Mutagenicity and irreversible binding of the hepatocarcinogen, 2,4-diaminotoluene.

Mutagenicity of 2,4-diaminotoluene (DAT) in the Salmonella mutagenicity assay was increased with liver fractions from phenobarbital (PB) or beta-naphthoflavone (BNF) treated rats. Substitutions of the hydrogens in the methyl group of 2,4-DAT with deuterium resulted in a decrease in mutagenicity. Incubation of rat liver microsomes with tritiated 2,4-DAT in the presence of NADPH led to the formation of irreversibly bound products to microsomal protein. The rates of binding were not increased using microsomes from PB or BNF-treated rats and was not altered by deuterium substitution in the methyl group. Addition of superoxide dismutase, glutathione (GSH) or rat liver supernatant reduced 2,4-DAT irreversible binding, whereas 2,4-DAT mutagenicity was unaffected by superoxide dismutase addition. Injection of tritiated 2,4-DAT 100 mg/kg to rats lead to its irreversible binding to liver protein and ribosomal RNA and to kidney protein in vivo, again protein binding was not increased after prior treatment with PB or BNF. No irreversible interaction of tritiated 2,4-DAT with DNA either in vitro or in vivo could be demonstrated.     

Plasma membrane proteins associated with undifferentiated and embryonicDaucus carota tissue

Summary Membranes and membrane proteins from undifferentiated cells and torpedo-stage embryos were compared. A comparison of marker enzyme profiles on linear sucrose gradients showed that the membrane vesicles obtained from 14-day-old embryos were consistently less dense than those obtained from undifferentiated carrot cells. The density of the endoplasmic reticulum, for instance, was 1.10g/cm³ in embryos and 1.12g/cm² in undifferentiated cells. Proteins and glycoproteins from endoplasmic reticulum-, Golgi apparatus-, and plasma membrane-enriched fractions were compared from undifferentiated carrot cells with 14-day-old embryos by 2D SDS-PAGE. When these two tissues were compared, extensive qualitative and quantitative changes in the steady-state endomembrane and plasma membrane proteins were observed. The plasma membrane was examined further by labeling the plasma membrane proteins with sulfosuccinimidylbiotin. Using this specific label, plasma membrane proteins of 54 kD, 41 kD, 16 kD, and 15 kD were found to be uniquely associated with the embryonic state. Conversely, a 70 kD protein and a 45 kD glycoprotein were found to be associated with only undifferentiated cells. These results demonstrate that proteins of the plasma membrane exhibit distinct changes as a result of somatic embryogenesis in carrot.Abbreviations conA concanavilin A - 2,4-D 2,4-dichlorophenoxyacetic acid - p protein - gp glycoprotein - kD kilodalton - SDS-PAGE sodium dodecylsulfate-polyacrylamide gel electrophoresis     

Chromaffin Granule Membrane-F-Actin Interactions and Spectrin-Like Protein of Subcellular Organelles: A Possible Relationship

The membrane of chromaffin granule, the secretory vesicle of adrenal medullary cells storing catecholamines, enkephalins, and many other components, interacts with F-actin. Using low shear falling ball viscometry to estimate actin binding to membranes, we demonstrated that mitochondrial and plasma membranes from chromaffin cells also provoked large increases in viscosity of F-actin solutions. Mitochondrial membranes also had the capacity to cause complete gelation of F-actin. In addition, vasopressin-containing granules from neurohypophysial tissue were shown to bind F-actin and to increase the viscosity of F-actin solutions. Using an antibody directed against human erythrocyte spectrin, it was found that a spectrin-like protein was associated with secretory granule membrane, mitochondrial membrane, and plasma membrane. The chromaffin granule membrane-associated spectrin-like protein faces the cytoplasmic side, is composed of two subunits (240 kD and 235kD ), the alpha-subunit (240 kD, pHi5 .5) being recognized by the antibody. Nonionic detergents such as Triton X-100 or Nonidet P40 failed to release fully active spectrin-like protein. In contrast, Kyro EOB , a different nonionic detergent, was found to release spectrin-like protein while keeping intact F-actin binding capacity, at least below 0.5% Kyro EOB concentration. Chromaffin cells in culture were stained with antispectrin antibody, showing the presence of spectrin-like protein in the cell periphery close to the cell membrane but also in the cytoplasm. We conclude that in living cells the interaction of F-actin with chromaffin granule membrane spectrin observed in vitro is important in controlling the potential function of secretory vesicles.     

