DEAE-Toyopearl Chromatography Research Articles

Identification of functional regions in the Rhodospirillum rubrum L-asparaginase by site-directed mutagenesis.

Site-directed mutagenesis of Rhodospirillum rubrum L-asparaginase (RrA) was performed in order to identify sites of the protein molecule important for its therapeutic and physico-chemical properties. Ten multipoint mutant genes were obtained, and five recombinant RrA variants were expressed in E. coli BL21(DE3) cells and isolated as functionally active highly purified proteins. Protein purification was performed using Q-Sepharose and DEAE-Toyopearl chromatography. Overall yield of the active enzymes was 70-80 %, their specific activity at pH 7.4 and 37 °C varied of 140-210 U/mg. L-Glutaminase activity did not exceed 0.01 % of L-asparaginase activity. All RrA mutants showed maximum enzyme activity at pH 9.3-9.5 and 53-58 °C. Km and Vmax values for L-asparagine were evaluated for all mutants. Mutations G86P, D88H, M90K (RrAH), G121L, D123A (RrАI) caused the loss of enzyme activity and confirmed the importance of these sites in the implementation of catalytic functions. Removal of four residues from C-terminal area of the enzyme (RrAK) resulted in the enzyme instability. Mutations D60K, F61L(RrАD), and R118H, G120R(RrАJ) led to the improvement of kinetic parameters and enzyme stabilization. Substitutions E149R, V150P (RrАB) improved antineoplastic and cytotoxic activity of the RrA. A64V, E67K substitutions, especially in combination with E149R, V150P (RrАE), considerably destabilized recombinant enzyme.

Inulin fructotransferase (DFA III-producing) from Leifsonia sp. T88-4

An inulin fructotransferase (DFA III-producing) [EC 2.4.1.93] from Leifsonia sp. T88-4 was purified and characterized, for the first time. The enzyme was purified from culture supernatant of the microorganism 32.4-fold with a yield of 11.3% using a DEAE–Toyopearl chromatography and two times of Super Q-Toyopearl chromatography. The enzyme showed maximum activity at pH 5.0 and 65 °C. This maximum temperature was highest in the inulin fructotransferase (DFA III-producing) reported to date. The enzyme activity was stable up to 60 °C after 30 min heat treatment. The molecular mass of the enzyme was estimated to be 44 kDa by SDS–PAGE and 74 kDa by gel filtration, and was considered to be a dimer. The N-terminal amino acid sequence (16 amino acid residues) was determined as METNVYDVTDVGVPGR. The K m value of the reaction at pH 5.0 and 60 °C was estimated to be 1.0 mM.

Thermostable inulin fructotransferase (DFA III-producing) from Arthrobacter sp. L68-1

An inulin fructotransferase (DFA III-producing) [EC 2.4.1.93] from Arthrobacter sp. L68-1 was purified and characterised. The enzyme was purified from culture supernatant of the microorganism 54.2-fold with a yield of 16.0% using DEAE-Toyopearl chromatography (repeated twice) and Super Q-Toyopearl chromatography. The enzyme showed maximum activity at pH 5.5–6.0 and 55 °C. The enzyme activity was stable up to 80 °C after 1 h heat treatment. This heat stability was the highest of the inulin fructotransferases (DFA III-producing) reported to date. The molecular mass of the enzyme was estimated to be 43 kDa by SDS-PAGE and 73 kDa by gel filtration, and was considered to be a dimer. The N-terminal amino acid sequence (18 amino acid residues) was determined as AEETKGGPFNSPNAYDVT.

Gene cloning and overproduction of low-specificity D-threonine aldolase from Alcaligenes xylosoxidans and its application for production of a key intermediate for parkinsonism drug.

