Application of preparative disk gel electrophoresis for antigen purification from inclusion bodies.

Specific antibodies are a reliable tool to examine protein expression patterns and to determine the protein localizations within cells. Generally, recombinant proteins are used as antigens for specific antibody production. However, recombinant proteins from mammals and plants are often overexpressed as insoluble inclusion bodies in Escherichia coli. Solubilization of these inclusion bodies is desirable because soluble antigens are more suitable for injection into animals to be immunized. Furthermore, highly purified proteins are also required for specific antibody production. Plastidic acetyl-CoA carboxylase (ACCase: EC 6.4.1.2) from Arabidopsis thaliana, which catalyzes the formation of malonyl-CoA from acetyl-CoA in chloroplasts, formed inclusion bodies when the recombinant protein was overexpressed in E. coli. To obtain the purified protein to use as an antigen, we applied preparative disk gel electrophoresis for protein purification from inclusion bodies. This method is suitable for antigen preparation from inclusion bodies because the purified protein is recovered as a soluble fraction in electrode running buffer containing 0.1% sodium dodecyl sulfate that can be directly injected into immune animals, and it can be used for large-scale antigen preparation (several tens of milligrams).

Human acetyl-CoA carboxylase 2 expressed in silkworm Bombyx mori exhibits posttranslational biotinylation and phosphorylation.

Biotin-dependent human acetyl-CoA carboxylases (ACCs) are integral in homeostatic lipid metabolism. By securing posttranslational biotinylation, ACCs perform coordinated catalytic functions allosterically regulated by phosphorylation/dephosphorylation and citrate. The production of authentic recombinant ACCs is heeded to provide a reliable tool for molecular studies and drug discovery. Here, we examined whether the human ACC2 (hACC2), an isoform of ACC produced using the silkworm BmNPV bacmid system, is equipped with proper posttranslational modifications to carry out catalytic functions as the silkworm harbors an inherent posttranslational modification machinery. Purified hACC2 possessed genuine biotinylation capacity probed by biotin-specific streptavidin and biotin antibodies. In addition, phosphorylated hACC2 displayed limited catalytic activity whereas dephosphorylated hACC2 revealed an enhanced enzymatic activity. Moreover, hACC2 polymerization, analyzed by native page gel analysis and atomic force microscopy imaging, was allosterically regulated by citrate and the phosphorylation/dephosphorylation modulated citrate-induced hACC2 polymerization process. Thus, the silkworm BmNPV bacmid system provides a reliable eukaryotic protein production platform for structural and functional analysis and therapeutic drug discovery applications implementing suitable posttranslational biotinylation and phosphorylation.

The human ACC2 CT-domain C-terminus is required for full functionality and has a novel twist.

Inhibition of acetyl-CoA carboxylase (ACC) may prevent lipid-induced insulin resistance and type 2 diabetes, making the enzyme an attractive pharmaceutical target. Although the enzyme is highly conserved amongst animals, only the yeast enzyme structure is available for rational drug design. The use of biophysical assays has permitted the identification of a specific C-terminal truncation of the 826-residue human ACC2 carboxyl transferase (CT) domain that is both functionally competent to bind inhibitors and crystallizes in their presence. This C-terminal truncation led to the determination of the human ACC2 CT domain-CP-640186 complex crystal structure, which revealed distinctions from the yeast-enzyme complex. The human ACC2 CT-domain C-terminus is comprised of three intertwined alpha-helices that extend outwards from the enzyme on the opposite side to the ligand-binding site. Differences in the observed inhibitor conformation between the yeast and human structures are caused by differing residues in the binding pocket.

Acetyl-CoA carboxylases 1 and 2 show distinct expression patterns in rats and humans and alterations in obesity and diabetes.

