Biotin Carboxyl Carrier Protein Subunit Research Articles

Nitrogen starvation–induced oxidative stress relieves PII-mediated inhibition of acetyl-CoA carboxylase (ACCase) activity and signals enhanced lipid synthesis in Synechococcus PCC 7942

The present study shows the existence of PII-acetyl-CoA carboxylase interaction in the cyanobacterium Synechococcus sp. PCC 7942 and the possible adverse impact of nitrogen starvation on this interaction. The in silico and in vitro analysis of PII-acetyl-CoA carboxylase interaction revealed that the biotin carboxyl carrier protein subunit of acetyl-CoA carboxylase enzyme actually interacts with the T-loop of PII protein. However, exposure of the cyanobacterium to nitrogen-starved condition showed a higher expression and activity of acetyl-CoA carboxylase at the intracellular level which denoted the impairment of PII-acetyl-CoA carboxylase interaction. A similar stimulatory effect of nitrogen starvation has also been noticed in the PII mutant of Synechococcus PCC 7942. Further, the physiological study reflected that nitrogen starvation–caused reactive oxygen species generation in the wild-type and PII mutant strains and lipid was increased in both strains of Synechococcus sp. PCC 7942. Proteomic analysis showed the upregulation of glycogen synthase, biotin carboxylase, and antioxidative enzymes and the deregulation of proteins involved in photosynthesis, energy metabolism, and protein synthesis. Interestingly, enhanced accumulation of transcripts of few tricarboxylic acid cycle genes was also noticed in the wild type. Although oxidative stress and lipid production were enhanced in both the test strains under nitrogen starvation, the impacts were more prominent in the mutant strain. Our results suggest that the nitrogen starvation–induced oxidative stress possibly relieved the PII-mediated inhibition of acetyl-CoA carboxylase and led to increased lipid synthesis in Synechococcus PCC 7942.

The BADC and BCCP subunits of chloroplast acetyl-CoA carboxylase sense the pH changes of the light–dark cycle

Acetyl-CoA carboxylase (ACCase) catalyzes the first committed step in the de novo synthesis of fatty acids. The multisubunit ACCase in the chloroplast is activated by a shift to pH 8 upon light adaptation and is inhibited by a shift to pH 7 upon dark adaptation. Here, titrations with the purified ACCase biotin attachment domain-containing (BADC) and biotin carboxyl carrier protein (BCCP) subunits from Arabidopsis indicated that they can competently and independently bind biotin carboxylase (BC) but differ in responses to pH changes representing those in the plastid stroma during light or dark conditions. At pH 7 in phosphate buffer, BADC1 and BADC2 gain an advantage over BCCP1 and BCCP2 in affinity for BC. At pH 8 in KCl solution, however, BCCP1 and BCCP2 had more than 10-fold higher affinity for BC than did BADC1. The pH-modulated shifts in BC preferences for BCCP and BADC partners suggest they contribute to light-dependent regulation of heteromeric ACCase. Using NMR spectroscopy, we found evidence for increased intrinsic disorder of the BADC and BCCP subunits at pH 7. We propose that this intrinsic disorder potentially promotes fast association with BC through a "fly-casting mechanism." We hypothesize that the pH effects on the BADC and BCCP subunits attenuate ACCase activity by night and enhance it by day. Consistent with this hypothesis, Arabidopsis badc1 badc3 mutant lines grown in a light-dark cycle synthesized more fatty acids in their seeds. In summary, our findings provide evidence that the BADC and BCCP subunits function as pH sensors required for light-dependent switching of heteromeric ACCase activity.

A green fluorescent protein-based assay for high-throughput ligand-binding studies of a mycobacterial biotin protein ligase

Biotin protein ligase (BirA) has been identified as an emerging drug target in Mycobacterium tuberculosis due to its essential metabolic role. Indeed, it is the only enzyme capable of covalently attaching biotin onto the biotin carboxyl carrier protein subunit of the acetyl-CoA carboxylase. Despite recent interest in this protein, there is still a gap in cost-effective high-throughput screening assays for rapid identification of mycobacterial BirA-targeting inhibitors. We present for the first time the cloning, expression, purification of mycobacterial GFP-tagged BirA and its application for the development of a high-throughput assay building on the principle of differential scanning fluorimetry of GFP-tagged proteins. The data obtained in this study reveal how biotin and ATP significantly increase the thermal stability (ΔTm=+16.5°C) of M. tuberculosis BirA and lead to formation of a high affinity holoenzyme complex (Kobs=7.7nM). The new findings and mycobacterial BirA high-throughput assay presented in this work could provide an efficient platform for future anti-tubercular drug discovery campaigns.

