Presence Of 2-mercaptoethanol Research Articles

O-Phthaldehyde fluorescence microassay for the determination of antibody concentration

o-Phthaldehyde (OPA) reagent reacts with primary amines in aqueous solution at room temperature. When the reaction occurs in the presence of 2-mercaptoethanol, a bright blue fluorescence is produced. We investigated the application of the OPA reagent in the routine determination of monoclonal antibody concentration. The OPA reagent produced maximal fluorescence within 30 min and was stable for 2 h. Buffers containing primary amines gave high background fluorescence; however, the OPA reagent gave reliable results with the most commonly used antibody solutions. The OPA reagent provided a rapid and simple measure of protein concentration from 1 μg to 1 mg and was readily adapted for use with a 96-well fluorescence reader.

Kinetic and electrophoretic characterization of NADP dependent dehydrogenases from root tissues of Norway spruce [Picea abies (L.) Karst.] employing a rapid one-step extraction procedure

The NADPH generating enzymes glucose-6-phosphate dehydrogenase (EC 1.1.1.49), 6-phosphogluconate dehydrogenase (EC 1.1.1.44), and isocitrate-dehydrogenase (NADP dependent; EC 1.1.1.42) have been characterized in spruce [Picea abies (L.) Karst.] roots. Interference from inherent phenolic compounds was minimized by complexation with borate and insoluble polyvinylpyrrolidone in the presence of 2-mercaptoethanol and NADP. A further addition of protective substances had no (bovine serum albumin) or even an inhibitory effect (ascorbate) on the enzyme activities. The enzymes were shown to be strictly NADP specific. The optimal pH values and the apparent Michaelis constants from spruce roots are in good agreement with data from different photosynthetic organisms and gametophytic tissues of conifers. Native electrophoresis and subsequent activity staining showed the same banding patterns for enzymes both from root and needle tissues. In addition, the applicability of a highly sensitive dot-blot assay for the accurate quantification of the extracted protein is shown.

Preparation and application of low molecular weight poly(vinyl chloride). IV. Preparation and characteristics of poly(vinyl chloride) with broad molecular weight distribution

AbstractPoly(vinyl chloride) (PVC) with a broad molecular weight distribution (BMD‐PVC) was prepared by suspension polymerization in the presence of PVC with relatively lower molecular weight (LMW‐PVC), which was prepared by suspension polymerization in the presence of 2‐mercaptoethanol as a chain‐transfer agent. It is elucidated using porosity measurement, scanning electron microscopy (SEM), and energy dispersion X‐ray microscopy (EDXM) that the resultant BMD‐PVC grains have an interesting internal structure at the level of primary particles. Discoloration time and fusion time of the BMD‐PVC was studied. Discoloration time and fusion time of BMD‐PVC is particularly dependent on the polymerization degree (Pw) of LMW‐PVC and LMW‐PVC content in the BMD‐PVC samples. © 1994 John Wiley & Sons, Inc.

Soluble factors produced by macrophages/monocytes inhibit lymphokine-activated killer activity in rat splenocyte cultures.

In the present study we investigated the inhibition of interleukin-2(IL-2)-induced lymphokine-activated killer (LAK) activity in rat splenocyte cultures in relation to the presence of 2-mercaptoethanol and macrophages/monocytes. The presence of 2-mercaptoethanol is necessary for induction of LAK activity in rat splenocyte cultures. Removal of macrophages/monocytes from rat splenocytes by plastic or nylon-wool adherence, or iron ingestion resulted in LAK induction by IL-2 in the absence of 2-mercaptoethanol. The effect of macrophages/monocytes on LAK activity was also studied in transwell co-cultures. In the absence of 2-mercaptoethanol, the induction of LAK activity was very low in macrophage/monocyte-depleted splenocytes with macrophages/monocytes in the upper compartment of a transwell culture. In contrast, in the presence of 2-mercaptoethanol a high level of LAK activity was induced in these transwell cultures, showing that 2-mercaptoethanol abolished the LAK-inhibiting capacity of macrophages/monocytes. In addition, established LAK activity was strongly inhibited when, after LAK induction, splenocytes were cultured with supernatant of unfractionated splenocytes, which were cultured with IL-2 but in the absence of 2-mercaptoethanol. Addition of 2-mercaptoethanol abrogated the inhibiting effect of the supernatant completely. These experiments demonstrate that rat macrophages/monocytes produce 2-mercaptoethanol-sensitive soluble LAK-inhibiting factors. Ultrafiltration of conditioned culture medium of macrophages/monocytes revealed the presence of LAK-inhibiting factors larger than 10 kDa. We concluded that 2-mercaptoethanol-sensitive soluble factors produced by macrophages/monocytes determine the level of LAK induction in rat splenocyte cultures.

