molecules in which one subunit had an active site mutation (R292A, N290A, K238Q, or E288K) and the other subunit had a wild-type active site resulted in 39-, 28-, 94-, and 285-fold decreases in the activity of these enzymes, respectively. The dominant negative effects of these mutant subunits were also detected in vivo by monitoring the rate of fatty acid biosynthesis by [(14)C]acetate labeling of cellular lipids. Expression of the mutant biotin carboxylase genes from an inducible arabinose promoter resulted in a significantly reduced rate of fatty acid synthesis relative to the same strain that expressed the wild type gene. Thus, both the in vitro and in vivo data indicate that both subunits of biotin carboxylase are required for activity and that the two subunits must be in communication during enzyme function.

Adenosine Triphosphate↗

PubMed2001

Identification of sequences within the gamma-carboxylase that represent a novel contact site with vitamin K-dependent proteins and that are required for activity.

The vitamin K-dependent (VKD) carboxylase converts clusters of Glu residues to gamma-carboxylated Glu residues (Glas) in VKD proteins, which is required for their activity. VKD precursors are targeted to the carboxylase by their carboxylase recognition site, which in most cases is a propeptide. We have identified a second tethering site for carboxylase and VKD proteins that is required for carboxylase activity, called the vitamin K-dependent protein site of interaction (VKS). Several VKD proteins specifically bound an immobilized peptide comprising amino acids 343-355 of the human carboxylase (CVYKRSRGKSGQK) but not a scrambled peptide containing the same residues in a different order. Association with the 343-355 peptide was independent of propeptide binding, because the VKD proteins lacked the propeptide and because the 343-355 peptide did not disrupt association of a propeptide factor IX-carboxylase complex. Analysis with peptides that overlapped amino acids 343-355 indicated that the 343-345 CVY residues were necessary but not sufficient for prothrombin binding. Ionic interactions were also suggested because peptide-VKD protein binding could be disrupted by changes in ionic strength or pH. Mutagenesis of Cys(343) to Ser and Tyr(345) to Phe resulted in 7-11-fold decreases in vitamin K epoxidation and peptide (EEL) substrate and carboxylase carboxylation, and kinetic analysis showed 5-6-fold increases in K(m) values for the Glu substrate. These results suggest that Cys(343) and Tyr(345) are near the catalytic center and affect the active site conformation required for correct positioning of the Glu substrate. The 343-355 VKS peptide had a higher affinity for carboxylated prothrombin (K(d) = 5 microm) than uncarboxylated prothrombin (K(d) = 60 microm), and the basic VKS region may also facilitate exiting of the Gla product from the catalytic center by ionic attraction. Tethering of VKD proteins to the carboxylase via the propeptide-binding site and the VKS region has important implications for the mechanism of VKD protein carboxylation, and a model is proposed for how the carboxylase VKS region may be required for efficient and processive VKD protein carboxylation.

Amino Acid Sequence↗

PubMed2002

Biotin uptake into human peripheral blood mononuclear cells increases early in the cell cycle, increasing carboxylase activities.

Cells respond to proliferation with increased accumulation of biotin, suggesting that proliferation enhances biotin demand. Here we determined whether peripheral blood mononuclear cells (PBMC) increase biotin uptake at specific phases of the cell cycle, and whether biotin is utilized to increase biotinylation of carboxylases. Biotin uptake was quantified in human PBMC that were arrested chemically at specific phases of the cell cycle, i.e., biotin uptake increased in the G1 phase of the cycle [658 +/- 574 amol biotin/(10(6) cells x 30 min)] and remained increased during phases S, G2, and M compared with quiescent controls [200 +/- 62 amol biotin/(10(6) cells x 30 min)]. The abundance of the sodium-dependent multivitamin transporter (SMVT, which transports biotin) was similar at all phases of the cell cycle, suggesting that transporters other than SMVT or splicing variants of SMVT may account for the increased biotin uptake observed in proliferating cells. Activities of biotin-dependent 3-methylcrotonyl-CoA carboxylase and propionyl-CoA carboxylase were up to two times greater in proliferating PBMC compared with controls. The abundance of mRNA encoding 3-methylcrotonyl-CoA carboxylase and propionyl-CoA carboxylase paralleled carboxylase activities, suggesting that PBMC respond to proliferation with increased expression of genes encoding carboxylases. Similarly, expression of the gene encoding holocarboxylase synthetase (which catalyzes binding of biotin to carboxylases) increased in response to proliferation, suggesting that cellular capacity to biotinylate carboxylases was increased. In summary, these findings suggest that PBMC respond to proliferation with increased biotin uptake early in the cell cycle, and that biotin is utilized to increase activities of two of the four biotin-requiring carboxylases.

