Mono-Q Column Research Articles

Characterization of Aryl Alcohol Oxidase Produced by Dye-Decolorizing Fungus, Geotrichum candidum Decl

Aryl alcohol oxidase (AAO) produced by Geotrichum candidum Dec1 (Dec1), a newly isolated decolorizing fungus, was purified by ultrafiltration and by using diethylaminoethyl (DEAE) Sephacel, Butyl-Toyopearl and Mono-Q columns. H 2O 2 produced by concomitant AAO oxidation of veratryl alcohol (VA) to veratraldehyde was consumed by a peroxidase (DyP) purified from Dec1 culture, leading to the decolorization of a dye in vitro. In the liquid culture of Dec1, the existence of H 2O 2 and veratraldehyde was confirmed during cultivation, when dye-decolorization and AAO activities were maintained. This indicates that VA produced by Dec1 was oxidized by AAO to veratraldehyde, generating H 2O 2, which supported dye-decolorizing activity of Decl in vivo. The prevention of polymerization of DyP oxidation products of a dye in the presence of AAO was shown.

Characterization of Aryl Alcohol Oxidase Produced by Dye-Decolorizing Fungus, Geotrichum candidum Dec1.

Aryl alcohol oxidase (AAO) produced by Geotrichum candidum Dec1 (Dec1), a newly isolated decolorizing fungus, was purified by ultrafiltration and by using diethylaminoethyl (DEAE) Sephacel, Butyl-Toyopearl and Mono-Q columns. H2O2 produced by concomitant AAO oxidation of veratryl alcohol (VA) to veratraldehyde was consumed by a peroxidase (DyP) purified from Dec1 culture, leading to the decolorization of a dye (in vitro). In the liquid culture of Dec1, the existence of H2O2 and veratraldehyde was confirmed during cultivation, when dye-decolorization and AAO activities were maintained. This indicates that VA produced by Dec1 was oxidized by AAO to veratraldehyde, generating H2O2, which supported dye-decolorizing activity of Decl in vivo. The prevention of polymerization of DyP oxidation products of a dye in the presence of AAO was shown.

Functional analysis of PSII-T protein of photosystem II complex from the thermophilic cyanobacterium, Thermosynechococcus (formerlySynechococcus) elongatus BP-1

PSII-T is a membrane-spanning protein with molecular mass of 4.7 kDa, which is associated with the PSII reaction center complex. From the thermophilic cyanobacterium, Thermosynechococcus (formerly Synechococcus) elongatus BP-1, we cloned and determined the nucleotide sequence of psbB and psbT genes which encode CP47 and a small membrane protein (PSII-T) in PSII complex, respectively. We disrupted psbT gene in T. elongatus with the chloramphenicol-resistant cartridge. The mutant could grow photoautotrophically like wild type. Thylakoids and oxygen-evolving PSII core complexes were successfully isolated from the mutant as well as wild type. There was no significant difference in the oxygen-evolving activities of cells, thylakoids, or PSII complexes between the mutant and wild type. This is in contrast with the lower activities of the other PSII mutants in T. elongatus. MonoQ column chromatography revealed that recovery of the dimeric PSII in the psbT-disrupted mutant was much less than wild type. These suggest that PSII-T is specifically involved in dimerization of PSII complex or stabilization of the PSII dimer.

Amphibian pepsinogens: purification and characterization of xenopus pepsinogens, and molecular cloning of Xenopus and bullfrog pepsinogens.

Two pepsinogens (Pg C and Pg A) were isolated from the stomach of adult Xenopus laevis by Q-Sepharose, Sephadex G-75, and Mono-Q column chromatographies. Autolytic conversion and activation of the purified Pgs into the pepsins were examined by acid treatment. We determined the amino acid sequences from the NH2-termini of Pg C, pepsin C, Pg A, and pepsin A. Based on the sequences, the cDNAs for Pg C and Pg A were cloned from adult stomach RNA, and the complete amino acid sequences of the Pg C and Pg A were predicted. In addition, a Pg A cDNA was cloned from the stomach of adult bullfrog Rana catesbeiana, and the primary structure of the Pg A was predicted. Molecular phylogenetic analysis showed that such anuran Pg C and Pg A belong to the Pg C group and the Pg A group in vertebrates, respectively. The molecular properties of Pg C and Pg A, such as size, sequences of the activation peptide and active site, profile of autolytic activation, and pH dependency of proteolytic activity of the activated forms, pepsin C and pepsin A, resemble those of Pgs found in other vertebrates. However, the hemoglobin-hydrolyzing activity of Xenopus pepsin C is completely inhibited in the presence of equimolar pepstatin, an inhibitor of aspartic proteinases. Thus, the Xenopus pepsin C differs significantly from other vertebrate pepsins C in its high susceptibility to pepstatin, and closely resembles A-type pepsins.