Genetic design of a gene for human metallothionein II and its expression as an active fusion protein in Escherichia coli

A synthetic DNA coding for human metallothionein II (hMT-II) was designed for efficient expression in Escherichia coli and cloned into a vector, pUEX2. Upon induction by heating, the gene for hMT-II was expressed as a fusion protein between β-galactosidase and hMT-II. The fusion protein accounted for approximately 24% of the total protein between β-galactosidase and hMT-II. The fusion protein accounted for approximatedly protein with a molecular mass of approximately 122 kDa was purified in homogeneity by washing the inclusion bodies with a solution of Triton X-100. The insoluble protein was easily solubilized in the presence of 2-mercaptoethanol and sodium dodecyl sulfate without any need for denaturants, such as urea or guanidine hydrochloride. The fused hMT-II protein bound to radioactive cadmium. A competition experiment with radioactive cadmium and various heavy metals showed that the hMT-II fusion protein had a higher affinity for Cu 2+, Co 2+, and Ag + ions than Cd 2+ and Zn 2+ ions. Thus, we succeeded in over expression of hMT-II as an active form in E. coli.

Solubilization of a novel isoflavone glycoside-hydrolyzing β-glucosidase from Lactobacillus casei subsp. rhamnosus

A novel isoflavone glycoside-hydrolyzing β-glucosidase produced by Lactobacillus casei subsp. rhamnosus IFO 3425 was solubilized by ultrasonic disruption of the cells in the presence of 2-mercaptoethanol and sorbitol as stabilizer. The β-glucosidase from L. casei subsp. rhamnosus specifically hydrolyzed soybean isoflavone glycosides, namely, daidzin and genistin, converting them to daidzein and genistein, respectively. By contrast, a commercial preparation of almond emulsin β-glucosidase could not hydrolyze these soybean isoflavone glycosides. The undesirably bitter and astringent isoflavone glycosides in soybean were decomposed for the first time with this novel β-glucosidase, an enzyme which has hitherto been considered difficult to solubilize, produced by a lactic acid bacterium.

Purification and properties of the enzyme arylamine N-acetyltransferase from the housefly Musca domestica

The enzyme arylamine N-acetyltransferase (ANAT) from the housefly (Musca domestica) has been purified. The M(r) of the purified enzyme was 27,600 +/- 1700 as estimated by gel filtration. SDS/PAGE yielded a value of 26,000 +/- 300, clearly indicating a monomeric structure. The purified enzyme had apparent Km values for acetyl-CoA and tyramine of 8.4 microM and 8.8 microM respectively, a pH optimum of 7.2 in 10 mM potassium phosphate buffer and an apparent pI of 5.8. ANAT activity showed a strong dependency on the presence of 2-mercaptoethanol during the purification stages. The enzyme could be completely inactivated by treatment with p-chloromercuribenzoate although the enzyme activity was protected by preincubation with acetyl-CoA. One or more cysteine residues are clearly required for catalytic activity, as demonstrated for the mammalian enzyme. In contrast, partial sequencing of the enzyme has yielded a number of peptide sequences, including the N-terminal sequence, which show no similarity with those reported for the mammalian and avian enzymes.

The molecular structure of corticotropin-induced secreted protein, a novel member of the thrombospondin family.