Adult↗

PubMed1974

Regulation of Soybean Net Photosynthetic CO(2) Fixation by the Interaction of CO(2), O(2), and Ribulose 1,5-Diphosphate Carboxylase.

Kinetic properties of soybean net photosynthetic CO(2) fixation and of the carboxylase and oxygenase activities of purified soybean (Glycine max [L.] Merr.) ribulose 1, 5-diphosphate carboxylase (EC 4.1.1.39) were examined as functions of temperature, CO(2) concentration, and O(2) concentration. With leaves, O(2) inhibition of net photosynthetic CO(2) fixation increased when the ambient leaf temperature was increased. The increased inhibition of CO(2) fixation at higher temperatures was caused by a reduced affinity of the leaf for CO(2) and an increased affinity of the leaf for O(2). With purified ribulose 1,5-diphosphate carboxylase, O(2) inhibition of CO(2) incorporation and the ratio of oxygenase activity to carboxylase activity increased with increased temperature. The increased O(2) sensitivity of the enzyme at higher temperature was caused by a reduced affinity of the enzyme for CO(2) and a slightly increased affinity of the enzyme for O(2). The similarity of the effect of temperature on the affinity of intact leaves and of ribulose 1,5-diphosphate carboxylase for CO(2) and O(2) provides further evidence that the carboxylase regulates the O(2) response of photosynthetic CO(2) fixation in soybean leaves. Based on results reported here and in the literature, a scheme outlining the stoichiometry between CO(2) and O(2) fixation in vivo is proposed.Oxygen competitively inhibited carboxylase activity with respect to CO(2), and CO(2) competitively inhibited oxygenase activity with respect to O(2). Within the limits of experimental error, the Michaelis constant (CO(2)) in the carboxylase reaction was identical with the inhibition constant (CO(2)) in the oxygenase reaction, and the Michaelis constant (O(2)) in the oxygenase reaction was identical with the inhibition constant (O(2)) in the carboxylase reaction. The Michaelis constant, (ribulose 1,5-diphosphate) was the same in both the carboxylase and oxygenase reactions. This equality of kinetic constants is consistent with the notion that the same enzyme catalyzes both reactions.

Journal Article↗

PubMed1993

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

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.

Acetyl-CoA Carboxylase↗

PubMed1992

Regulatory phosphorylation of Sorghum leaf phosphoenolpyruvate carboxylase. Identification of the protein-serine kinase and some elements of the signal-transduction cascade.