Isolation and characterization of a 60 kDa 2,4-D-binding protein from the shoot apices of peach trees (Prunus persica L.); it is a homologue of protein disulfide isomerase

To obtain a candidate auxin-binding protein (ABP), a soluble 60 kDa protein was isolated from an extract of shoot apices of peach trees (Prunus persica L.) by affinity chromatography on a 2,4-dichlorophenoxyacetic acid (2,4-D)-linked Sepharose4B column. The 60 kDa polypeptide, designated Pp60, was purified as a single band on SDS-PAGE by column chromatography. Its dissociation constant (Kd) for [14C]-2,4-D was calculated to be 3.5 x 10(-5) M. The binding of Pp60 for [14C]-2,4-D was inhibited by naphthalene-1-acetic acid (NAA) and p-chlorophenoxyisobutyric acid (PCIB) as well as 2,4-D. Indole-3-acetic acid (IAA) had little effect on the binding. These results suggested that Pp60 is a protein that has an affinity for 2,4-D, NAA, and PCIB in vitro. The partial amino acid sequences of Pp60 showed high homology to those of protein disulfide isomerase (EC 5.3.4.1). Immunoblot analysis demonstrated that Pp60 exists ubiquitously in shoots and leaves. In fruit, expression of Pp60 is restricted at an early stage of development.     

Metabolic activation of indole-containing prostaglandin D2 receptor 1 antagonists: impacts of glutathione trapping and glucuronide conjugation on covalent binding

Some DP1 receptor antagonists from an indole-containing series were shown to cause in vitro covalent binding to protein in rat and human liver microsomes. Glutathione trapping experiments along with in vitro labeling assays confirmed that the presence of a strong electron withdrawing group was necessary to abrogate in vitro covalent binding, leading to the discovery of MK-0524. Hepatocyte incubations and in vivo studies showed that acyl-glucuronide formation did not translate into covalent binding.     

Evaluation of the specific dicyclohexylcarbodiimide binding sites in brown adipose tissue mitochondria

1. The content of the membrane sector of the ATPase complex (Fo) in brown adipose tissue mitochondria was determined by means of specific [14C]-DCCD binding. 2. The specific DCCD binding to the F0 protein was distinguished from the nonspecific binding to the other membrane proteins and phospholipids by: (a) Scatchard plot analysis of the equilibrium binding data, (b) SDS-polyacrylamide gel electrophoresis of the 14C-labelled membrane proteins, (c) partial purification of the chloroform-methanol extractable DCCD-binding protein. It was found that the specific DCCD binding was present in three polypeptides of a relative molecular weight of 9000, 16 000 and 32 000. In brown adipose tissue mitochondria the specific binding was 10-times lower than in heart or liver mitochondria. The binding to the other membrane proteins and to phospholipids was quite similar in all mitochondrial preparations studied. 3. The decreased quantity of the specific binding sites in brown adipose tissue mitochondria demonstrated that the reduction of F0 parallels the reduction of the F1-ATPase and revealed that in these mitochondrial membranes the ratio between the respiratory chain enzymes and the ATPase complex is 10- to 20- times higher than in heart or liver mitochondria.     

A covalent tandem dimer of the mitochondrial ADP/ATP carrier is functional in vivo

The adenine nucleotide carrier, or Ancp, is an integral protein of the inner mitochondrial membrane. It is established that the inactive Ancp bound to one of its inhibitors (CATR or BA) is a dimer, but different contradictory models were proposed over the past years to describe the organization of the active Ancp. In order to decide in favor of a single model, it is necessary to establish the orientations of the N- and C-termini and thus the parity of the Ancp transmembrane segments (TMS). According to this, we have constructed a gene encoding a covalent tandem dimer of the Saccharomyces cerevisiae Anc2p and we demonstrate that it is stable and active in vivo as well as in vitro. The properties of the isolated dimer are strongly similar to those of the native Anc2p, as seen from nucleotide exchange and inhibitor binding experiments. We can therefore conclude that the native Anc2p has an even number of TMS and that the N- and C-terminal regions are exposed to the same cellular compartment. Furthermore, our results support the idea of a minimal dimeric functional organization of the Ancp in the mitochondrial membrane and we can suggest that TMS 1 of one monomer and TMS 6 of the other monomer in the native dimer are very close to each other.     