The dtaAX gene encoding a pyridoxal 5'-phosphate (pyridoxal-P)-dependent low-specificity D-threonine aldolase was cloned from the chromosomal DNA of Alcaligenes xylosoxidans IFO 12669. It contains an open reading frame consisting of 1,134 nucleotides corresponding to 377 amino acid residues. The predicted amino acid sequence displayed 54% identity with that of D-threonine aldolase from gram-positive bacteria Arthrobacter sp. DK-38, but showed no significant similarity with those of other known pyridoxal-P enzymes. This gram-negative bacterial enzyme was highly overproduced in recombinant Escherichia coli cells, and the specific activity of the enzyme in the cell extract was as high as 18 U/mg (purified enzyme 38.6 U/mg), which was 6,000 times higher than that from the wild-type Alcaligenes cell extract. The recombinant enzyme was thus feasibly purified to homogeneity by ammonium sulfate fractionation and DEAE-Toyopearl chromatography steps. The recombinant low-specificity D-threonine aldolase was shown to be an efficient biocatalyst for resolution of L-beta-3,4-methylenedioxyphenylserine, an intermediate for production of a therapeutic drug for Parkinson's disease.

Epitope mapping for the anti-rabbit cholesteryl ester transfer protein monoclonal antibody that selectively inhibits triglyceride transfer

Among the monoclonal antibodies (Mab) against rabbit plasma cholesteryl ester transfer protein (CETP), Mab 14-8F cross-reacted with human CETP and selectively inhibited triglyceride transfer but not cholesteryl ester transfer (Ko, K. W. S., T. Ohnishi, and S. Yokoyama. 1994. J. Biol. Chem. 269: 28206;-28213). The epitope of this antibody was studied by using synthetic fragment peptides of rabbit and human CETP. Mab 14-8F reacted with the peptide R451-Q473 of human CETP near the carboxyl-terminal and not with the peptides representing any other regions, and inhibited the binding of human CETP to the goat antibody against its carboxyl-terminal peptide R451-S476. The experiments with a series of the fragment peptides in this region revealed that the epitope requires the segment 465-473 (EHLLVDFLQ) of human CETP or 485-493 (KHLLVDFLQ) of rabbit CETP (core epitope) though neither peptide by itself binds to the antibody. Both peptides needed extension at least by one residue beyond either amino- or carboxyl-end in order to show the reactivity to the antibody, but the effect was not highly residue-specific at least at the amino-end. Circular dichroism analysis demonstrated the increase of helical conformation by the extension of the "core epitope" peptides to either direction. Thus, the epitope is dependent on conformation of the core epitope induced by the presence of an additional residue(s) in either end. The core epitope occupies the central 64% of the reported linear epitope of Mab TP2, a widely used anti-human CETP monoclonal antibody that inhibits both cholesteryl ester and triglyceride transfer.Therefore, we conclude that the limited interaction of Mab with a common lipid interaction site causes selective inhibition of the transfer of triglyceride that has presumably lower priority than cholesteryl ester for the CETP reaction.

Purification and Characterization of Phytase from Bran of Triticum aestivum L.cv. Nourin #61.

Two phytase isozymes (PHY1 and PHY2) were purified homogeneously from bran of Triticum aestivum L. cv. Nourin #61 by (NH4)2SO4 fractionation, methanol fractionation, Sephacryl S-200 HR gel filtration chromatography, DEAE-TOYOPEARL chromatography, CM-TOYOPEARL chromatography and second Sephacryl S-200 HR gel filtration chromatography. Molecular weights of the two enzymes were 71,000 and 66,000 by gel filtration, and 68,000 and 66,000 by SDS-PAGE, respectively. Optimum pH and temperatures were 6.0 and 45°C for PHY1, and 5.5 and 50°C for PHY2. The activity of both phytases was stable at pHs between pH 4.0-7.0 and below 40°C. The Km values for myo-inositol hexakisphosphate (IHP) were 0.48 μM for PHY1 and 0.77 μM for PHY2. The Ki values for Pi were 2.69 mM for PHY1 and 6.59 mM for PHY2. Both phytases showed relatively high specificity for IHP.