The phosphoenolpyruvate (PPrv) carboxylase isozyme involved in C4 photosynthesis undergoes a day/night reversible phosphorylation process in leaves of the C4 plant, Sorghum. Ser8 of the target enzyme oscillates between a high (light) and a low (dark) phosphorylation status. Both in vivo and in vitro, phosphorylation of dark-form carboxylase was accompanied by an increase in the apparent Ki of the feedback inhibitor L-malate and an increase in Vmax. Feeding detached leaves various photosynthetic inhibitors, i.e. 3-(3,4-dichlorophenyl)-1,1-dimethylurea, gramicidin and DL-glyceraldehyde, prevented PPrv carboxylase phosphorylation in the light, thus suggesting that the cascade involves the photosynthetic apparatus as the light signal receptor, and presumably has the electron transfer chain and the Calvin-Benson cycle as components in the signal-transduction chain. Two protein-serine kinases capable of phosphorylating PPrv carboxylase in vitro have been partially purified from light-adapted leaves. One was isolated on a calmodulin-Sepharose column; it was calcium-dependent but did not require calmodulin for activity. The other was purified on a blue-dextran-agarose column and the only Me2+ required for activity was Mg2+. In reconstituted phosphorylation assays, only the latter caused the expected decrease in malate sensitivity of PPrv carboxylase suggesting that this protein is the genuine PPrv-carboxylase-kinase. Desalted extracts from light-adapted leaves possessed a considerably greater phosphorylation capacity with immunopurified dephosphorylated PPrv carboxylase as substrate than did dark extracts. This light stimulation was insensitive to type 2A protein phosphatase inhibitors, okadaic acid and microcystin-LR, which suggests that the kinase is a controlled step in the cascade which leads to phosphorylation of PPrv carboxylase. The higher phosphorylation capacity of light-adapted leaf tissue was nullified by pretreatment with the cytosolic protein synthesis inhibitor, cycloheximide. Thus, protein turnover is involved as part of the mechanism controlling the activity of the kinase purified on blue-dextran-agarose. However, no information is available with respect to the specific nature of the link between the above-mentioned light transducing steps and the protein kinase that achieves the physiological response. Finally, the in vivo phosphorylation site (Ser8) in the N-terminal region of the C4 type Sorghum PPrv carboxylase is also present in a non-photosynthetic form of the Sorghum enzyme (Ser7), as deduced by cDNA sequence analysis.

Amino Acid Sequence↗

PubMed1996

Normalization by insulin treatment of low mitochondrial glycerol phosphate dehydrogenase and pyruvate carboxylase in pancreatic islets of the GK rat.

The enzyme activity of the mitochondrial glycerol phosphate dehydrogenase (mGPD) in the pancreatic islet has been reported to be less than one-half of normal in the Goto-Kakizaki (GK) rat, a genetic model of NIDDM. In the current study, mGPD enzyme activity and the amount of mGPD protein, as judged by Western analysis, were 35-40% of normal in the islets of these animals. With the exception of pyruvate carboxylase, the activities of other enzymes were not abnormal. The assayable activity and amount of pyruvate carboxylase protein were decreased approximately 50% in the islets of the GK rats. Because mGPD, which is the key enzyme of the glycerol phosphate shuttle, and pyruvate carboxylase, which is the key component of the pyruvate malate shuttle, have been proposed to be essential for stimulus-secretion coupling in the pancreatic beta-cell, an important question is whether the decreases in these enzymes have a causal role in the hyperglycemia or whether the diabetic syndrome caused the decreases. To attempt to differentiate between these two possibilities, GK rats were treated with insulin to normalize their blood sugars. The activities of both mGPD and pyruvate carboxylase were also normalized by insulin treatment. An incidental discovery of this study was the identification of a high level of propionyl-CoA carboxylase activity and a lesser amount of methylcrotonyl-CoA carboxylase activity in pancreatic islets. These enzymes were normal in the islets of the GK rats. This is the first report on the presence of these two carboxylases in the islet and of low pyruvate carboxylase activity in the islet in NIDDM. We conclude that the decreased mGPD and pyruvate carboxylase in the pancreatic islet of the GK rat result from the diabetic syndrome.

Animals↗

PubMed1991

Acetyl-coenzyme A carboxylase messenger ribonucleic acid metabolism in liver, adipose tissues, and mammary glands during pregnancy and lactation.

In the rat, the acetyl-coenzyme A carboxylase gene exists as a single copy per haploid chromosome set. However, multiple forms of acetyl-coenzyme A carboxylase mRNA exist, the relative abundance of which varies in a tissue-specific manner under different physiological conditions. In the mammary gland, the major acetyl-coenzyme A carboxylase mRNA species are of the class 2 type, which are products of promoter II. In parametrial white adipose tissue, the main form of species of acetyl-coenzyme A carboxylase is of the class 1 type, which are produced by promoter I. Pregnancy and lactation affect the amounts of these acetyl-coenzyme A carboxylase mRNA. Although the mammary gland acetyl-coenzyme A carboxylase mRNA species increase dramatically upon parturition, the parametrial white adipose tissue forms decrease precipitously at the same time and are not expressed at all during the lactation period. In the liver of these animals, the only form of acetyl-coenzyme A carboxylase mRNA that is expressed is the FL56 form; this form shows a modest decrease during pregnancy that is slowly reversed during lactation. These observations indicate that the changes in lipogenesis that occur during pregnancy and lactation are determined by the transcriptional activity of the acetyl-coenzyme A carboxylase gene. In order to analyze the complex transcriptional activity of this gene in a meaningful way, it is necessary to examine the metabolism of individual isoforms of acetyl-coenzyme A carboxylase mRNA.