BACKGROUND: Acetyl-CoA carboxylases (ACC) 1 and 2 are central enzymes in lipid metabolism. To further investigate their relevance for the development of obesity and type 2 diabetes, expression of both ACC isoforms was analyzed in obese fa/fa Zucker fatty and Zucker diabetic fatty rats at different ages in comparison to Zucker lean controls. METHODS: ACC1 and ACC2 transcript levels were measured by quantitative real-time polymerase chain reaction in metabolically relevant tissues of Zucker fatty, Zucker diabetic fatty and Zucker lean control animals. Quantitative real-time polymerase chain reaction was also applied to measure ACC tissue distribution in human tissues. For confirmation on a protein level, quantitative mass spectrometry was used. RESULTS: Disease-related transcriptional changes of both ACC isoforms were observed in various tissues of Zucker fatty and Zucker diabetic fatty rats including liver, pancreas and muscle. Changes were most prominent in oxidative tissues of diabetic rats, where ACC2 was significantly increased and ACC1 was reduced compared with Zucker lean control animals. A comparison of the overall tissue distribution of both ACC isoforms in humans and rats surprisingly revealed strong differences. While in rats ACC1 was mainly expressed in lipogenic and ACC2 in oxidative tissues, ACC2 was predominant in oxidative and lipogenic tissues in humans. CONCLUSION: Our data support a potential role for both ACC isoforms in the development of obesity and diabetes in rats. However, the finding of fundamental species differences in ACC1 and ACC2 tissue expression might be indicative for different functions of both isoforms in humans and rats and raises the question to which degree these models are predictive for the physiology and pathophysiology of lipid metabolism in humans.

A biotin-based protein tagging system.

Biotin protein ligase (BPL) mediates covalent attachment of biotin to a specific lysine residue of biotin carboxyl carrier protein (BCCP) of biotin-dependent enzymes. We recently found that the biotinylation reaction from thermophilic archaeon Sulfolobus tokodaii has a unique characteristic that the enzyme BPL forms a tight complex with the product, biotinylated BCCP (169 amino acid residues). In the current work, we attempted to apply this characteristic to a novel protein tagging system. Thus, the N terminus of S. tokodaii BCCP was truncated and the interaction of the resulting BCCP, BCCPDelta100 and BCCPDelta17 (with 69 and 152 residues, respectively), with BPL was investigated by surface plasmon resonance (SPR). It was found that the binding of BPL to the biotinylated BCCPDelta100 is extremely tight with a dissociation constant (K(D)) of 1.2 nM, whereas that to the unbiotinylated counterpart was moderate with a K(D) of 3.3 microM. Furthermore, chimeric proteins of glutathione S-transferase (GST) and green fluorescence protein (GFP) with BCCPDelta100 fused to their C terminus were prepared. The resulting fusion proteins were successfully biotinylated and captured on the BPL-modified SPR sensor chip or BPL-modified magnetic beads. The function of GST and GFP was hardly impaired on fusion with BCCPDelta100 and biotinylation of the latter.

Biotinoyl domain of human acetyl-CoA carboxylase: Structural insights into the carboxyl transfer mechanism.