Intramitochondrial fatty acylation of a cytoplasmic imported protein in animal cells

Mitochondrial digitonin particles from mouse liver (and also from other tissues) incorporate [3H]myristic acid into a 52-kilodalton (kDa) protein in an energy-dependent manner. The 52-kDa N-myristylated protein is located inside the mitochondrial inner membrane since it is protected against proteolytic degradation in intact mitoplasts. Disruption of mitochondrial inner membrane by sonication results in severalfold higher labeling of the 52-kDa protein, further confirming that the enzyme system for protein fatty acylation as well as the 52-kDa target protein are compartmentalized inside the mitochondrial inner membrane matrix. The results of in vitro labeling of submitochondrial fractions suggest that both the 52-kDa target protein and the enzyme system for fatty acylation are in the matrix fraction, although the N-myristylated protein is found loosely associated with the inner membrane. Finally, immunoprecipitation of cytoplasmic free polysome translation products and in vitro transport of proteins into isolated mitochondria show that the 52-kDa protein is of cytoplasmic translation origin. These results demonstrate that the intramitochondrial N-myristylation of the 52-kDa protein is not translationally linked.     

Patterns of protein synthesis in dividing and auxin-starved soybean cell suspensions. Differential expression of a major group of Mr-17000 peptides

Auxin starvation of soybean cell suspensions results in the arrest of cell growth after about 4 days. The addition of 4 M 2,4-D enables cells to divide again after a lag phase of 1 day. By comparing the patterns of in vivo and in vitro protein synthesis, we have identified two sets of polypeptides whose synthesis is positively or negatively regulated by auxin. Several major peptide bands (17, 26, 31, 35, 38 kD) are characteristic of auxin-starved cells. The 17 kD peptide group, containing one major (75%) component, accounts for 6% and 25% of the radiolabeling in vivo of proteins from respectively dividing and auxin-starved cells. Our results suggest the 17 kD major component to be a direct translation product whose synthesis is regulated by the abundance or the activity of the relevant mRNA. The soybean suspension culture system here described provides a model to study auxin-mediated control of gene expression.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - pmsf phenylmethanesulfonyl fluoride - tlck N-p-tosyl-L-lysine chloromethylketone - tpck N-tosyl-L-phenylalanine chloromethylketone - SDS-PAGE SDS-polyacrylamide gel electrophoresis - IEF isoelectrofocusing - BSA bovine serum albumin - pI isoelectric point - pcv packed cell volume     

激素对枸杞体细胞胚发生及可溶性蛋白质含量和组分的影响

以宁夏枸杞无菌苗叶片为材料,对体细胞胚发生过程中激素作用、可溶性蛋白质变化及体细胞胚发生频率进行了研究。发现不同激素处理、可溶性蛋白质含量、组分和体细胞胚发生频率均有一定的差异,三者之间存在着相关性。结果如下：（１）ＭＳ＿１、ＭＳ＿２和ＭＳ＿３三种培养基上继代的愈伤组织难以诱导形成体细胞胚,为非胚性愈伤组织;而从ＭＳ＿１、ＭＳ＿２和ＭＳ＿３分别转到ＭＳ＿０和ＭＳ＿４培养基上的愈伤组织体细胞胚发生频率高,属于胚性愈伤组织。其中以ＭＳ＿３转至ＭＳ＿０后的体细胞胚发生频率最高。（２）可溶性蛋白质ＳＤＳ－ＰＡＧＥ分析表明：非胚性愈伤组织有特异性蛋白质６７ｋＤ,胚性愈伤组织有特异性蛋白质３５ｋＤ和４４ｋＤ;蛋白质３３ｋＤ和６７ｋＤ受２,４－Ｄ调控,蛋白质４９ｋＤ、５７ｋＤ受６ＢＡ调控,而蛋白质３７ｋＤ、５１ｋＤ受２,４－Ｄ和６ＢＡ协同调控。     

Functional definition of outer membrane proteins involved in preprotein import into mitochondria