A Family of Negative Regulators Targets the Committed Step of de Novo Fatty Acid Biosynthesis

Acetyl-CoA carboxylase (ACCase) catalyzes the committed step of de novo fatty acid biosynthesis. In prokaryotes, green algae, and most plants, this enzyme is a heteromeric complex requiring four different subunits for activity. The plant complex is recalcitrant to conventional purification schemes and hence the structure and composition of the full assembly have been unclear. In vivo coimmunoprecipitation using subunit-specific antibodies identified a novel family of proteins in Arabidopsis thaliana annotated as biotin/lipoyl attachment domain containing (BADC) proteins. Results from yeast two-hybrid and coexpression in Escherichia coli confirmed that all three BADC isoforms interact with the two biotin carboxyl carrier protein (BCCP) isoforms of Arabidopsis ACCase. These proteins resemble BCCP subunits but are not biotinylated due to a mutated biotinylation motif. We demonstrate that BADC proteins significantly inhibit ACCase activity in both E. coli and Arabidopsis. Targeted gene silencing of BADC isoform 1 in Arabidopsis significantly increased seed oil content when normalized to either mass or individual seed. We conclude the BADC proteins are ancestral BCCPs that gained a new function as negative regulators of ACCase after initial loss of the biotinylation motif. A functional model is proposed.

Molecular cloning and expression analysis of a novel BCCP subunit gene from Aleurites moluccana.

Aleurites moluccana L. is grown as a roadside tree in southern China and the oil content of its seed is higher than other oil plants, such as Jatropha curcas and Camellia oleifera. A. moluccana is considered a promising energy plant because its seed oil could be used to produce biodiesel and bio-jet fuel. In addition, the bark, leaves, and kernels of A. moluccana have various medical and commercial uses. Here, a novel gene coding the biotin carboxyl carrier protein subunit (BCCP) was cloned from A. moluccana L. using the homology cloning method combined with rapid amplification of cDNA end (RACE) technology. The isolated full-length cDNA sequence (designated AM-accB) was 1188 bp, containing a 795-bp open reading frame coding for 265 amino acids. The deduced amino acid sequence of AM-accB contained a biotinylated domain located between amino acids 190 and 263. A. moluccana BCCP shows high identity at the amino acid level to its homologues in other higher plants, such as Vernicia fordii, J. curcas, and Ricinus communis (86, 77, and 70%, respectively), which all contain conserved domains for ACCase activity. The expression of the AM-accB gene during the middle stage of development and maturation in A. moluccana seeds was higher than that in early and later stages. The expression pattern of the AM-accB gene is very similar to that of the oil accumulation rate.

Identification and characterization of two putative genes encoding acetyl-coenzyme A carboxylase subunits that are possibly associated with internal browning during cold storage of ‘Hass’ avocados (Persea americana Mill.)

Cold storage is widely used to extend the postharvest life of ‘Hass’ avocados (Persea americana Mill.). However, prolonged low-temperature storage results in the development of chilling-induced physiological disorders. Additionally, the response of avocados to cold storage depends on their stage of maturity, as late-harvest avocados are more susceptible to developing physiological disorders than those that are harvested early in the season. To understand the molecular mechanisms that cause physiological disorders in cold-stored ‘Hass’ avocados, we sought to identify and characterize the lipid metabolism-related genes. In this work, we focused our analysis on the genes that encode a multi-subunit acetyl-CoA carboxylase enzyme (MS-ACCase), which is one of the key enzymes in fatty acid biosynthesis. Two avocado MS-ACCase subunits were identified by in silico analysis, a biotin carboxylase (BC) and a biotin carboxyl carrier protein (BCCP). Transcriptional qPCR analyses of both identified gene subunits were performed in avocados that had been harvested both early and late in the season after 40d of storage at 0 and 5°C and subsequent ripening at 20°C. For both harvest dates, PamACCase-BC and PamACCase-BCCP transcripts increased in ripe fruit after cold storage at 5°C; this increase was significant for PamACCase-BCCP. A similar expression pattern was observed in ripe avocados from early-harvested fruit after cold storage at 0°C; however, significant down-regulation of PamACCase-BC and PamACCase-BCCP expression was observed in ripe late-harvested avocados, suggesting that these fruit have senesced. A control study involving late-harvested avocados demonstrated that the expression of these genes did not change throughout ripening. Thus we suggest that the MS-ACCase BCCP subunit might be transcriptionally regulated during physiological disorder development in cold-stored ‘Hass’ avocados.