Purification and characterization of a cysteine proteinase from eggs of the cotton boll worm, Helicoverpa armigera

High proteolytic activities were detected from oocytes of the cotton boll worm, Helicoverpa armigera at pH 3–4. The proteolytic activities can be inhibited by TosLysCH 2Cl, iodoacetate, E-64, chymostatin, but not by TosPheCH 2Cl, EDTA, iPr 2P-F and pepstatin. It is suggested that a cysteine proteinase might exist in the oocytes. By DEAE-cellulose and DEAE-Toyopearl chromatography, a proteinase was purified from the oocytes. The molecular mass of the proteinase was estimated at 30 kDa by SDS-PAGE. This proteinase effectively hydrolyzed both bovine serum albumin and bovine hemoglobin at pH 3–4. The most effective temperature for the proteinase is 30–50°C. The activity of the proteinase can be inhibited by TosLysCH 2Cl, iodoacetate, iPr 2P-F, E-64, chymostatin and leupeptin. Because E-64 is a specific inhibitor of cysteine proteinase, the purified proteinase is likely to be a cysteine proteinase. Also, this proteinase is inhibited by iPr 2P-F, a specific inhibitor of serine proteinase, which suggests that serine residue is also necessary for its activiation of the proteinase.

Cloning, sequencing, high expression, and crystallization of the thermophile Thermus aquaticus glycerol kinase.

Glycerol kinase (EC 2.7.1.30) is a key enzyme of glycerol uptake and metabolism in bacteria. Using PCR, we amplified and cloned a glycerol kinase gene, glpK, from Thermus aquaticus. The complete gene has 1488 base pairs, coding for a protein of 496 amino acids with a predicted molecular weight of 54,814. The amino acid sequence deduced from T. aquaticus glpK was found to have identities of 97 and 81%, respectively, with those of Thermus flavus and Bacillus subtilis glpK genes. After overproduction in Escherichia coli, the expressed enzyme was easily purified to homogeneity by DEAE-Toyopearl chromatography. The purified enzyme has been crystallized by the hanging drop vapor diffusion method at 22 degrees C. Comparison of the amino acid sequence with that of the B. subtilis enzyme showed that Ser and Lys are replaced by Ala and Arg, as was seen in mesophile and thermophile enzymes.

Separation and amino acid composition of three troponin components from bovine muscle

Three troponin components were isolated from bovine skeletal muscle, and their molecular weights and amino acid composition were studied. Crude troponin prepared from bovine muscle was purified by DEAE-Toyopearl chromatography. The purified troponin was dissociated in the order of tropopins C, I and T by CM-Toyopearl chromatography in the presence of 6 M urea. The molecular weights of troponins C, I and T were 19,500, 23,300 and 40,400, respectively, as determined with SDS-PAGE. The separation of troponin into three components was also achieved using reverse-phase HPLC; however, the elution order of troponins T and C was contrary to that of the cation-exchange chromatography described above. In this study, the amino acid composition of the three troponin components from bovine skeletal muscle was first determined. The amino acid composition of the three troponin components among bovine, rabbit and chicken skeletal muscles showed stronger similarity than that between bovine skeletal and cardiac muscle with a different muscle type. We considered that this method of troponin preparation from bovine muscle must be a very useful technique for investigating the changes in troponin components, especially troponin T, during ageing of beef.

Overexpression and feasible purification of thermostable L-2-halo acid dehalogenase ofPseudomonas sp. YL

The gene encoding thermostable L-2-halo acid dehalogenase of Pseudomonas sp. YL was isolated, and its overexpression system was constructed. Gene library was prepared from Sau3AI fragments of total DNA from Ps. sp. YL, pUC118 as a vector and Escherichia coli JM109 as a host. The recombinant cells resistant to bromoacetate, a germicide, were isolated and shown to produce L-2-halo acid dehalogenase. Subsequently, subcloning was carried out with pKK223-3 as a vector, and the length of DNA inserted was reduced to 1.1 kbp. One of the subclones showed very high activity, and the amount of the dehalogenase produced corresponded to about 30% of the soluble protein. From 5 g (wet weight) of cells, 105 mg of dehalogenase was efficiently purified by heat treatment and DEAE-Toyopearl chromatography. This overexpression system provides a large amount of the thermostable enzyme to enable us to study the properties, structure and application of the enzyme.