CISP (corticotropin-induced secreted protein) is a secreted protein recently purified in our laboratory from the conditioned medium of ACTH-treated bovine adrenocortical cells. Partial amino acid sequencing of CISP revealed homology with thrombospondins (TSPs), a family of adhesive proteins and in particular with TSP2. We report here the characterization of the molecular structure of CISP. Analysis of CISP by polyacrylamide gel electrophoresis in the absence or presence of SDS indicated an apparent molecular mass approximately equal to 600 kDa for the unreduced protein and an apparent molecular mass of 195 kDa after reduction by 2-mercaptoethanol. The sedimentation coefficient of CISP determined by ultracentrifugation on sucrose gradients was shifted from 9.7 S in the absence to 5.7 S in the presence of 2-mercaptoethanol. These data are consistent with a trimeric organization of the CISP molecule in which 195-kDa monomers would be linked together by disulfide bonds. The trimeric structure of CISP could be observed by rotary shadowing/electron microscopy, where CISP appeared to be composed of three equally electron-dense nodules and of a fourth nodule formed by the close association of three smaller fragments. The overall size of the molecule was 60 nm. We also observed that CISP is sulfated and glycosylated. Using glycosylation inhibitors, we could determine that CISP is synthesized as a 175-kDa core protein, is then matured into a 190-kDa high-mannose form and secreted as a 195-kDa mature protein. Inhibition of sulfation by chlorate did not prevent CISP secretion, whereas inhibition of glycosylation by tunicamycin blocked it. Taken together, these data indicate that the TSP2-related CISP molecule presents both structural and functional properties very similar to those of TSP1. CISP differs greatly, however, from TSP1 by the inducibility of its synthesis by cAMP.

Purification and properties of cytosol 5'-nucleotidase from black rockfish (Sebastes inermis) muscle

A cytosol 5'-nucleotidase from black rockfish white muscle was purified to homogeneity using two steps of affinity chromatography on concanavalin A and 5'-AMP Sepharose columns. The isolated enzyme with an apparent molecular weight of 242 000 in the absence of 2-mercaptoethanol had a single band on SDS-PAGE stained with silver. In the presence of 2-mercaptoethanol two bands were observed with estimated molecular weights of 148 000 and 94 000, suggesting a heterodimeric form, being different from the membrane-bound isoform which has a homotetrameric form. This was confirmed by gel filtration which yielded a single peak with enzyme activity at 250 000

Determination of proline by reversed-phase high-performance liquid chromatography with automated pre-column o-phthaldialdehyde derivatization

A simple and sensitive fluorometric HPLC method was developed for the analysis of proline in biological samples. The HPLC apparatus consisted of an autosampler, a binary solvent delivery system, a 3-μm reversed-phase C 18 column (150 × 4.6 mm I.D.) guarded by a 40-μm reversed-phase C 18 column (50 × 4.6 mm I.D.), a fluorescence detector, and a computer workstation. Proline was oxidized to 4-amino-1-butanol in the presence of chloramine-T and NaBH 4 in a 60°C water bath, which took 11 min. 4-Amino-1-butanol was automatically derivatized with o-phthaldialdehyde in the presence of 2-mercaptoethanol. The proline derivative was separated on a 25-min gradient program, employing solvent A (0.1 M sodium acetate-0.5% tetrahydrofuran-9% methanol; pH 7.2) and solvent B (methanol). Fluorescence was monitored with excitation at 340 nm and emission at 450 nm. The conversion of proline to 4-amino-1-butanol was quantitative, reproducible, and linear with proline concentrations ranging from 25 to 500 μ M commonly present in biological samples. The described method can be readily used for quantifying proline in biomedical research.

Isolation and in vitro translation of messenger RNA from the American cockroach.

Total RNA was extracted from the whole body of American cockroach (Periplaneta americana) using chaotropic salt guanidine isothiocyanate in the presence of 2-mercaptoethanol (2-ME). Polyadenylated mRNA was isolated by oligothymidylic acid-cellulose chromatography and mRNA was translated using a rabbit reticulocyte lysate system. The translation, as judged by the incorporation of 35S-methionine, was obtained with poly(A)+ RNA, where an approximately 9.5-fold increase in label incorporation over control was achieved. Analysis of translation products by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) in combination with autoradiography showed that many proteins with apparent molecular weights ranging from 12 to 200 kD were synthesized, and no labelled proteins were found with negative RNA control and poly(A)- RNA. Immunoprecipitation studies performed using polyclonal and monoclonal antibodies revealed that synthesized proteins of MW 90, 78, 72, 49, 45, and 26 kD corresponded with previously identified principal and major allergens of American cockroach from our laboratory. In addition, the allergenicity of the translation mixtures was also confirmed by fluoroallergosorbent test (FAST) inhibition studies with IgE antibodies of human reaginic serum pool.