Acetyl-CoA carboxylase (ACC) catalyzes the first step in fatty acid biosynthesis: the synthesis of malonyl-CoA from acetyl-CoA. As essential regulators of fatty acid biosynthesis and metabolism, ACCs are regarded as therapeutic targets for the treatment of metabolic diseases such as obesity. In ACC, the biotinoyl domain performs a critical function by transferring an activated carboxyl group from the biotin carboxylase domain to the carboxyl transferase domain, followed by carboxyl transfer to malonyl-CoA. Despite the intensive research on this enzyme, only the bacterial and yeast ACC structures are currently available. To explore the mechanism of ACC holoenzyme function, we determined the structure of the biotinoyl domain of human ACC2 and analyzed its characteristics and interaction with the biotin ligase, BirA using NMR spectroscopy. The 3D structure of the hACC2 biotinoyl domain has a similar folding topology to the earlier determined domains from E. coli and P. shermanii. However, the local structures near the biotinylation sites have notable differences that include the geometry of the consensus "Met-Lys-Met" (MKM) motif and the absence of "thumb" structure in the hACC2 biotinoyl domain. Observations of the NMR signals upon the biotinylation indicate that the biotin group of hACC2 does not affect the structure of the biotinoyl domain, while the biotin group for E. coli ACC interacts directly with the thumb residues that are not present in the hACC2 structure. These results imply that, in the E. coli ACC reaction, the biotin moiety carrying the carboxyl group from BC to CT can pause at the thumb of the BCCP domain. The human biotinoyl domain, however, lacks the thumb structure and does not have additional noncovalent interactions with the biotin moiety; thus, the flexible motion of the biotinylated lysine residue must underlie the "swinging arm" motion. The chemical shift perturbation and the cross saturation experiments of the human ACC2 holo-biotinoyl upon the addition of the biotin ligase (BirA) showed the interaction surface near the MKM motif, the two glutamic acids (Glu 926, Glu 953), and the positively charged residues (several lysine and arginine residues). This study provides insight into the mechanism of ACC holoenzyme function and supports the swinging arm model in human ACCs.

A unique biotin carboxyl carrier protein in archaeon Sulfolobus tokodaii.

Biotin carboxyl carrier protein (BCCP) is one subunit or domain of biotin-dependent enzymes. BCCP becomes an active substrate for carboxylation and carboxyl transfer, after biotinylation of its canonical lysine residue by biotin protein ligase (BPL). BCCP carries a characteristic local sequence surrounding the canonical lysine residue, typically -M-K-M-. Archaeon Sulfolobus tokodaii is unique in that its BCCP has serine replaced for the methionine C-terminal to the lysine. This BCCP is biotinylated by its own BPL, but not by Escherichia coli BPL. Likewise, E. coli BCCP is not biotinylated by S. tokodaii BPL, indicating that the substrate specificity is different between the two organisms.

In vitro phosphorylation and inactivation of rat liver acetyl-CoA carboxylase purified by avidin affinity chromatography.

Acetyl-CoA carboxylase (EC 6.4.1.2) has been isolated from rat liver by an avidin-affinity chromatography technique. This preparation has a specific activity of 1.17 +/- 0.06 U/mg and appears as a major (240,000 dalton) and minor (140,000 dalton) band on SDS-polyacrylamide gel electrophoresis. Enzyme isolated by this technique can incorporate 1.09 +/- 0.07 mol phosphate per mol enzyme (Mr = 480,000) when incubated with the catalytic subunit of the cyclic AMP-dependent protein kinase at 30 degrees C for 1 h. The associated activity loss under these conditions is 57 +/- 4.0% when the enzyme is assayed in the presence of 2.0 mM citrate. Less inactivation is observed when the enzyme is assayed in the presence of 5.0 mM citrate. The specific protein inhibitor of the cyclic AMP-dependent protein kinase blocks both the protein kinase stimulated phosphorylation and inactivation of acetyl-CoA carboxylase. The phosphorylated, inactivated rat liver carboxylase can be partially dephosphorylated and reactivated by incubation with a partially purified protein phosphatase. Preparations of acetyl-CoA carboxylase also contained an endogenous protein kinase(s) which incorporated 0.26 +/- 0.11 mol phosphate per mol carboxylase (Mr = 480,000) accompanied by a 26 +/- 9% decline in activity. We have additionally confirmed that the rat mammary gland enzyme, also isolated by avidin affinity chromatography, can be both phosphorylated and inactivated upon incubation with the cyclic AMP-dependent kinase.

Affinity labelling of rat liver acetyl-CoA carboxylase by a 2',3'-dialdehyde derivative of ATP.