Investigating the Role of Protein UnkG from the Pseudomonas putida UW4 in the Ability of the Bacterium to Facilitate Plant Growth

A previous study showed that overexpressing protein UnkG decreased the ability of the plant growth-promoting bacterium Pseudomonas putida UW4 to facilitate plant growth and an unkG knockout mutant of P. putida UW4 displayed increased plant growth promotion. When activities of wild-type and the UnkG overexpressing strain, including growth rates, carbon utilization, cell size, 3-indoleacetic acid production, and 1-aminocyclopropane-1-carboxylate deaminase activity, were measured, there were no apparent differences between the strains. Monitoring proteome-level changes to the wild-type and overexpressing transformant by means of two-dimensional difference in-gel electrophoresis followed by mass spectrometry identification of the altered proteins, 1839 protein spots were detected and 16 of the 84 protein spots with changed expression levels were identified. Proteins with increased expression included arginine deiminase, dihydrodipicolinate synthase, azurin, flavoprotein (α-subunit), ferredoxin-NADP reductase, ATP-dependent Hs1 protease (ATP-binding subunit), UDP-N-acetyl muramate-L-alanine ligase, biotin carboxyl carrier protein subunit of acetyl-CoA carboxylase, and Fis two-component transcriptional regulator. Proteins with decreased expression included glutaminase-asparaginase, arginine/ornithine ABC transporter, cell division protein FtsZ and glutamyl-tRNA synthetase. The functions of three of the 16 proteins could not be identified. The results are consistent with UnkG being detrimental to plant growth because it acts as a regulatory protein that negatively affects several key cellular functions related to the energy balance of the bacterium.

Chloroplast acetyl-CoA carboxylase activity is 2-oxoglutarate–regulated by interaction of PII with the biotin carboxyl carrier subunit

The PII protein is a signal integrator involved in the regulation of nitrogen metabolism in bacteria and plants. Upon sensing of cellular carbon and energy availability, PII conveys the signal by interacting with target proteins, thereby modulating their biological activity. Plant PII is located to plastids; therefore, to identify new PII target proteins, PII-affinity chromatography of soluble extracts from Arabidopsis leaf chloroplasts was performed. Several proteins were retained only when Mg-ATP was present in the binding medium and they were specifically released from the resin by application of a 2-oxoglutarate-containing elution buffer. Mass spectroscopy of SDS/PAGE-resolved protein bands identified the biotin carboxyl carrier protein subunits of the plastidial acetyl-CoA carboxylase (ACCase) and three other proteins containing a similar biotin/lipoyl-binding motif as putative PII targets. ACCase is a key enzyme initiating the synthesis of fatty acids in plastids. In in vitro reconstituted assays supplemented with exogenous ATP, recombinant Arabidopsis PII inhibited chloroplastic ACCase activity, and this was completely reversed in the presence of 2-oxoglutarate, pyruvate, or oxaloacetate. The inhibitory effect was PII-dose-dependent and appeared to be PII-specific because ACCase activity was not altered in the presence of other tested proteins. PII decreased the V(max) of the ACCase reaction without altering the K(m) for acetyl-CoA. These data show that PII function has evolved between bacterial and plant systems to control the carbon metabolism pathway of fatty acid synthesis in plastids.

Immunoprecipitation analysis of pathological autoantibodies in Graves' patients' sera using biotinylated human thyrotropin receptor labeled with 125I-neutravidiny.