Purification and Properties of Extracellular Amylase from the Hyperthermophilic Archaeon Thermococcus profundus DT5432

A hyperthermophilic archaeon, Thermococcus profundus DT5432, produced extracellular thermostable amylases. One of the amylases (amylase S) was purified to homogeneity by ammonium sulfate precipitation, DEAE-Toyopearl chromatography, and gel filtration on Superdex 200HR. The molecular weight of the enzyme was estimated to be 42,000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The amylase exhibited maximal activity at pH 5.5 to 6.0 and was stable in the range of pH 5.9 to 9.8. The optimum temperature for the activity was 80(deg)C. Half-life of the enzyme was 3 h at 80(deg)C and 15 min at 90(deg)C. Thermostability of the enzyme was enhanced in the presence of 5 mM Ca(sup2+) or 0.5% soluble starch at temperatures above 80(deg)C. The enzyme activity was inhibited in the presence of 5 mM iodoacetic acid or 1 mM N-bromosuccinimide, suggesting that cysteine and tryptophan residues play an important role in the catalytic action. The amylase hydrolyzed soluble starch, amylose, amylopectin, and glycogen to produce maltose and maltotriose of (alpha)-configuration as the main products. Smaller amounts of larger maltooligosaccharides were also produced with a trace amount of glucose. Pullulan; (alpha)-, (beta)-, and (gamma)-cyclodextrins; maltose; and maltotriose were not hydrolyzed.

Purification and properties of mannanase from Bacillus subtilis

Extracellular mannanase from Bacillus subtilis NM-39, an isolate from Philippine soil, was purified about 240-fold with a yield of 7.3% by ammonium sulphate fractionation, DEAE-Toyopearl chromatography and Sephacryl S-200 gel filtration. Its M r was 38 kDa and it had a pI of 4.8 and optimum activity at pH 5.0 and 55°C. It was stable at pH 4 to 9 and below 55°C. The amino acid composition of the enzyme was in the order Gly>Glx>Ser and Asx>Ala.

Nicotinamide Mononucleotide Adenylyltransferase Activity in Human Erythrocytes

Nicotinamide mononucleotide adenylyltransferase (NMN-AT) activity has not hitherto been demonstrated in human red blood cells, owing to its low activity. Since it is usually located in the nucleus, the possibility of finding it in human erythrocytes was excluded. Here we report the first demonstration and characterization of NMN-AT in human red blood cells, by an HPLC method. The enzyme is Mg2+ dependent, with a Km of 0.303 mM for nicotinamide mononucleotide and 0.103 mM for ATP, and a Vmax of 346 amolg Hb−1 h−1. The crude preparation is also active on nicotinic acid mononucleotide, producing nicotinic acid adenine dinucleotide. NMN-AT activity is inhibited by nicotinic acid mononucleotide, and nicotinic acid adenylyltransferase is inhibited by nicotinamide mononucleotide. Fiftyfold purification of NMN-AT was achieved by DEAE-Toyopearl chromatography, and the kinetic characteristics were determined. The partially purified preparation maintained its nicotinic acid adenylyltransferase activity. These findings are discussed in light of the regulation of NAD metabolism in human red blood cells.

Isolation of Carp Myosin Rod and Its Structural Stability.

Carp myosin rod was isolated from the chymotryptic digest of myosin by using DEAE-Toyopearl chromatography. The carp rod thus prepared was very similar to rabbit rod in its molecular mass, viscosity, sedimentation velocity, and amino acid composition. Changes in tryptophan fluorescence intensity indicated that carp rod more easily unfolded upon addition of urea or guanidine-HCI, and thus was structurally less stable than rabbit rod. It was found that the addition of 8-anilino-1-naphthalene sulfonate (ANS) to carp rod solution resulted in no change in fluorerescence intensity, providing no information on its structural stability. However, ANS was an excellent probe for detecting the structural change of myosin subfragment-1 (S-1) portion; namely carp S-1 required a lower urea concentration than rabbit S-1 for inducing an increase in the ANS fluorescence intensity.