PSII-I protein plays an essential role in dimerization of photosystem II

PSII-I is a small membrane-spanning protein, which is associated with the PSII reaction center complex. It is universally found in cyanobacteria as well as green plants, although its function is still not known. In this communication, We attempted to elucidate the role of the PSII-I protein in the thermophilic cyanobacterium, Thermosynechococcus (formerly Synechococcus) elongatus strain BP-1 by targeted disruption and biochemical analysis. The psbI gene encoding PSII-I was cloned by PCR and used for gene disruption. The psbI-disruptant could grow autotrophically like wild type. Oxygen-evolving activity of PSII particles was approximately 20% lower in the mutant than in wild type. MonoQ column chromatography of the crude PSII particles revealed that the PSII dimer is missing in the psbI-disruptant. The molecular size of the monomer PSII from the mutant was confirmed by gel filtration chromatography. These suggest that PSII-I is specifically involved in dimerization of PSII complex or stabilization of the PSII dimer. The crude PSII from the psbI-disruptant exhibited lower efficiency of light-harvesting than that of wild type. These findings imply that dimerization supports structural integrity of the functional PSII or in supramolecular interaction between PSII and antennae complexes.

Purification of germinal-vesicle-breakdown-inducing proteins from Tetrahymena pyriformis

Summary Germinal-vesicle-breakdown (GVBD) of Xenopus laevis oocytes was induced by injecting 132,000 g supernatant of Tetrahymena pyriformis homogenates into oocytes. Using induction of GVBD as a bioassay system, we succeeded in purifying two GVBD-inducing proteins from the 132,000 g supernatant by liquid chromatography with an anion-exchange MonoQ column, ultra-filtration, and electroelution from bands on a native-PAGE gel. A protein of 40 kDa induced GVBD in the absence of protein synthesis of Xenopus oocytes whereas another protein of 56 kDa induced GVBD only in the presence of the oocyte protein synthesis. These proteins did not react with the PSTAIR antibody, which is specific for cdc2 homologues.

Structural and Functional Characterization of Platelet Receptor-mediated Factor VIII Binding

Optimal rates of factor X (FX) activation require occupancy of receptors for factor IXa (FIXa), factor VIII (FVIII), and FX on the activated platelet surface. The presence of FVIII and FX increases 5-fold the affinity of FIXa for the surface of activated platelets, and the presence of FVIII or FVIIIa generates a high affinity, low capacity specific FX-binding site on activated platelets. We have now examined the effects of FX and active site-inhibited FIXa (EGR-FIXa) on the binding of both FVIII and FVIIIa to activated platelets and show the following: (a) von Willebrand factor inhibits FVIII binding (K(i) = 0.54 nM) but not FVIIIa binding; (b) thrombin and the thrombin receptor activation peptide (SFLLRN amide) are the most potent agonists required for FVIII-binding site expression, whereas ADP is inert; (c) FVa does not compete with FVIIIa or FVIII for functional platelet-binding sites; and (d) Annexin V is a potent inhibitor of FVIIIa binding (IC(50) = 10 nM) to activated platelets. The A2 domain of FVIII significantly increases the affinity and stoichiometry of FVIIIa binding to platelets and contributes to the stability of the FX-activating complex. Both FVIII and FVIIIa binding were specific, saturable, and reversible. FVIII binds to specific, high affinity receptors on activated platelets (n = 484 +/- 59; K(d) = 3.7 +/- 0.31 nM) and FVIIIa interacts with an additional 300-500 sites per platelet with enhanced affinity (K(d) = 1.5 +/- 0.11 nM). FVIIIa binding to activated platelets in the presence of FIXa and FX is closely coupled with rates of F-X activation. The presence of EGR-FIXa and FX increases both the number and the affinity of binding sites on activated platelets for both FVIII and FVIIIa, emphasizing the validity of a three-receptor model in the assembly of the F-X-activating complex on the platelet surface.