The role of plant mitochondrial outer membrane proteins in the process of preprotein import was investigated, as some of the principal components characterized in yeast have been shown to be absent or evolutionarily distinct in plants. Three outer membrane proteins of Arabidopsis thaliana mitochondria were studied: TOM20 (translocase of the outer mitochondrial membrane), METAXIN, and mtOM64 (outer mitochondrial membrane protein of 64 kD). A single functional Arabidopsis TOM20 gene is sufficient to produce a normal multisubunit translocase of the outer membrane complex. Simultaneous inactivation of two of the three TOM20 genes changed the rate of import for some precursor proteins, revealing limited isoform subfunctionalization. Inactivation of all three TOM20 genes resulted in severely reduced rates of import for some but not all precursor proteins. The outer membrane protein METAXIN was characterized to play a role in the import of mitochondrial precursor proteins and likely plays a role in the assembly of beta-barrel proteins into the outer membrane. An outer mitochondrial membrane protein of 64 kD (mtOM64) with high sequence similarity to a chloroplast import receptor was shown to interact with a variety of precursor proteins. All three proteins have domains exposed to the cytosol and interacted with a variety of precursor proteins, as determined by pull-down and yeast two-hybrid interaction assays. Furthermore, inactivation of one resulted in protein abundance changes in the others, suggesting functional redundancy. Thus, it is proposed that all three components directly interact with precursor proteins to participate in early stages of mitochondrial protein import.     

In vitro macromolecular binding of 2-(2-chlorophenyl)-2-(4-chlorophenyl)-1,1-dichloroethane (o,p'-DDD) in the mouse lung and liver

The activation and covalent binding of 14C-labelled 2-(2-chlorophenyl)-2-(4-chlorophenyl)-1,1-dichloroethane (o,p'-DDD) in mouse lung and liver S-9 preparations were examined in vitro. These results showed an oxidative cytochrome P-450 mediated transformation of o,p'-DDD to metabolite(s) that bind covalently to proteins, phospholipids and to added naked DNA in both lung and liver. The apparent Km-values for the covalent binding of o,p'-DDD to protein were 0.25 microM and 3.30 microM in lung and liver, respectively. Addition of glutathione to the incubation medium decreased the binding of o,p'-DDD more efficiently in the liver than in the lung. Thus, the selective lung binding of o,p'-DDD previously observed in vivo seems to result from an in situ activation. The tissue selectivity in vivo is suggested to be due to the low apparent Km in the lung favouring bioactivation at low, ecotoxicologically relevant doses, as well as to a less pronounced protection by glutathione in the lung.     

Induction of Stress Shock Proteins DnaK and GroEL by Phenoxyherbicide 2,4-D in Burkholderia sp. YK-2 Isolated from Rice Field

The purpose of this work was to investigate the induction of stress shock proteins in Burkholderia sp. YK-2 in response to the phenoxyherbicide 2,4-dichlorophenoxyacetic acid (2,4-D). The stress shock proteins, which contribute to the resistance of the cytotoxic effect of 2,4-D, were induced at different 2,4-D concentrations in exponentially growing cultures of Burkholderia sp. YK-2. This response involved the induction of a 43-kDa DnaK and 41-kDa GroEL proteins, characterized by SDS-PAGE and Western blot by use of the anti-DnaK and anti-GroEL monoclonal antibodies. The total stress shock proteins were analyzed by 2-D PAGE. Survival of Burkholderia sp. YK-2 with time in the presence of different concentrations of 2,4-D was monitored, and viable counts paralleled the induction of the stress shock proteins in this strain. Received: 1 November 1999 / Accepted: 24 November 1999     

Interactions of atrazine and 2,4-D with human serum albumin studied by gel and capillary electrophoresis, and FTIR spectroscopy.

The herbicides 6-chloro-N-ethyl-N'-(1-methylethyl)-1,3,5-triazine-2,4-diamine (atrazine) and 2,4-dichlorophenoxyacetic acid (2,4-D) are widely used in agricultural practice to fight dicotyledon weeds mainly in maize, cereals, and lucerne. As a result, these compounds are found not only in the plants, soil, and water, but also in the cultivated ground in the following years as well as in agricultural products such as fruits, milk, butter, and sugar beet. The toxicological effects of herbicides occur in vivo, when transported to the target organ through the bloodstream. It has been suggested that human serum albumin (HSA) serves as a carrier protein to transport 2,4-D to molecular targets. This study was designed to examine the interaction of atrazine and 2,4-D with HSA in aqueous solution at physiological pH with herbicide concentrations of 0.0001-1 mM, and final protein concentration of 1% w/v. Gel and capillary electrophoresis, UV-visible and Fourier transform infrared spectroscopic methods were used to determine the drug binding mode, the drug binding constant, and the protein secondary structure in aqueous solution. Structural analysis showed that different types of herbicide-HSA complexes are formed with stoichiometric ratios (drug/protein) of 3:1 and 11:1 for atrazine and 4.5:1 and 10:1 for 2,4-D complexes. Atrazine showed a weak binding affinity (K=3.50 x 10(4) M(-1)), whereas two bindings (K(1)=2.50 x 10(4) M(-1) and K(2)=8.0 x 10(3) M(-1)) were observed for 2,4-D complexes. The herbicide binding results in major protein secondary structural changes from that of the alpha-helix 55% to 45--39% and beta-sheet 22% to 24--32%, beta-anti 12% to 10--22% and turn 11% to 12--15%, in the drug-HSA complexes. The observed spectral changes indicate a partial unfolding of the protein structure, in the presence of herbicides in aqueous solution.     