Partial purification and characterization of an endo-α-N-acetylgalactosaminidase from the culture medium of Streptomyces sp. OH-11242

For the purification of a new type of endo-alpha-N-acetylgalactosaminidase from the culture medium of Streptomyces sp. OH-11242 (endo-GalNAc-ase-S) [Iwase, Ishii, Ishihara, Tanaka, Omura & Hotta (1988) Biochem. Biophys. Res. Commun. 151, 422-428], a method for assaying enzyme activity was established. Using purified pig gastric mucus glycoprotein (PGM) as the substrate, oligosaccharides liberated from PGM were pyridylaminated, and the reducing terminal sugars of oligosaccharides larger than Gal beta 1-3GalNAc were analysed by h.p.1.c. The crude enzyme of endo-GalNAc-ase-S was prepared as an 80% (w/v) ammonium sulphate precipitate from the concentrated culture medium. The enzyme was partially purified by gel chromatofocusing and subsequent DEAE-Toyopearl chromatography. Endo-enzyme activity eluted around pI 4.8 on a gel chromatofocusing column and eluted with 0.19-0.25 M-NaCl on a DEAE-Toyopearl column. In the enzyme fraction obtained, no exo-glycosidases or proteases could be detected. The molecular mass of the enzyme was estimated as 105 kDa by gel filtration, and the optimum pH was 5.5. Endo-GalNAc-ase-S hydrolysed the O-glycosidic linkage between GalNAc and Ser (Thr) in 3H-labelled and unlabelled asialofetuin, liberating both the disaccharide (Gal beta 1-3GalNAc) and the tetrasaccharide [Gal beta 1-3 (Gal beta 1-4GlcNAc beta 1-6)GalNAc]. When endo-alpha-N-acetylgalactosaminidase from Alcaligenes sp. (endo-GalNac-ase-A) was incubated with 3H-labelled and unlabelled asialofetuin, only the disaccharide (Gal beta 1-3GalNAc) was liberated.

Reconstitution of Na+/H(+)-antiporter of bovine renal brush-border membrane into proteoliposomes and detection of a 110 kDa protein cross-reactive with antibodies against a human Na+/H(+)-antiporter partial peptide in antiport-active fractions after partial fractionation.

Bovine renal brush-border membranes were solubilized by 1.6% sodium cholate. Na+/H(+)-antiporter was recovered in the supernatant after centrifugation at 160,000 x g for 1 h and was successfully reconstituted into proteoliposomes by a cholate-dialysis procedure. The reconstituted Na+/H(+)-antiporter showed a pH-gradient dependent and amiloride-sensitive 22Na+ uptake very similar to that of brush-border membrane vesicles. Factors affecting the efficiency of reconstitution as well as the stability of the solubilized antiporter at various temperatures were studied. Sodium cholate-solubilized brush-border membrane proteins were fractionated by Sephacryl S-400 and DEAE-Toyopearl chromatography, and fractions containing reconstitutively active Na+/H(+)-antiporter were identified. A 110 kDa peptide cross-reactive with a polyclonal antibody against a C-terminal peptide (22-amino acid residues) of human Na+/H(+)-antiporter was consistently found on the immunoblot of the active fractions. A closely similar peptide was also detected in human placental membranes by this antibody. These results strongly suggest that the 110 kDa protein is responsible for Na+/H(+)-antiporter activity.

Purification of solanesyl-diphosphate synthase from Micrococcus luteus. A new class of prenyltransferase.

The activity of solanesyl-diphosphate synthase from Micrococcus luteus is stimulated by a high molecular mass fraction (HMF) which is separated from cell-free extracts of the same bacterium by DEAE-Toyopearl chromatography followed by Sephadex G-100 chromatography. By employing HMF in the assay procedure, solanesyl-diphosphate synthase was able to be purified to homogeneity and was found to be a homodimer with a monomeric molecular mass of 34 kDa. In contrast to hexaprenyl- and heptaprenyl-diphosphate synthases, which are composed of two easily dissociable components that are inactive unless combined, the homogeneously purified solanesyl-diphosphate synthase itself showed a catalytic activity, though weak, catalyzing the synthesis of both (all-E)-nonaprenyl-(solanesyl-) and (all-E)-octaprenyl diphosphate. HMF does not affect the stability of solanesyl-diphosphate synthase or Km values for isopentenyl diphosphate and farnesyl diphosphate, but it markedly increases Vmax values in a time-dependent manner. Several lines of evidence indicate that HMF contains a factor which binds to polyprenyl products and removes them out of the active site of enzyme to facilitate and maintain the turnover of catalysis.