SYNCRIP, a Cytoplasmic Counterpart of Heterogeneous Nuclear Ribonucleoprotein R, Interacts with Ubiquitous Synaptotagmin Isoforms

Synaptotagmins (Syts) are a large family of membrane proteins consisted of at least 12 isoforms. They are categorized in neuron-specific isoforms (I-V, X, and XI) and ubiquitous isoforms (VI-IX) based on their expression patterns. Syt-I, a neuron-specific and abundant isoform, has been well characterized and postulated to be the exocytotic Ca(2+) sensor. However, the functions of other isoforms remain obscure. Here, we report that ubiquitous isoforms of synaptotagmins, Syt-VII, Syt-VIII, and Syt-IX, interacted with a cytoplasmic RNA-binding protein, SYNCRIP (Synaptotagmin-binding, cytoplasmic RNA-interacting protein), through their C2B domains. SYNCRIP was originally found in the Syt-II C2AB domain bound fraction from the mouse brain lysate. cDNA cloning of SYNCRIP cDNA revealed that the protein was highly homologous to heterogeneous nuclear ribonucleoprotein R (hnRNP R) recently identified. SYNCRIP protein was ubiquitously and constantly expressed in various tissues of mice parallel to hnRNP R. SYNCRIP indeed bound RNA with preference to poly(A) RNA; however, in contrast to the nuclear localization of hnRNP R, SYNCRIP was distributed predominantly in the cytoplasm as judged by both biochemical fractionation and immunohistochemical studies. In vitro binding experiments showed the potential interaction of SYNCRIP with C2B domains of Syts except for those of Syt-V, -VI, and -X. Furthermore, the interaction between SYNCRIP and Syt-VII, -VIII, or -IX was revealed by co-immunoprecipitation experiments using COS cells transiently expressing each Syt isoform. These findings suggested that SYNCRIP was a target of ubiquitous type of Syts and implied the involvement of ubiquitous Syts in the regulation of dynamics of the cytoplasmic mRNA.

Isolation and characterization of Dorin M, a lectin from plasma of the soft tick Ornithodoros moubata

A lectin with high hemagglutinating activity, which we have named Dorin M, was identified in the plasma of the soft tick Ornithodoros moubata. The activity of the plasma lectin could be efficiently inhibited by sialic acid, N-acetyl- d-hexosamines and sialoglycoproteins. Dorin M was purified to homogeneity using two different isolation systems: affinity chromatography on a column of bovine submaxillary mucin conjugated to Sepharose 4B with specific elution by N-acetyl- d-glucosamine and chromatography on Blue-Sepharose followed by anion exchange FPLC on a MonoQ column. The purified lectin is a glycoprotein which, in the native state, forms aggregates with molecular mass of about 640 kDa. Non-reducing SDS PAGE revealed that the lectin consists of two noncovalently bound subunits migrating closely around 37 kDa. Dorin M is a glycoprotein, probably modified by N-type glycosylation. After chemical deglycosylation, only one band of about 32 kDa was detected. Dorin M is the first lectin purified from ticks.

Cloning of phosphatase I gene from a psychrophile, Shewanella sp., and some properties of the recombinant enzyme.

Psychrophilic phosphatase I from Shewanella sp. is a cold enzyme that was found as a novel protein-tyrosine-phosphatase (PTPase, EC 3. 1.3.48) with a histidine as its catalytic residue [Tsuruta and Aizono (1999) J. Biochem. 125, 690-695]. Here, we determined the nucleotide sequence of a DNA fragment (2,004 bp) containing the phosphatase I gene by cloning with polymerase chain reaction (PCR) and inverted PCR techniques. The deduced amino acid sequence, of the enzyme contained a conserved region of protein-serine/threonine-phosphatase (PPase). The 38.5 kDa-recombinant protein expressed in Escherichia coli was purified to homogeneity by glutathione-Sepharose 4B column chromatography, treatment with endoproteinase and Mono-Q column chromatography. The recombinant enzyme had a specific activity of 49.4 units and, like native psychrophilic phosphatase I, exhibited high catalytic activity at low temperature and PTPase activity.