Intracellular aflatoxin B1-binding proteins in rat liver

Intracellular aflatoxin B1 binding in rat liver was studied under both in vitro and in vivo conditions. Binding in vivo appeared similar to that observed in vitro except that some covalent adduct formation was detected. Participation of previously described carcinogen-binding proteins such as the Ah receptor, h2-5S protein, 4-5S receptor for 3-methylcholanthrene and the Z-protein fraction was discounted on the grounds of competition binding studies and gel-permeation chromatography. The molecular weight of 45,000 was estimated for the major aflatoxin B1-binding component. Aflatoxin B1 co-eluted with the glutathione S-transferases during gel-permeation and separation of the various isozymes by cation-exchange chromatography indicated interactions with the YaYa and YaYc-forms. These proteins, however, account for less than 20% of the total intracellular aflatoxin binding. A protein of apparent monomeric structure appears to form the major in vitro/in vivo complex with aflatoxin B1.     

Mechanism of organic anion transport across the apical membrane of choroid plexus

The mechanism and membrane localization of choroid plexus (CP) organic anion transport were determined in apical (or brush border) membrane vesicles isolated from bovine choroid plexus and in intact CP tissue from cow and rat. Brush border membrane vesicles were enriched in Na(+),K(+)-ATPase (20-fold; an apical marker in CP) and demonstrated specific, sodium-coupled transport of proline, glucose, and glutarate. Vesicular uptake of the anionic herbicide 2, 4-dichlorophenoxyacetic acid (2,4-D) was markedly stimulated by an inward sodium gradient but only in the presence of glutarate, indicating the presence of apical dicarboxylate/organic anion exchange. Consistent with this interpretation, an imposed outward glutarate gradient stimulated 2,4-D uptake in the absence of sodium. Under both conditions, uptake was dramatically slowed and overshoot was abolished by probenecid. Likewise, apical accumulation of 2,4-D by intact bovine choroid plexus tissue in vitro was stimulated by external glutarate in the presence of sodium. Glutarate stimulation was abolished by 5 mM LiCl. Identical findings were obtained using rat CP tissue, which showed both sodium/glutarate-stimulated 2,4-D (tissue/medium (T/M) approximately 8) and p-aminohippurate (T/M = 2) transport. Finally, since the renal exchanger (rROAT1) has been cloned in rat kidney, a rROAT1-green fluorescent protein construct was used to analyze exchanger distribution directly in transiently transfected rat CP. As predicted by the functional studies, the fluorescently tagged transporter was seen in apical but not basolateral membranes of the CP.     

A Pea Plasma Membrane Protein Exhibiting Blue Light-Induced Phosphorylation Retains Photosensitivity following Triton Solubilization

Phosphorylation of a polypeptide of approximately 120 kD in pea (Pisum sativum L.) plasma membranes in response to blue light has been shown to be involved in phototropic curvature, but the relationship of this protein to the kinase and photoreceptor acting upon it is uncertain. Using two-phase aqueous partitioning to isolate right-side-out plasma membrane vesicles, we have obtained evidence suggesting that the photoreceptor, kinase, and substrate are localized to the plasma membrane fraction. Latent phosphorylation accessible through Triton X-100 or freeze/thaw treatments of purified plasma membrane vesicles indicates that at least the kinase moiety is present on the internal face of the plasma membrane. Effects of solubilization of vesicles on fluence-response characteristics and on phosphorylation levels provide evidence that the receptor, kinase, and protein substrate are present together in individual mixed detergent micelles, either as a stable complex or as domains of a single polypeptide. In vivo blue-light irradiation results in a small but significant decrease in mobility of the 120-kD phosphorylated protein on sodium dodecylsulfate gel electrophoresis. This mobility shift is evident on Coomassie-stained gels and on western blots probed with polyclonal antibodies raised against the 120-kD protein. Among the plasma membrane proteins bound to the reactive nucleotide analog fluorosulfonylbenzoyladenine (FSBA), a distinct protein band at 120 kD can be detected on blots probed with anti-FSBA antibodies. This band exhibits an in vivo light-dependent mobility shift identical to that observed for the protein band and antibodies specific for the 120-kD protein, implying that the 120-kD protein has an integral nucleotide binding site and consistent with the possibility that the substrate protein is also a kinase.     