Acetyl-CoA Carboxylase↗

PubMed1989

Regulation of acetyl CoA carboxylase mRNA in rat liver by high carbohydrate diet and insulin.

Acetyl CoA carboxylase contents in liver cytosol of rats refed a high carbohydrate diet or injected with insulin were measured by an immunoassay method in order to evaluate the effects of dietary carbohydrate and insulin treatment on the control in the amount of acetyl CoA carboxylase. Acetyl CoA carboxylase was purified 1,552 folds with a specific activity of 3.88 units/mg protein from livers of rats refed a high carbohydrate diet for 3 days following a 3-day fasting and the antibody was generated against the purified acetyl CoA carboxylase in a rabbit. Treatment of insulin (1.5 units/100g BW) and a high carbohydrate diet increased the amount of acetyl CoA carboxylase in liver cytosol by 3 times and 10 times, respectively, when compared to the enzyme content found in the control. The synthetic ratio of acetyl CoA carboxylase to total cytosolic proteins was 4 times higher in the insulin-treated group and 10 times higher in the high carbohydrated diet-treated group than the control group. The polysomal RNA contents in liver cytosols were 279% of the control in the insulin-treated group and 365% of the control in the high carbohydrate diet group. Also, the nascent chain of acetyl CoA carboxylase in polysome were 158% of the control in the insulin-treated group and 311% of the control in the high carbohydrate treated group. From these results, it is assumed that the increase of acetyl CoA carboxylase content in the rat liver cells by insulin treatment, or high carbohydrate diet refeeding has resulted from the increased polysomal acetyl CoA carboxylase mRNA, which is directly related to the biosynthesis of this enzyme.

Acetyl-CoA Carboxylase↗

PubMed1989

Heterogeneity at the 5' end of rat acetyl-coenzyme A carboxylase mRNA. Lipogenic conditions enhance synthesis of a unique mRNA in liver.

Multiple forms of acetyl-CoA carboxylase mRNA were previously detected in the mammary gland (Lopez-Casillas, F., Luo, X., Kong, I.-S., and Kim, K.-H. (1989) Gene, in press). We have now established that the rat liver also contains heterogeneous acetyl-CoA carboxylase mRNA populations that differ in the 5'-untranslated region. In addition, the liver contains a unique form of acetyl-CoA carboxylase mRNA in which the 5'-nontranslated end differs from the species in mammary gland. The 5' end of this unique species was characterized using a procedure for cloning minute amounts of primer extension products (pAU clones). This procedure should also be useful for obtaining full length clones of other mRNAs. The DNA sequence of pAU clones indicates that this liver-specific acetyl-CoA carboxylase mRNA has a 315-base long untranslated region. The first 242 nucleotides replace the 5' end of the predominant acetyl-CoA carboxylase mRNA found in the mammary gland (FL56 type). Under lipogenic conditions the unique liver acetyl-CoA carboxylase mRNA increases and is the major species of acetyl-CoA carboxylase mRNA. Livers from rats fed a normal diet and the mammary glands of lactating rats do not contain detectable amounts of the pAU type mRNA. On the other hand, the epididymal adipose tissue from these animals contains mainly the pAU type and only minimal amounts of the FL56 type. The multiple forms of acetyl-CoA carboxylase mRNA appear to be generated by differential splicing. In addition, transcription appears to be physiologically regulated by the use of tissue-specific acetyl-CoA carboxylase gene promoters.

Acetyl-CoA Carboxylase↗

PubMed1988

Regulation of acetyl-coenzyme A carboxylase. II. Effect of fasting and refeeding on the activity, phosphate content, and aggregation state of the enzyme.