Collagenolytic protease complex fromParalithodes camtschatica hepatopancreas: Separation into individual components

Nine proteases, the main components of the collagenolytic complex from hepatopancreas ofParalithodes camtschatica crab have been isolated by gel filtration and FPLC, including ion-exchange chromatography on DEAE-Toyo-Pearl 650M and Mono-Q columns and hydrophobic interaction chromatography on a Phenyl-Superose column. Molecular weights of the resultant proteases were 36, 35 (2 proteins), 32, 28, 25 (3 proteins), and 23 kD (according to SDS-PAAG electrophoresis and HPLC on a Zorbax GF-250 column). N-terminal analysis showed that six proteases were trypsin-like enzymes homologous to digestive trypsins from other sources. One protease (23 kD) was related toAstacus fluviatilis metalloprotease and two others (32 and 35 (I) kD possess unique N-terminal sequences.

Jararhagin ECD-Containing Disintegrin Domain: Expression in Escherichia coli and Inhibition of the Platelet–Collagen Interaction

Jararhagin, a hemorrhagin from Bothrops jararaca venom, is a soluble snake venom component comprising metalloproteinase and disintegrin cysteine-rich domains and, therefore, is structurally closely related to the membrane-bound ADisintegrin And Metalloproteinase (ADAMs) protein family. Its hemorrhagic activity is associated with the effects of both metalloproteinase and disintegrin domains; the metalloproteinase enzymatically damages the endothelium and the disintegrin domain inhibits platelet–collagen interactions. The expression of whole jararhagin or its disintegrin domain has never been attempted before. The aim of this study was to investigate whether we could express the disintegrin domain of jararhagin and to verify whether this domain displays an inhibitory effect on the platelet–collagen interaction. Therefore, the cDNA fragment coding for the disintegrin plus cysteine-rich domains of jararhagin was cloned into the pET32a vector, used to transform the Escherichia coli AD494(DE3)pLysS strain. The thioredoxin–disintegrin fusion protein was recovered from the soluble extract of the cells, yielding up to 50 mg/liter culture. The fusion protein was isolated using polyhistidine binding resin which resulted in a main band of 45 kDa recognized by anti-native jararhagin antibodies. Antibodies raised in rabbits against the fusion protein had high enzyme-linked immunosorbent assay titers against native jararhagin and detected a band of 52 kDa on Western blots of whole B. jararaca venom demonstrating that these antibodies recognize the parent jararhagin molecule. Treatment of the fusion protein with enterokinase, followed by further capture of the enzyme, resulted in a band of 30 kDa, the expected size for jararhagin-C. Further purification of the cleaved disintegrin using FPLC Mono-Q columns resulted in one fraction capable of efficiently inhibiting collagen-induced platelet aggregation in a dose-dependent manner (IC50 of 8.5 μg/ml).

Expression and purification of the recombinant protective antigen of Bacillus anthracis.

Protective antigen (PA) is a major component of the vaccine against anthrax. The structural gene for the 83-kDa PA was expressed as fusion protein with 6x Histidine residues in Escherichia coli. Expression of PA in E. coli under the transcriptional regulation of the T5 promoter yielded an insoluble protein aggregating to form inclusion bodies. The inclusion bodies were solubilized in 6 M guanidine-HCl and the protein was purified under denaturing conditions using nickel nitrilotriacetic acid (Ni-NTA) affinity chromatography. The denatured protein was renatured by gradual removal of the denaturant while immobilized on the Ni-NTA column. The protein was then purified using Mono-Q column on FPLC. The yield of the purified recombinant PA (rPA) from this procedure was 2 mg/liter of the culture. The rPA had an apparent molecular mass of 83 kDa as determined by SDS-PAGE. Antisera to native PA recognized the fusion protein. The rPA was biologically as well as functionally active. Thus, the recombinant PA may be used to develop an effective recombinant vaccine against anthrax.