In the present article we describe a method for the direct immunoprecipitation analysis of pathological autoantibodies against TSH receptor (TSHR) in sera of patients with Graves' disease. For this purpose the fusion TSH receptor (TSHR-BIO-6HIS) was constructed. This fusion consists of the N-terminal 725 amino acids of the human TSHR linked to the 87-amino acid C-terminal domain of the biotin carboxyl carrier protein subunit of E. coli acetyl-CoA carboxylase (this domain directs the efficient posttranslational biotinylation of the protein) followed by 6 histidine sequence. TSHR-BIO-6HIS was produced in HeLa cells using recombinant vaccinia virus. The expressed receptor was complete active and was biotinylated with a high efficiency (about 90%). Biotinylated TSHR-BIO-6HIS was immobilized on Ni-NTA agarose and selectively labeled with a biotin binding protein-- 125I-neutravidin. The 125I-neutravidin labeled TSHR-BIO-6HIS, freed of the excess of nonbound radioactivity, was eluted from Ni-NTA agarose and used for the detection of pathological autoantibodies in 50 Graves' disease, 10 Hashimoto's disease, 10 insulin-dependent diabetes mellitus and 50 normal sera. 46 of 50 (92%) Graves' disease sera were positive in immunoprecipitation assay, as they have bound 125I-TSHR more effectively than the normal sera. There was a clear positive correlation between the immunoprecipitating activity and TSH-binding inhibiting activity of different Graves' sera (r = 0.69, P < 0.001). These findings pave the way for the development of a new practical assay, capable of detecting all pathological autoantibodies to the TSHR, particularly those which bind but do not affect the hormone-receptor interaction.

Kinetic Partitioning Between Alternative Protein–Protein Interactions Controls a Transcriptional Switch

Proteins can perform completely distinct functions in response to the particular partners that they bind to. Consequently, determination of the mechanism of functional regulation in such systems requires elucidation of the mechanism switching between binding partners. The central protein of the Escherichia coli biotin regulatory system, BirA, switches between its function as a metabolic enzyme or a transcriptional repressor in response to binding either the biotin carboxyl carrier protein subunit of acetyl-CoA carboxylase or a second BirA monomer. These two protein–protein interactions are structurally mutually exclusive. The results of earlier studies suggest that the system is regulated by kinetic partitioning between the two protein–protein interactions. In this work, sedimentation velocity was employed to monitor the partitioning directly. The results indicate similar equilibrium parameters governing formation of the two protein–protein interactions. Kinetic analysis of the sedimentation velocity data indicated that holoBirA dimerization is governed by very slow forward and reverse rate constants. The slow kinetics of holoBirA dimerization combined with fluctuations in the intracellular apoBCCP pool are critical determinants in partitioning BirA between its distinct biological functions.

Is Dimerization Required for the Catalytic Activity of Bacterial Biotin Carboxylase?

Acetyl-coenzyme A carboxylases (ACCs) have crucial roles in fatty acid metabolism. The biotin carboxylase (BC) subunit of Escherichia coli ACC is believed to be active only as a dimer, although the crystal structure shows that the active site of each monomer is 25 A from the dimer interface. We report here biochemical, biophysical, and structural characterizations of BC carrying single-site mutations in the dimer interface. Our studies demonstrate that two of the mutants, R19E and E23R, are monomeric in solution but have only a 3-fold loss in catalytic activity. The crystal structures of the E23R and F363A mutants show that they can still form the correct dimer at high concentrations. Our data suggest that dimerization is not an absolute requirement for the catalytic activity of the E. coli BC subunit, and we propose a new model for the molecular mechanism of action for BC in multisubunit and multidomain ACCs.

ビオチンプロテインリガーゼの結晶構造から見えたビオチンの活性化

Biotin protein ligase (BPL) catalyses the synthesis of an activated form of biotin, biotinyl-5'-AMP, from substrates biotin and ATP followed by biotinylation of the biotin carboxyl carrier protein subunit of acetyl-CoA carboxylase. In order to visualize the structural feature of the BPL reaction, crystal structures of BPL from Pyrococcus horikoshii OT3 have been determined in an unliganded form and three liganded forms with biotin, ADP and the reaction intermediate biotinyl-5'-AMP. The exact locations of the ligands and the active site residues allow us to propose a general scheme for the first step of the reaction carried out by BPL.