Separation and Characterization of Homo and Hetero-oligomers of the Intermediate Filament Proteins Desmin and Vimentin

Affinity chromatography on single-stranded (ss)DNA-cellulose in conjunction with gel permeation chromatography in the presence of urea was employed to separate the intermediate filament (IF) protein complement of catalytically oxidized BHK-21 cell Triton cytoskeletons into disulfide-cross-linked homo- and heterodimers of desmin and vimentin and uncross-linked homodimers. The same separation was performed on a desmin-vimentin mixture under autoxidizing conditioning in 6 M-urea to obtain the respective cross-linked collision complexes of both proteins. In 5 M-urea, the oxidation products were identifified as dimers that were physically indistinguishable from uncross-linked homodimers, suggesting that they were in the form of partially denatured face-to face pairs. Heterodimers derived from intact IFs were identical to those derived from collision complexes. In the presence of 2-mercaptoethanol, heterodimers were unstable and transformed spontaneously into homodimers. After removal of urea, all cross-linked dimers were totally unable to polymerize into filaments; however, in the presence of 2-mercaptoethanol they showed a normal assembly competence. This inability of oxidized homo- and heterodimers to polymerize, together with the relatively low yield of cross-linked dimers obtained from cytoskeletons, is probably due to the introduction of steric strain into the dimers by disulfide bond formation. Substantial amounts of cross-linked heterodimers could also be isolated from IFs reconstituted from mixtures of desmin and vimentin in their homodimeric or tetrameric forms. Taken together, these results suggest that the cross-linked dimers isolated from cytoskeletons arise from a reaction between subfilament strands of IFs rather than from disulfide bond formation within pre-existing dimers and that the heterotypic IFs of BHK-21 cells are largely formed from homodimers and tetramers, respectively, rather than from heterodimers. The differential capacity of desmin and vimentin to interact with ssDNA has also been exploited to distinguish between homotypic and heterotypic protofilaments, the latter consisting of one homodimer of each protein species. This distinction could be made on the basis of characteristic differences in the sedimentation behavior of the respective protein-DNA complexes.

Purification, characterisation, and carbohydrate specificity of the lectin of Ficus cunia

A lectin, isolated from the seeds of Ficus cunia and purified by affinity chromatography on fetuin-Sepharose, was homogeneous in PAGE, GPC, HPLC, and immunodiffusion, and had mol wt of 3200–3500. In SDS-PAGE and HPLC in the absence and presence of 2-mercaptoethanol, the lectin gave a single band or peak corresponding to M r 3300–3500, thus indicating it to be a monomer. The lectin agglutinated human erythrocytes regardless of blood group, bound to Ehrlich ascites cells and to human rat spermatozoa, and was thermally stable; its activity was enhanced by Ca 2+. The lectin is a metalloprotein that was inactivated by dialysis with EDTA followed by acetic acid, but reactivated by the addition of Ca 2+. The lectin contained 2.0% of carbohydrates, large proportions of acidic amino acids, but little methionine. In hapten-inhibition assays, chitin oligosaccharides [(1 → 4)-linked β-GlcNAc] and N-acetyl-lactosamine were inhibitors of which N,N′,N″,N″′-tetra-acetylchitotetraose was the most potent. Among the macromolecules tested that contain either multiple N-acetyl-lactosamine and/or (1 → 4)/(1 → 6)-linked β-GlcNAc, asialofetuin glycopeptide was the most potent inhibitor. Thus, an N-acetyl group and substitution at C-1 of d-GlcN are necessary for binding.