Purification and preliminary characterisation of a thiamine-binding protein from spruce seeds

A thiamine-binding protein was isolated from spruce seeds (Picea abies L.), Karst.) in a nearly homogeneous form using a combination of ammonium sulphate fractionation, DEAE-cellulose ion-exchange chromatography, high performance gel filtration on TSK G3000SW column and fast protein ion-exchange chromatography with MonoQ column. SDS-polyacrylamide gel electrophoresis revealed the basic subunit of 23 kDa. However, the native protein was an oligomer with a molecular mass of about 130 kDa as estimated by gel filtration on analytical Superdex-200 column. The estimated isoelectric point was about 5.1. Thiamine was bound with a capacity of 8.5 nmol per mg protein, suggesting a simple 1:1 molar stoichiometry of thiamine-protein interaction. The dissociation constant of the complex was 8 μM in 0.05 M phosphate buffer at optimal pH 8.6. Several thiamine analogues were also bound to this protein but always with a lower affinity than thiamine. From this chemical probing, the binding site on the spruce protein seems to fit the model, previously deduced for the purified buckwheat-seed protein and generalised in a study of seed extracts of species sampled from major classes of Spermatophyta.

Purification and characterization of protease produced by Staphylococcus aureus isolated from a diseased chicken

A protease produced by Staphylococcus aureus, isolated from a chicken suffering from dermatitis, was purified by successive precipitation with ammonium sulfate, ion-exchange chromatography on Q-Sepharose FF, Sp-Sepharose FF and Mono-Q columns. By Mono-Q column chromatography, two proteases (protease 1 and 2) were obtained. The molecular weights of protease 1 and 2 were estimated at 23.1 and 22.7 kDa, respectively, by SDS-polyacrylamide gel electrophoresis. Their isoelectric points were 5.85 and 5.55, respectively, and they possessed antigenic similarity when examined by the immunoblotting. The N-terminal amino acid sequences of both the proteases were identical (RAQYVNQLKNFKIRETQ). The activities of both the proteases were strongly increased by reducing agents such as L-cysteine and sodium thioglycolate. Their activity was inhibited by thiol protease inhibitors, but was not inhibited by metalloprotease or serine protease inhibitors. From the results, it seems likely that these proteases, produced by S. aureus from diseased chickens, might belong to the thiol protease group.

Glucose Transporter Glut3 Is Targeted to Secretory Vesicles in Neurons and PC12 Cells

In rat brain and cultured neuroendocrine PC12 cells, Glut3 is localized at the cell surface and, also, in a distinct population of homogenous synaptic-like vesicles. Glut3-containing vesicles co-purify with "classical" synaptic vesicles, but can be separated from the latter by sucrose gradient centrifugation. Unlike classical synaptic vesicles, Glut3-containing vesicles possess a high level of aminopeptidase activity, which has been identified as aminopeptidase B. This enzyme has recently been shown to be a marker of the secretory pathway in PC12 cells (Balogh, A., Cadel, S., Foulon, T., Picart, R., Der Garabedian, A., Rousselet, A., Tougard, C., and Cohen, P. (1998) J. Cell Sci. 111, 161-169). We, therefore, conclude that Glut3 is targeted to secretory vesicles in both neurons and PC12 cells.

CDNA Cloning and Expression of Acutin,a Thrombin-Like Enzyme fromAgkistrodonacutus

Acutin, a thrombin-like enzyme was purified fromAgkistrodonacutus venom in three steps by DEAE-Sepharose CL-6B, Superose 12 column on FPLC and Mono-Q column chromatographies. Its first 15 N-terminal amino acid residues sequence 〈VIGGVECDINEHRFL〉 was then determined and the acutin cDNA was isolated from venom gland total RNA using RT-PCR. Determination of its nucleotide sequence allowed elucidation of the amino acid sequence of mature peptide for the first time. The mature acutin has 233 amino acids and its amino acid sequence exhibits significant homology with those of thrombin-like enzymes from crotaline snakes venoms. Based on the homology, the catalytic residues and disulfide bridges of acutin were deduced to be as follows: catalytic residues, His41, Asp84and Ser179; and disulfide bridges, Cys7-Cys139, Cys26-Cys42, Cys74-Cys231, Cys118-Cys185, Cys150-Cys164, Cys175-Cys200. The recombinant acutin has been expressed in E. coli and purified by affinity column. The renatured recombinant acutin is reported for the first time to have the activity of clotting fibrinogen and arginine-esterase.

Actinobacillus actinomycetemcomitans toxin induces both cell cycle arrest in the G2/M phase and apoptosis.