Acetyl-CoA carboxylase isolated from freeze-clamped livers of fed rats has relatively low phosphate content (5.0 mol of Pi/mol of subunit) and high specific activity (3.5 units/mg in the absence of citrate). The enzyme from rats fasted for 12, 18, 24, and 48 h exhibited decreasing specific activities of 2.75, 1.85, 1.7, and 0.9 units/mg, respectively. Citrate activated all preparations of carboxylase, with most activation observed with the least active preparation. There was no significant change in the sensitivity of the enzyme to citrate since half-maximal activation was observed at 0.2 mM for carboxylase from fed as well as fasted rats. With the decrease in activity as a function of fasting, there was a concomitant increase in the phosphate content of carboxylase, with values of 5.3, 5.6, 6.7, and 7.6 mol of Pi/mol of subunit obtained for preparations from rats fasted for 12, 18, 24, and 48 h, respectively. Refeeding the fasted rats resulted in increased specific activity of carboxylase (3.4 units/mg) and decreased phosphate content (5.1 mol of Pi/mol of subunit). Moreover, dephosphorylation by [acetyl-CoA carboxylase]-phosphatase 2 activated the carboxylase from 48-h fasted rats to a value of 2.9 units/mg, assayed in the absence of citrate, indicating that the low activity of carboxylase from fasted rats was due to its increased phosphate content. Superose 6 chromatography showed that the enzyme exists in two polymeric forms, a highly active polymer of greater than or equal to 40 subunits and less active octamer. The former predominates in livers of fed rats, whereas the latter predominates in livers of fasted rats. The octamer could be converted to the highly active polymer by dephosphorylation. These observations indicate that fasting/refeeding results in phosphorylation/dephosphorylation of acetyl-CoA carboxylase with concomitant depolymerization/polymerization of the protein and ultimately decreasing or increasing its specific activity.

Acetyl-CoA Carboxylase↗

PubMed1982

Purification and properties of a kinase which phosphorylates and inactivates acetyl-CoA carboxylase.

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.

Acetyl-CoA Carboxylase↗

PubMed1981

[Multiple molecular forms of ribulose diphosphate carboxylase from mung bean leaves].

Ribulose diphosphate carboxylase from Phaseolus aureus leaves is represented by four molecular forms differing in their carboxylase and oxygenase activities, molecular weights, sedimentation coefficients, electrophoretic mobilities in polyacrylamide gel, chromatographic behaviour upon elution on Sephadex G-200 and DEAE-cellulose, pH optima of carboxylase activity and pI values. In some parameters, e. g. molecular weight of the whole molecule (530 000), molecular weights of the large and the pH optima of the carboxylase and oxygenase activities (8.5 and oxygenase activities (2.4 and 0.09 units per mg of protein, respectively) and the pH optima of the carboxylase and oxygenase activities (8.5 and 9.3, respectively) form II differs from form I, which has a lower molecular weight (480 000), a higher oxygenase and a lower carboxylase activities (0.2 and 1.5 units per mg of protein, respectively) and a higher pH optimum of the carboxylase activity (9.0). Both forms I and II are composed of eight large and eight small subunits. The molecular weight of the large subunits of form I is lower than that of form II and is equal to 48 000. The respective pI values for forms I and II are 6.8 and 6.4. Forms III and IV as well as forms I and II produce distinct bands under ion-exchange chromatography on DEAE-cellulose. The carboxylase and oxygenase activities of form III are 0.7 and 0.04 units per mg of protein, respectively. Form IV has a higher oxygenase and a lower carboxylase activity (0.09 and 0.25 units per mg of protein, respectively). The corresponding molecular weights of forms III and IV are 530 000 and 480 000. The molecular weights of the large and small subunits of form II are 53 000 and 12 000, respectively; those of form IV -- 48 000 and 12 000, respectively.

Carboxy-Lyases↗

PubMed1999

Inhibition of biotin carboxylase by a reaction intermediate analog: implications for the kinetic mechanism.