Nuclear matrix of the lower eukaryote Physarum polycephalum and the mammalian epithelial LLC‐PK1 cell line

Agarose-encapsulated nuclear matrix preparations of the lower eukaryote Physarum polycephalum and the mammalian renal epithelial LLC-PK1 cell line were analyzed after various experimental protocols with respect to the protein composition. The effect of the mode of deproteinization (2 M NaCl, 0.25 M ammonium sulfate or 25 mM lithium diiodosalicylate), presence of 2-mercaptoethanol, Ca2+, Cu2+, chelating agents, the sequence of protein extraction and nuclease digestion, the use of RNase, the temperature at which the experimental manipulations were performed and the use of hypotonic or isotonic conditions was investigated. No significant differences in the final nuclear matrix composition could be observed, regardless of the experimental procedure applied. In Physarum, the major nuclear matrix proteins range over 12-70 kDa with prominent bands at 24, 31, 37 and 45 kDa; the proteins of the matrix in LLC-PK1 cells extend predominantly over 40-80 kDa. Furthermore, no essential differences in the protein composition could be observed when type I and type II nuclear matrices from the highly differentiated LLC-PK1 cell line were compared. The same was found for analogous matrix preparations of Physarum. Therefore, in both systems a distinction between type I/II matrix is questionable. Immunoblotting of the matrix preparations with a variety of antibodies against intermediate filament proteins and with antinuclear autoantibodies revealed the presence of intermediate filament proteins as components of the nuclear matrix. We conclude that the nuclear matrix represents a much more stable and reproducible structure than has been proposed so far, largely independent of changes in the preparation protocol.

Kinetic Fluorimetric Method for the Determination of Tobramycin by Stopped-Flow Mixing Methodology

Abstract The reaction of tobramycin with o-phthaldialdehyde (OPA) in the presence of 2-mercaptoethanol was studied kinetically by using stopped-flow mixing methodology. New data on the kinetics of this reaction were exploited to develop a simple, fast method for tobramycin determination. The linear range (0.05–30.0 μg mL−1), detection limit (0.03 μg mL−1), precision (&RSD 1.92) and selectivity of the proposed kinetic method are quite acceptable. The method was satisfactorily applied to the analysis of pharmaceutical preparations with no pretreatment.

Monoclonal antibodies against urease from Canavalia ensiformis

Monoclonal antibodies against purified urease (EC 3.5.1.5) from Canavalia ensiformis were raised by hybridoma technology using Sp2/0 myeloma cells as a fusion partner. All culture wells exhibited hybrid growth and 25% of these ( ie 45 culture wells) contained anti-urease activity. Two positive hybrid cells were cloned twice by the limiting dilution method and three hybridoma clones (B6F, C4F and B18) secreting monoclonal antibodies were selected ar random for purification and characterisation purposes. All three cell lines secreted monoclonal antibodies of IgM class which were purified by gel filtration chromatography on Sephacryl S-200 column with a final recovery of 85% and a purification factor of about 18. The purified preparations were apparently homogenous on native PAGE running with a M r of 920 000 Da. mAbs were highly specific for jack bean urease as determined by Western blotting. The affinity constants (K) for these mAbs ranged fro 10 8 to 10 9 1 mol −1. mAb B6F inhibited about 65% of urease activity whereas C4F and B18 stimulated the enzyme activity slightly by 20%. The presence of 2-mercaptoethanol in incubation mixtures protected urease from inactivation by B6F. Urease inactivation by B6F could be reversed by addition of 2-mercaptoethanol which reactivated most of the partially inactive enzyme. Gel filtration chromatography of purified urease exhibited two protein peaks with M r values of 290 000 and 90 000 Da which revealed antibody activity. This result suggests that the mAb B6F recognizes the trimeric as well as the monomeric forms of urease.

Purification, subunit structure and inhibitor profile of cathepsin A

Cathepsin A (EC 3.4.16.1), a lysosomal carboxypeptidase, has been purified 1374-fold from pig kidney. Purification steps included concanavalin A-Sepharose and phenyl-Sepharose chromatography and chromatofocusing. The specific activity (16.9 U/mg) of the purified enzyme was significantly higher than previously reported values. The enzyme preparation appeared homogeneous when analyzed by non-denaturing polyacrylamide gel electrophoresis was free of detectable protease contamination. The molecular mass ( M r = 97 000), isoelectric point (5.0), and sensitivity to inhibitors were consistent with reported properties of cathepsin A. However, the previously reported three-peptide chain structure was not observed. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis in the presence or absence of 2-mercaptoethanol demonstrated that the enzyme is composed of two M r 47 000 subunits, each of which dissociate in the presence of 2-mercaptoethanol into two polypeptide chains of 19 000 and 31 000.