Crystal Structures of Biotin Protein Ligase from Pyrococcus horikoshii OT3 and its Complexes: Structural Basis of Biotin Activation

Biotin protein ligase (EC 6.3.4.15) catalyses the synthesis of an activated form of biotin, biotinyl-5'-AMP, from substrates biotin and ATP followed by biotinylation of the biotin carboxyl carrier protein subunit of acetyl-CoA carboxylase. The three-dimensional structure of biotin protein ligase from Pyrococcus horikoshii OT3 has been determined by X-ray diffraction at 1.6A resolution. The structure reveals a homodimer as the functional unit. Each subunit contains two domains, a larger N-terminal catalytic domain and a smaller C-terminal domain. The structural feature of the active site has been studied by determination of the crystal structures of complexes of the enzyme with biotin, ADP and the reaction intermediate biotinyl-5'-AMP at atomic resolution. This is the first report of the liganded structures of biotin protein ligase with nucleotide and biotinyl-5'-AMP. The structures of the unliganded and the liganded forms are isomorphous except for an ordering of the active site loop upon ligand binding. Catalytic binding sites are suitably arranged to minimize the conformational changes required during the reaction, as the pockets for biotin and nucleotide are located spatially adjacent to each other in a cleft of the catalytic domain and the pocket for biotinyl-5'-AMP binding mimics the combination of those of the substrates. The exact locations of the ligands and the active site residues allow us to propose a general scheme for the first step of the reaction carried out by biotin protein ligase in which the positively charged epsilon-amino group of Lys111 facilitates the nucleophilic attack on the ATP alpha-phosphate group by the biotin carboxyl oxygen atom and stabilizes the negatively charged intermediates.

The Biotin Repressor: Modulation of Allostery by Corepressor Analogs

The Escherichia coli biotin repressor functions in biotin retention and regulation of biotin biosynthesis. Biotin retention is accomplished via the two-step biotinylation of the biotin-dependent enzyme, acetyl-CoA carboxylase. In the first step of this reaction the substrates biotin and ATP are utilized in synthesis of the activated biotin, biotinyl-5′-AMP, while in the second step this activated biotin is transferred to a unique lysine residue of the biotin carboxyl carrier protein subunit of the carboxylase. Regulation of biotin biosynthesis is accomplished through binding of the repressor to the transcription control region of the biotin biosynthetic operon. The adenylated or activated biotin functions as the corepressor in this DNA binding process. The activated biotin is a mixed anhydride and thus labile. In efforts to develop tools for structural and thermodynamic studies of the biotin regulatory interactions, two analogs of the adenylate, a sulfamoyl derivative and an ester derivative, have been synthesized and functionally characterized. Results of fluorescence measurements indicate that both analogs bind with high affinity to the repressor and that both are inactive in biotin transfer to the acceptor protein. Functional studies of their corepressor properties indicate that while the sulfamoyl is a weak allosteric activator, the ester closely mimics the physiological corepressor in activation of assembly of the transcription repression complex. Results of these studies also provide further insight into the allosteric mechanism of the biotin repressor.

Stabilization of the Biotinoyl Domain of Escherichia coli Acetyl-CoA Carboxylase by Interactions between the Attached Biotin and the Protruding “Thumb” Structure

We previously reported (Chapman-Smith, A., Forbes, B. E., Wallace, J. C., and Cronan, J. E., Jr. (1997) J. Biol. Chem. 272, 26017-26022) that the biotinylated (holo) species of the biotin carboxyl carrier protein (BCCP) biotinoyl domain is much more resistant to chemical modification and proteolysis than the unbiotinylated (apo) form. We hypothesized that the increased stability was due to a conformational change engendered by interaction of the domain with biotin protein ligase, the enzyme that attaches the biotin moiety. We now report that a BCCP-87 species to which the biotin moiety was attached by chemical acylation rather than by biotin protein ligase showed the characteristically greater stability of the holo biotinoyl domain. This result demonstrates that our hypothesis was incorrect; the attached biotin is solely responsible for the increased stability. The bacterial and chloroplast multisubunit acetyl-CoA carboxylases are unusual in that the highly symmetrical and conserved structure of the biotinoyl domain of the BCCP subunit is disrupted by a structured loop called the "thumb" that protrudes from body of the domain. Prior structural work showed that the thumb interacts with uriedo ring of the attached biotin moiety. We have tested whether the thumb-biotin interactions are responsible for the greater holo form stability by examination of two BCCP-87 species that lack the thumb. These BCCP species were produced in both the apo and holo forms, and their sensitivities to trypsin digestion were compared. The holo forms of these proteins were found to be only marginally more stable than their apo forms and much more sensitive to trypsin digestion than the wild type holo-BCCP-87. Therefore, removal of the thumb has an effect similar to lack of biotinylation, indicating that thumb-biotin interactions are responsible for most (but not all) of the increased stability of the holo biotinoyl domain. In the course of these experiments we demonstrated that treatment of Escherichia coli with the peptide deformylase inhibitor, actinonin, results in the expected (but previously unreported) accumulation of an N-formylated protein species.