The first committed step in long-chain fatty acid synthesis is catalyzed by the multienzyme complex acetyl CoA carboxylase. One component of the acetyl CoA carboxylase complex is biotin carboxylase which catalyzes the ATP-dependent carboxylation of biotin. The Escherichia coli form of biotin carboxylase can be isolated from the other components of the acetyl CoA carboxylase complex such that enzymatic activity is retained. The synthesis of a reaction intermediate analog inhibitor of biotin carboxylase has been described recently (Organic Lett. 1, 99-102, 1999). The inhibitor is formed by coupling phosphonoacetic acid to the 1'-N of biotin. In this paper the characterization of the inhibition of biotin carboxylase by this reaction-intermediate analog is described. The analog showed competitive inhibition versus ATP with a slope inhibition constant of 8 mM. Noncompetitive inhibition was found for the analog versus biotin. Phosphonoacetate exhibited competitive inhibition with respect to ATP and noncompetitive inhibition versus bicarbonate. Biotin was found to be a noncompetitive substrate inhibitor of biotin carboxylase. These data suggested that biotin carboxylase had an ordered addition of substrates with ATP binding first followed by bicarbonate and then biotin.

Adenosine Triphosphate↗

PubMed1970

Immunochemical studies with soluble and mitochondrial pyruvate carboxylase activities from rat tissues.

1. Pyruvate carboxylase (EC 6.4.1.1), purified from rat liver mitochondria to a specific activity of 14 units/mg, was used for the preparation of antibodies in rabbits. 2. Tissue distribution studies showed that pyruvate carboxylase was present in all rat tissues that were tested, with considerable activities both in gluconeogenic tissues such as liver and kidney and in tissues with high rates of lipogenesis such as white adipose tissue, brown adipose tissue, adrenal gland and lactating mammary gland. 3. Immunochemical titration experiments with the specific antibodies showed no differences between the inactivation of pyruvate carboxylase from mitochondrial or soluble fractions of liver, kidney, mammary gland, brown adipose tissue or white adipose tissue. 4. The antibodies were relatively less effective in reactions against pyruvate carboxylase from sheep liver than against the enzyme from rat tissues. 5. Pyruvate carboxylase antibodies did not inactivate either propionyl-CoA carboxylase or acetyl-CoA carboxylase from rat liver. 6. It is concluded that pyruvate carboxylase in lipogenic tissues is similar antigenically to the enzyme in gluconeogenic tissues and that the soluble activities of pyruvate carboxylase detected in many rat tissues do not represent discrete enzymes but are the result of mitochondrial damage during tissue homogenization.

Adipose Tissue↗

PubMed1989

Vitamin K-dependent carboxylase: affinity purification from bovine liver by using a synthetic propeptide containing the gamma-carboxylation recognition site.

The vitamin K-dependent carboxylase catalyzes the posttranslational modification of specific glutamic acid residues to form gamma-carboxyglutamic acid residues within the vitamin K-dependent proteins. This enzyme recognizes the gamma-carboxylation recognition site on the propeptide of the precursor forms of the vitamin K-dependent blood coagulation proteins. To purify this enzyme to homogeneity, the carboxylase from bovine liver microsomes was solubilized with 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS), the protein was fractionated with ammonium sulfate, and then the enzyme was isolated by affinity chromatography using a synthetic peptide based upon the structure of the prothrombin propeptide. Elution with 10 mM propeptide yielded a single major band on SDS gel electrophoresis with a molecular weight of 77,000. In the presence of high concentrations of propeptide, only minimal carboxylase activity was measurable. Antibodies to the protein inhibited the carboxylase activity in crude preparations. In an alternative affinity purification strategy the propeptide was coupled through an NH2-terminal cysteine to an activated thiol-Sepharose column. The carboxylase-propeptide complex was eluted at 25 degrees C by reductive cleavage of the enzyme-propeptide complex in the presence of detergent and phospholipids. The eluted protein (Mr, 77,000) contained both stable vitamin K-dependent carboxylase and vitamin K epoxidase activity. The protein, purified by either method, was detected as a single band (Mr, 77,000) in a Western blot using anti-carboxylase antibodies. A 10,000-fold purification of carboxylase activity from crude microsomes was estimated. Purified bovine liver vitamin K-dependent carboxylase should facilitate the study of its structure and of the mechanism of action of vitamin K as a cofactor in the reaction catalyzed by this enzyme.

Amino Acid Sequence↗