Isolation of Streptomyces sp. producing glucose-tolerant .BETA.-glucosidases and properties of the enzymes.

Stveptomyces sp. No. 35, taxonomically closed to S. thermodiastaticus, was isolated from domestic soil. It showed β-glucosidase activity only in the cells. The enzyme synthesis was induced by cellobiose. Repeated DEAE-Toyopearl chromatography of a sonic extract of the cells showed three activity peaks, F1, F2, and F3, which were eluted in the relative amount of 50 :50:1 in this order by an NaCl concentration gradient. The enzymes were purified to homogeneity. Molecular weights of F1 F2, and F3 were 100,000, 105,000, and 130,000 by gel filtration, and 52,000, 50,000, and 52,000, respectively, by sodium dodecyl sulfate-polyacrylamide-gel electrophesis. F1 and F2 hydrolyzed PNP-β-glucoside and cellobiose nearly at the same rate, while F3 hydrolyzed the former slightly. F1 and F2 were inhibited by glucose, giving Kis of 56 and 40 mm, respectively. On the other hand, F3 was activated twofold by 0.1 m glucose and inhibited 50% by 1 m glucose. Xylose also similarly activated F3. All the enzymes had β-galactosida...

Purification of Mouse Ren 2 Prorenin Produced in Chinese Hamster Ovary Cells1

Renin is produced from a larger, inactive precursor, prorenin, by endoproteolytic removal of the amino-terminal prosegment. In this study, we have transfected Chinese hamster ovary cells with the expression plasmid of mouse Ren 2 preprorenin, and have purified mouse Ren 2 prorenin from the incubation medium of these cells by DEAE-Toyopearl chromatography, Blue-Toyopearl chromatography, and isoelectric focusing. Prorenin thus purified has a molecular mass of 42 kDa as determined by SDS-PAGE and an isoelectric point of 6.5. Amino-terminal sequencing has demonstrated that the purified prorenin has the amino-terminus predicted from the nucleotide sequence of mouse Ren 2 preprorenin cDNA.

Three isozymes of catechol 1,2-dioxygenase (pyrocatechase), alpha alpha, alpha beta, and beta beta, from Pseudomonas arvilla C-1.

Three isozymes of catechol 1,2-dioxygenase (pyrocatechase) from Pseudomonas arvilla C-1 were separated using DEAE-Toyopearl chromatography. The specific activities of each isozyme were similar to one another. The molecular weights of isozymes 1, 2, and 3 were estimated to be approximately 67,000, 64,000, and 59,000, respectively, from gel filtration. On sodium dodecyl sulfate-polyacrylamide gel electrophoresis, isozymes 1 and 3 gave a single protein band, corresponding to Mr = 32,000 and 30,000, respectively, and isozyme 2 gave two bands corresponding to Mr = 32,000 and 30,000. These results indicated that isozymes 1 and 3 were homodimers, while isozyme 2 was a heterodimer. The NH2-terminal sequences up to 20 residues of these three isozymes confirmed that isozymes 1, 2, and 3 consisted of beta beta, alpha beta, and alpha alpha, respectively, based on our previous data (Nakai, C., Kagamiyama, H., Saeki, Y., and Nozaki, M. (1979) Arch. Biochem. Biophys. 195, 12-22). Properties of these isozymes such as absorption spectrum, iron content, substrate specificity, and kinetic constants were similar to one another. Subunit exchange between the different isozymes and dissociation of the isozymes into subunits was not observed under nondenaturing conditions. Available evidence indicates that these isozymes exist naturally in the bacterium and were not due to artifacts caused by purification.