The multisubunit acetyl-CoA carboxylase is strongly associated with the chloroplast envelope through non-ionic interactions to the carboxyltransferase subunits

The committed step for de novo fatty acid biosynthesis is the carboxylation of acetyl-CoA catalyzed by acetyl-CoA carboxylase (ACCase). Plastidial ACCase from most plants is a multisubunit complex composed of multiple copies of four different polypeptides, biotin carboxyl carrier protein (BCCP), biotin carboxylase (BC), and carboxyltransferase (α-CT and β-CT). Immunoblot analyses revealed these four proteins were mostly (69% of total) associated with a 17,000 g insoluble fraction from lysed pea chloroplasts. Under the same conditions only 8% of ribulose-1,5-bisphosphate carboxylase was associated with this insoluble fraction. BCCP and biotin carboxylase BC subunits freely dissociated from 17 kg insoluble fractions under high ionic strength conditions, whereas α-CT and β-CT subunits remained tightly associated. Both CT subunits were highly enriched in envelope versus stroma and thylakoid preparations whereas BC and BCCP subunits were predominantly stromal-localized due to partial dissociation. Rapid solubilization of intact chloroplasts with Triton X-100 followed by centrifugation at 30 kg resulted in a pellet that was up to 8-fold enriched in ACCase activity and 21-fold enriched in BC activity. Triton-insoluble 30 kg pellets were reduced in lipid and chlorophyll content but enriched in chloroplast DNA due to the isolation of nucleoid particles. However, ACCase was not directly associated with nucleoids since enzymatic digestion of DNA or RNA had no effect on the association with Triton-insoluble matter. The amount of Triton-insoluble ACCase was similar in chloroplasts isolated from dark- or light-adaptated leaves suggesting transitory starch granules were also not involved in this association. It is proposed that ACCase is associated with envelope membranes through interactions with an unidentified integral membrane protein.

Isolation of radiochemically pure 125I-labeled human thyrotropin receptor and its use for the detection of pathological autoantibodies in sera from Graves' patients.

We report a method for the purification and radioactive labeling of human TSH receptor (TSHR). The method is based on the construction of a fusion TSHR (TSHR-Xa-BIO) which consists of the N-terminal 725 amino acids of human TSHR linked to the 4-amino acid Xa protease cleavage site and the 87-amino acid C-terminal domain of the biotin carboxyl carrier protein subunit of Escherichia coli acetyl-CoA carboxylase (the C-terminal domain directs the efficient posttranslational biotinylation of the protein). TSHR-Xa-BIO was produced in HeLa cells using recombinant vaccinia virus. The expressed protein was fully functional and was biotinylated with an efficiency of about 90%. Streptavidin-agarose-immobilized TSHR-Xa-BIO was labeled with 125I using the chloramine T oxidation procedure and specifically eluted from the solid phase after cleavage with protease Xa. Isolated native radiochemically pure 125I-labeled TSHR specifically interacted with pathological autoantibodies in the sera of patients with Graves' disease, and thus could be useful for the detection of these autoantibodies by immunoprecipitation analysis.

Expression of a biotinylated human thyrotropin receptor in HeLa cells using recombinant vaccinia virus and its application for the detection of Graves' autoantibodies.