The interaction of rat liver acetyl-CoA carboxylase with a 2',3'-dialdehyde derivative of ATP (oATP) has been studied. The degree of the enzyme inactivation has been found to depend on the oATP concentration and the incubation time. ATP was proved to be the only substrate which protected the inactivation. Acetyl-CoA did not effect inactivation, while HCO3- accelerated the process. Ki values for oATP in the absence and presence of HCO3- were 0.35 +/- 0.04 and 0.5 +/- 0.06 mM, and those of the modification constant (kmod) were 0.11 and 0.26 min-1 respectively. oATP completely inhibited the [14C]ADP in equilibrium ATP exchange and did not effect the [14C]acetyl-CoA in equilibrium malonyl-CoA exchange. Incorporation of approximately 1 equivalent of [3H]oATP per acetyl-CoA carboxylase subunit has been shown. No recovery of the modified enzyme activity has been observed in Tris or beta-mercaptoethanol containing buffers, and treatment with NaB3H4 has not led to 3H incorporation. The modification elimination of the ATP triphosphate chain. The results indicated the affinity modification of acetyl-CoA carboxylase by oATP. It was shown that the reagent apparently interacted selectively with the epsilon-amino group of lysine in the ATP-binding site to form a morpholine-like structure.

Stabilization of an acetyl-coenzyme A carboxylase complex from Pseudomonas citronellolis.

Using stabilizing conditions the acetyl-CoA carboxylase (EC 6.4.1.2) of Pseudomonas citronellolis has been isolated as a complex containing four different polypeptide chains with molecular weights of 53 000, 36 000, 33 000 and 25 000. Evidence is presented to suggest that these polypeptide chains correspond to distinct biotin carboxylase, transcarboxylase and biotin carboxyl carrier protein subunits in analogy with similar subunits of Escherichia coli acetyl-CoA carboxylase, an unstable complex in vitro.

Preliminary X-ray crystallographic analysis of biotin carboxylase isolated from Escherichia coli.

Acetyl CoA carboxylase catalyzes the first committed step in the biosynthesis of long chain fatty acids. In Escherichia coli, the enzyme consists of three subunits that are isolated separately and display distinct functional properties. Here we report the crystallization and preliminary X-ray analysis of one of these components, namely biotin carboxylase. The crystals are grown by microdialysis against 10 mM potassium phosphate (pH 7.0), 1 mM EDTA, 2 mM DTT and 1 mM NaN3 at 4 degrees C. They belong to the space group P2(1)2(1)2(1) with unit cell dimensions of a = 61.9 A, b = 96.1 A and c = 180.6 A and contain one dimer per asymmetric unit. The crystals diffract to a nominal resolution of 2.2 A. From a mechanistic standpoint, biotin carboxylase is especially interesting in that it is the smallest protein within its class and is one of only two carboxylases that can utilize free biotin as a substrate.

Biotin carboxyl carrier protein co-purifies as a contaminant in core-streptavidin preparations.

We have successfully cloned and expressed core-streptavidin in Escherichia coli. Core-streptavidin was expressed in shaker flask culture as a soluble protein, isolated by periplasmic extraction, purified by immobilized metal affinity chromatography column, and analyzed for its size, thermal stability, and biotin-binding activity. In Western blots using streptavidin-horseradish peroxidase (HRP) as a probe, we identified a contaminant that co-purified with core-streptavidin, identified as biotin carboxyl carrier protein (BCCP). Although BCCP cannot be detected on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, it appears as a prominent band in Western blot when probed with streptavidin peroxidase conjugate. Based on the results from in vitro gel digestion, mass spectrometry and Mascot database search results, we confirmed the presence of BCCP. It was found that BCCP can complex with core-streptavidin and can dissociate when heated above 80 degrees C. BCCP could be successfully removed and recovered by using core-streptavidin immobilized magnetic beads under mild conditions. In addition, the enriched fractions of core-streptavidin oligotetramers were separated, which may be the by-products of BCCP binding to core-streptavidin in various ratios. Finally, enzyme linked immunosorbent assay results have shown that the amount of biotin-HRP binding to core-streptavidin was higher compared to commercially available streptavidin.