High-affinity binding of 2,3,7,8-tetrachlorodibenzo-p-dioxin in cell nuclei from rat liver

The intranuclear binding of radioactive 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) in rat liver has been studied both in vivo and in vitro. Following the intravenous administration of [1,6-3H]TCDD, a maximum uptake by cell nuclei could be observed at 2 h after injection with a concurrent decrease in the cytosolic uptake. Using linear sucrose density gradient centrifugation, dextran-coated charcoal adsorption assay, DEAE-Sepharose ion-exchange chromatography, competition, enzymatic and saturation studies, a high-affinity binding protein for TCDD in liver cell nuclei could be demonstrated both in vivo and after an exchange in vitro of intravenously administered unlabelled 2,3,7,8- tetrachlorodibenzofuran (TCDBF) for [3H]TCDD. Sucrose density gradient analysis showed a size of 4-5 S for both the cytosolic and nuclear TCDD binding entity. The specific binding of [3H]TCDD to nuclear components was heat labile and saturable and had an equilibrium dissociation constant of 1.05 nM. Based on a differential susceptibility to specific hydrolases, i.e. DNAase, RNAase, trypsin and pronase, the binding entity appears to be a 4-5 S salt-extractable protein.     

Distribution and macromolecular binding of benzo[a]pyrene and two polychlorinated biphenyl congeners in female mice

PCBs are complete rodent carcinogens and their potent tumor promoting activity has been reported, but their tumor-initiating activity remains controversial. Macromolecular binding of PCB metabolites has been demonstrated in vitro, but this issue remains unclear in vivo. The purpose of this study was to determine the binding affinity of 4-chlorobiphenyl and 3,3',4,4'-tetrachlorobiphenyl to proteins and DNA in vivo. C57/BL6 female mice were treated intraperitoneally with hepatic enzyme inducers (phenobarbital and beta-naphthoflavone) and then with 14C-labelled polychlorinated biphenyls or benzo[a]pyrene. The short-term distribution of labeled compounds into liver, lungs and kidneys and into different sub-cellular fractions of these tissues was assessed and the DNA and proteins from the 700 x g pellet were further purified to assess covalent binding. All compounds were distributed in low amounts into the liver, kidneys and lungs, with the greatest accumulation in the liver, and the lowest in lungs. In all tissues, test compounds were mostly found in cytosols and organellar pellets (10,000 x g), and lower amounts were present in nuclear pellets (700 x g) and microsomes. In lungs and kidneys, only benzo[a]pyrene showed significant covalent binding to proteins. In the liver, protein binding indices were significant for all compounds (P<0.05), but no significant binding of the test compounds to DNA could be demonstrated with this approach. Our results suggest that at the 24 h time point, all compounds were activated to electrophilic intermediates prone to macromolecular binding. Hepatic proteins apparently act as a sink for PCB-derived electrophiles, thus preventing detectable levels of covalent binding to hepatic DNA or to proteins in less metabolically active tissues.     

The purified recombinant precursor of rat mitochondrial dimethylglycine dehydrogenase binds FAD via an autocatalytic reaction

The precursor of the rat mitochondrial flavoenzyme dimethylglycine dehydrogenase (Me(2)GlyDH) has been produced in Escherichia coli as a C-terminally 6-His-tagged fusion protein, purified by one-step affinity chromatography and identified by ESI-MS/MS. It was correctly processed into its mature form upon incubation with solubilized rat liver mitoplasts. The purified precursor was mainly in its apo-form as demonstrated by immunological and fluorimetric detection of covalently bound flavin adenine dinucleotide (FAD). Results described here definitively demonstrate that: (i) covalent attachment of FAD to Me(2)GlyDH apoenzyme can proceed in vitro autocatalytically, without third reactants; (ii) the removal of mitochondrial presequence by mitochondrial processing peptidase is not required for covalent autoflavinylation.