We have prepared a biotinylated thyrotropin receptor (TSHR-BIO), and characterized its activity in cells and when bound to solid phase (streptavidin agarose). TSHR-BIO consists of the N-terminal 725 amino acids of the human thyrotropin (TSH) receptor linked to the 87-amino acid C-terminal domain of the biotin carboxyl carrier protein subunit of Escherichia coli acetyl-CoA carboxylase. The C-terminal domain directs the efficient post-translational biotinylation of the protein. TSHR-BIO was expressed using a vaccinia virus expression system. HeLa cells infected with recombinant virus produced large amounts of TSH receptor of approximately 120,000 molecules per cell. Vaccinia virus produced TSHR-BIO was fully functional interacting with TSH (Kd of 2.3+/-0.1 x 10(-10) M) and coupling to cyclic adenosine monophosphate (cAMP) second messenger system. The expressed protein was biotinylated with high efficiency; more than 90% of TSHR-BIO was bound to streptavidin. We have shown the application of streptavidin agarose immobilized TSHR-BIO for the detection of thyroid-binding inhibiting immunoglobulines in unfractionated sera. There was a good positive correlation between the results obtained in this assay and the commercially available TRAK assay performed with solubilized porcine TSH receptor (r = 0.71; p < 0.001, in 45 sera of patients with Graves' disease and 17 normal sera).

Covalent modification of an exposed surface turn alters the global conformation of the biotin carrier domain of Escherichia coli acetyl-CoA carboxylase.

We have studied the apo (unbiotinylated) and holo (biotinylated) forms of BCCP87, an 87-residue COOH-terminal peptide comprising the biotin carrier domain of the biotin carboxyl carrier protein subunit of Escherichia coli acetyl-CoA carboxylase. The apo protein spontaneously formed disulfide-linked dimers and was modified readily by sulfhydryl reagents, whereas the holo protein remained monomeric and was unreactive toward sulfhydryl reagents unless a protein denaturant was present. These data indicated that the single cysteine residue of the domain (Cys-116) was much more reactive in the apo form of the protein. Incubation of apoBCCP87 with biotin ligase for different times, followed by reaction with fluorescein-5-maleimide, clearly showed that the loss of Cys-116 reactivity was the result of modification with biotin. In addition, reaction of Cys-116 with 5,5'-dithiobis(2-nitrobenzoic acid) showed that apoBCCP87 denatured at lower urea concentrations than holoBCCP87. We also found that apoBCCP87 was at least 10-fold more sensitive than the holo form to proteolysis by a range of proteases. Identification of the cleavage sites indicated that the differences in protease sensitivity could not be attributed to shielding of susceptible bonds by the biotin moiety of the holo protein. These data indicate that a conformational change accompanies biotinylation of the biotin domain. Thus, modification of a beta-turn protruding from the protein surface results in alteration of the overall structure of this protein domain.

The pea chloroplast membrane-associated protein, IEP96, is a subunit of acetyl-CoA carboxylase.

Two forms of acetyl-CoA carboxylase (ACCase) have been characterized in pea (Pisum sativum L.) leaves; a heteromeric chloroplast enzyme and a homomeric, presumably cytosolic enzyme. The biotin carboxylase (BC), biotin carboxyl carrier protein (BCCP), and beta-carboxyltransferase (CT) subunits of the plastidial-ACCase have recently been characterized and cloned. To further characterize the carboxyltransferase, an improved assay for CT was developed and used to follow its partial purification. CT activity co-purifies with ACCase activity during gel permeation chromatography. However, upon anion-exchange chromatography or native PAGE, CT separates from the BC and BCCP subunits of plastidial-ACCase and ACCase activity is lost. In addition, it is demonstrated that a previously sequenced pea chloroplast cDNA of unknown function (IEP96) with a predicted molecular weight of 91 kDa encodes the alpha-CT subunit of the MS-ACCase. Antibodies raised against the first 404 amino acids of IEP96 protein detected a polypeptide with molecular weight of 91 kDa that co-eluted during gel permeation chromatography with plastidial CT and ACCase activities. These antibodies also immunoprecipitated the activities of both ACCase and CT with the concomitant precipitation of the beta-CT subunit. Furthermore, antibodies against beta-CT immunoprecipitated the IEP96 protein. Two-dimensional PAGE and DEAE purification of ACCase protein demonstrated that the beta-CT forms a tight association with the IEP96 protein. Pea leaf was fractionated into soluble and membrane fractions and the alpha-CT subunit was primarily associated with the membrane fraction. Together, these data demonstrate that IEP96 is the alpha-CT subunit of pea chloroplast ACCase.