Graminicide insensitivity correlates with herbicide-binding co-operativity on acetyl-CoA carboxylase isoforms.

The sensitivity of grass species to important classes of graminicide herbicides inhibiting ACCase (acetyl-CoA carboxylase) is associated with a specific inhibition of the multifunctional ACCase located in the plastids of grasses. In contrast, the multisubunit form of ACCase found in the chloroplasts of dicotyledonous plants is insensitive and the minor cytosolic multifunctional isoforms of the enzyme in both types of plants are also less sensitive to inhibition. We have isolated, separated and characterized the multifunctional ACCase isoforms found in exceptional examples of grasses that are either inherently insensitive to these graminicides, or from biotypes showing acquired resistance to their use. Major and minor multifunctional enzymes were isolated from cell suspension cultures of Festuca rubra and the 'Notts A1'-resistant biotype of Alopecurus myosuroides, and their properties compared with those isolated from cells of wild-type sensitive A. myosuroides or from sensitive maize. Purifications of up to 300-fold were necessary to separate the two isoforms. The molecular masses (200-230 kDa) and K(m) values for all three substrates (ATP, bicarbonate and acetyl-CoA) were similar for the different ACCases, irrespective of their graminicide sensitivity. Moreover, we found no correlation between the ability of isoforms to carboxylate propionyl-CoA and their sensitivity to graminicides. However, insensitive purified forms of ACCase were characterized by herbicide-binding co-operativity, whereas, in contrast, sensitive forms of the enzymes were not. Our studies on isolated individual isoforms of ACCase from grasses support and extend previous indications that herbicide binding co-operativity is the only kinetic property that differentiates naturally or selected insensitive enzymes from the typical sensitive forms usually found in grasses.

Analytical and micropreparative peptide mapping by high performance liquid chromatography/electrospray mass spectrometry of proteins purified by gel electrophoresis.

We report the use of microbore reverse-phase high performance liquid chromatography connected on-line to an electrospray mass spectrometer for the separation/detection of peptides derived by proteolytic digestion of proteins separated by polyacrylamide gel electrophoresis. A small fraction (typically 10% of the total) of the peptides eluting from the column was diverted through a flow-splitting device into the ion source of the mass spectrometer, whereas the majority of the peptide samples was collected for further analyses. We demonstrate the feasibility of obtaining reproducible peptide maps from submicrogram amounts of protein applied to the gel and good correlation of the signal detected by the mass spectrometer with peptide detection by UV absorbance. Furthermore, independently verifiable peptide masses were determined from subpicomole amounts of peptides directed into the mass spectrometer. The method was used to analyze the 265-kDa and the 280-kDa isoforms of the enzyme acetyl-CoA carboxylase isolated from rat liver. The results provide compelling evidence that the two enzyme isoforms are translation products of different genes and suggest that these approaches may be of general utility in the definitive comparison of protein isoforms. We furthermore illustrate that knowledge of peptide masses as determined by this technique provides a major advantage for error-free data interpretation in chemical high-sensitivity peptide sequence analysis.

Non-specific acetyl-CoA carboxylase and methylmalonyl-CoA carboxyltransferase in Streptomyces noursei var. polifungini.

1. Acetyl-CoA carboxylase (EC 6.4.1.2) and methylmalonyl-CoA carboxyltransferase (EC 2.1.3.1) have been isolated from mycelia of Streptomyces noursei var. polifungini, and purified about 50-fold. 2. Both enzymes carboxylate acetyl-CoA and propionyl-CoA; the respective Km values are 1.1 and 1.6 mM with acetyl-CoA carboxylase and 2.5 and 1.25 mM with carboxyltransferase. 3. The activities of both enzymes are inhibited by free fatty acids. Almost total inhibition of methylmalonyl-CoA carboxyltransferase was observed by 0.1 mM-butyrate or 0.1 mM-C14-C18 acids. Acetyl-CoA carobxylase was affected to the same extent by these compounds at concentration of about 1 mM. 4. The role of both carboxylating enzymes is biosynthesis of the antibiotic is discussed.