Purification and characterization of a Fuc alpha 1–>2Gal beta 1–> and GalNAc beta 1–>-specific lectin in root tubers of Trichosanthes japonica.

A prominent lectin in the root tubers of Trichosanthes japonica was purified by affinity chromatography on a porcine stomach mucin-Sepharose column and termed TJA-II. The molecular mass of the native lectin was determined to be 64 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and TJA-II was separated into two different subunits of 33 and 29 kDa in the presence of 2-mercaptoethanol. The respective subunits contained mannose, N-acetylglucosamine, fucose, and xylose. It was determined by equilibrium dialysis to have two equal binding sites per molecule, the association constant toward tritium-labeled Fuc alpha 1-->2Gal beta 1-->3GlcNAc beta 1-->3Gal beta 1-->4GlcOT being K alpha = 3.05 x 10(5) M-1. The precise carbohydrate binding specificity of immobilized TJA-II was studied using various tritium-labeled oligosaccharides. A series of oligosaccharides possessing Fuc alpha 1-->2Gal beta 1--> or GalNAc beta 1--> groups at their nonreducing terminals showed stronger binding ability than ones with Gal beta 1-->GlcNAc (Glc) groups, indicating that TJA-II fundamentally recognizes a beta-galactosyl residue and the binding strength increases on substitution of the hydroxyl group at the C-2 position with a fucosyl or acetylamino group. This lectin column is useful for fractionating oligosaccharides or glycoproteins containing blood group type 1H, type 2H, and Sd antigenic determinants.

Inhibitory effect of sulfhydryl group on Ca 2+-ATPase activity in the plasma membrane-rich fraction from bovine parotid gland

1. 1. The present study demonstrated that the Ca 2+-ATPase activity of the plasma membrane-rich fraction from bovine parotid gland was decreased by the addition of reducing agents. 2. 2. Ca 2+-ATPase activity staining on SDS-PAGE gels was lost in the presence of 2-mercaptoethanol. 3. 3. Among all the reducing agents tested, GSH was the most effective in inhibiting Ca 2+-ATPase. 4. 4. The Ca 2+-ATPase activity decreased by the GSH was restored by the addition of an oxidizing reagent. However, oxidation with an oxidizing reagent subsequent to alkylation of the reduced enzyme with iodoacetamide resulted in no restoration of activity. 5. 5. The decrease of Ca 2+-ATPase activity by GSH is due to a decrease in the V ax of the enzyme. 6. 6. These results suggest that the disulfide bond in this enzyme protein is necessary to maintain the activity of this enzyme.

Purification of Two Superoxide Dismutase Isozymes and Their Subcellular Localization in Needles and Roots of Norway Spruce (Picea abies L.) Trees

Two isozymes of superoxide dismutase (SOD; EC 1.15.1.1) were purified from Norway spruce (Picea abies L.) needles to apparent electrophoretic homogeneity. Purification factors were 354 for SOD I and 265 for SOD II. The native molecular mass of both purified enzymes was approximately 33 kD, as determined by gel filtration. The subunit molecular weights, as estimated by sodium dodecyl sulfate polyacrylamide gel electrophoresis, were 20,000 for SOD I and 16,000 for SOD II in the presence of 2-mercaptoethanol, and 15,800 and 15,000, respectively, in its absence. These results indicate that the native enzymes were homodimers whose subunits contained intrachain disulfide bonds. Isoelectric points determined by nondenaturing isoelectric focusing were 4.5 and 5.5 for SOD I and II, respectively. NH(2)-terminal sequence analysis of the first 22 to 23 amino acids revealed 70 to 75% sequence identity with chloroplastic CuZn SODs from other plant species for SOD I, and 75% sequence identity with the cytosolic CuZn SOD from Scots pine for SOD II. SOD I was the major activity in needles and it was associated with chloroplasts. SOD II activity was dominant in roots.