We found that the culture supernatant of the periodontopathic bacterium Actinobacillus actinomycetemcomitans had a cytotoxic effect on several cell lines. In this study, we purified the toxin from the culture supernatant of A. actinomycetemcomitans Y4 by a four-step procedure: ammonium sulfate precipitation, POROS HQ/M column chromatography, polymyxin B matrix column chromatography, and Mono-Q column chromatography. The purified toxin gave two major bands of protein with molecular masses of 80 and 85 kDa upon sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The mechanism of cell death of the B-cell hybridoma cell line HS-72 was examined by observing changes in nuclear morphology, an increase in the proportion of fragmented DNA, and the typical ladder pattern of degraded chromosomal DNA, indicating the induction of apoptosis. Overexpression of human Bcl-2 suppressed apoptosis in HS-72 cells, indicating that the toxin from A. actinomycetemcomitans induces apoptosis by a Bcl-2-inhibitable mechanism. Flow cytometric analysis revealed that the toxin caused cell cycle arrest in the G2/M phase and apoptosis in HS-72 cells. In addition, aurintricarboxylic acid, a DNA endonuclease inhibitor, markedly decreased the percentage of apoptotic cells but had no effect on cell cycle arrest in the G2/M phase. Taken together, these findings suggest that the toxin from A. actinomycetemcomitans could mediate the development of periodontal diseases through cell cycle arrest in the G2/M phase and apoptosis in B lymphocytes of periodontal tissue.

Intra- and Extracellular Expression of Rabbit Reticulocyte 15-Lipoxygenase in the Baculovirus/Insect Cell System

Rabbit reticulocyte 15-lipoxygenase was expressed as intracellular enzyme and as export protein in High Five cells. While intracellular expression in the baculovirus/insect cell system was already reported for various mammalian lipoxygenases, we have developed a strategy for secretion of this cytosolic enzyme using the signal sequence of human interleukin-2. Expression levels of 10 mg/liter (intracellular strategy) and 18 mg/liter (extracellular strategy) were obtained. The recombinant enzyme expressed as intracellular protein was purified to apparent homogeneity by anion-exchange chromatography on a Mono-Q column with an overall recovery of 80% enzyme activity. For the final enzyme preparation, a specific linoleic acid oxygenase activity of 16.7 micromol 13-hydroperoxyoctadeca-9,11-dieic acid formation mg-1 min-1 was determined, which corresponds to a molecular turnover number of 21 s-1. Similar turnover rates have been reported for the native rabbit 15-lipoxygenase. Extracellularly expressed recombinant 15-lipoxygenase exhibited a heterogeneity in anion-exchange chromatography. Three major peaks of 15-lipoxygenase activity were eluted from a Mono-Q column and the relative amounts of these isoforms varied from batch to batch of enzyme expression. One of the major isoenzymes which cochromatographed with the native 15-lipoxygenase was purified to homogeneity from the cell-free culture supernatant and exhibited a specific activity of 5.1 micromol 13-hydroperoxyoctadeca-9,11-dienoic acid formation mg-1 min-1 (turnover rate of 6.1 s-1). The recombinant enzyme species were characterized with respect to their protein-chemical and enzymatic properties and no differences to the native rabbit 15-lipoxygenase were detected.

Binding of Inhibiting Adducts of Ketones and NAD+to Alcohol Dehydrogenase fromDrosophila melanogaster

Drosophilaalcohol dehydrogenase (DADH) can be converted with NAD+and ketone into more negatively charged isoforms. Completely modified ADH isoforms are inactive, but activity is regained after native polyacrylamide gel electrophoresis depending on the ketone used. When unmodified ADH is incubated with NAD+and acetylacetone, more negatively charged bands appear. Modified ADH isoforms bind to a Mono-Q column, while unmodified ADH and NAD+do not. A covalently bound adduct is also formed between NAD+and ketone in the absence of enzyme. These adducts can be purified by ion-exchange FPLC and have been characterized by mass spectrometry and UV spectroscopy and also form inactive isoforms after incubation with unmodified enzyme. The effect of temperature increase on unmodified and modifiedDrosophila melanogastervariants was determined by circular dichroism experiments. Unfolding temperatures (Tm) of the modified ADH isoforms were 5 to 20° higher than those of the unmodified ones, indicating that the enzyme molecule becomes more compact upon adduct binding.