Coenzyme A esters of 2-aryloxyphenoxypropionate herbicides and 2-arylpropionate antiinflammatory drugs are potent and stereoselective inhibitors of rat liver acetyl-CoA carboxylase.

The CoA esters of diclofop, haloxyfop and fluazifop are up to 425-fold more potent than the corresponding unconjugated herbicides as inhibitors of rat liver acetyl-CoA carboxylase (EC 6.4.1.2); the most potent inhibitor is (R)-fluazifopyl-CoA2 (Ki = 0.03 microM). The binding site is stereoselective for (R)-diclofop, the herbicidally active enantiomer, and for (R)-diclofopyl-CoA. The CoA esters of the antiinflammatory drugs ibuprofen and fenoprofen also strongly inhibit this carboxylase. (S)-Ibuprofenyl-CoA (Ki = 0.7 microM), the CoA ester of the enantiomer with antiinflammatory activity, is 15-fold more potent as an inhibitor than (R)-ibuprofenyl-CoA. These results suggest that some of the biological effects of these herbicides and antiinflammatory drugs in animals may be due to the inhibition of acetyl-CoA carboxylase by their acyl-CoA derivatives.

Rat-liver fatty-acid-synthesizing complex.

Under conditions favoring lipogenesis, a high-molecular-weight species of acetyl-CoA carboxylase was isolated that did not co-sediment with the in vitro polymerized enzyme. Assays for ATP-citrate lyase, acetyl-CoA carboxylase, and fatty acid synthetase indicated that all three enzymes were associated together as a high-molecular-weight complex and that under low-lipogenic conditions the level of these enzymes decreased. Phosphorylation of the isolated complex shifted it toward a lower molecular weight.

Isolation and identification of acetyl-CoA carboxylase from rainbow trout (Salmo gairdneri) liver.

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)

Alterations of hepatic acetyl-CoA carboxylase by 2,3,7,8-tetrachlorodibenzo-p-dioxin.

The effect of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) on acetyl-CoA carboxylase (ACC) activity and synthesis was examined. Male Wistar rats received a single i.p. injection of TCDD (53 micrograms/kg), and nine days later body weight, liver weight, hepatic lipid, ACC activity and mass were determined and compared to pair-fed controls. Body weights of TCDD-treated animals decreased, while liver weights increased resulting in an increase in liver to body weight ratios. ACC activity was decreased by 65%, however sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western analysis using a biotin specific probe revealed that ACC protein levels were not appreciably changed. In addition, there was a large increase in exogenous lipid material in TCDD-treated livers as determined by osmium tetroxide staining. These data suggest that the decrease in ACC activity may be due to direct inhibition of the enzyme by negative allosteric interactions with free fatty acids released from adipose tissue that subsequently accumulate in liver.

[Properties and biosynthesis of acetyl-CoA-carboxylase from chicken liver with administration of nicotinic acid during stimulation of lipogenesis]. / Svoistva i biosintez atsetil-CoA-karboksilazy pecheni tsypliat pri vvedenii nikotinovoi kisloty na fone stimuliatsii lipogeneza.

Acetyl-CoA-carboxylase is isolated and purified to a homogeneous state from the chicken liver with alimentary lipogenesis stimulation. Under the action of nicotinic acid in vivo the specific enzyme activity is shown to decrease considerably followed by some variations in its properties. According to the results obtained during ultracentrifugation and PAAG electrophoresis nicotinic acid causes partial enzyme deaggregation with simultaneous increase of its phosphorylation. The latter is accompanied by a rise in the content of phosphate labile to alkali on acetyl-CoA-carboxylase subunits. Nicotinic acid in vivo has practically no effect on acetyl-CoA-carboxylase synthesis and decay rate. Its inhibiting action is induced by stimulation of enzyme phosphorylation.