Cibacron Blue Chromatography Research Articles

Purification and Characterization of the Lectin from Taro (Colocasia esculenta) and Its Effect on Mouse Splenocyte Proliferation In Vitro and In Vivo

Lectins are proteins found in a wide range of organisms, with the ability to bind reversibly to specific carbohydrates. They can display important biological activities, such as the activation of the cell cycle in lymphocytes. Storage proteins with lectin activity have been reported in tuberous plant species, such as Colocasia esculenta, popularly known as taro. A simple strategy based on Cibacron Blue chromatography was used to purify a 12 kDa polypeptide 1.3-fold, with a recovery of 30 %. The purified protein was identified as tarin by mass spectrometry, which indicated that it was present in G1a/G1d isoforms. Tarin exhibited both agglutinating activity against hamster erythrocytes and mitogenic activity in vitro and in vivo toward mouse splenocytes. Optimum cellular proliferation in vitro was achieved by 625 ng of the crude extract or 500 ng of the purified tarin. Total mouse splenocyte proliferation measured after 5 days of intraperitoneal inoculation of purified tarin was increased 3.3-fold in comparison to the control group. Half of the proliferating cells were identified as B lymphocytes by flow cytometry. These results show that this is an efficient and simple strategy to purify tarin and aid in establishing this protein as a new therapeutic drug, able to promote cell proliferation in a murine model.

Probing the Role of the Extreme N‐terminus in Rhodobacter sphaeroides ADPGlucose Pyrophosphorylase

The allosteric enzyme ADPGlucose Pyrophosphorylase (ADPG PPase) catalyzes the rate‐limiting step in glucan biosynthesis. The enzyme from Rhodobacter sphaeroides is perhaps the most highly regulated ADPG PPase and is activated by F6P, FBP, and pyruvate. Given the diverse sequences present in the ADPG PPase family at the N‐terminus and the lack of structural information about this region, it was of interest to probe the role of the extreme N‐terminus by truncation mutagenesis. Three truncation constructs have been generated (initiating at the third, fifth, and ninth codons) and the altered proteins have been successfully expressed and purified to homogeneity using anion exchange, hydrophobic interaction, and Cibacron Blue chromatography. Truncation 1 displayed a Vmax ~20% of wild‐type and S0.5 and A0.5 values for substrates and activators ~2–3 fold higher than wild type with ~2‐fold higher activation by activators. Truncation 3 exhibited only ~1% of wild type activity and also an extremely diminished response to activators. The N‐terminus is clearly important for optimal activity and regulation. Complete characterization of the altered proteins is in progress. Supported in part by NSF grant 0448676.

Preparation from bovine colostrum of a fraction rich in latent transforming growth factor-β2 and cleared of most allergenic proteins by one-step Cibacron Blue chromatography

A strategy was developed for the preparation of a latent transforming growth factor-β2 (latent TGF-β2) rich fraction from bovine colostrum. Different conventional chromatography techniques were tested to investigate some physicochemical properties of latent TGF-β2 complexes to optimize their recovery. Three latent TGF-β2 forms were detected by gel filtration (600, 150–250 and 65–150 kDa). Cation exchange chromatography showed that the p I of latent TGF-β2 was above pH 7; hydrophobic interaction chromatography showed that the hydrophobicity of latent TGF-β2 was much higher than that of other whey proteins. Affinity chromatography on Cibacron Blue 3G was used to separate latent TGF-β2 and immunoglobulin G from the most immunogenic proteins, α-lactalbumin, β-lactoglobulin and albumin. Optimum conditions were determined: preparation of a fraction 26 times richer in TGF-β2 than in serocolostrum was obtained with a good yield (up to 70%) of TGF-β2, 80% of which was in the latent form.

Proteins related to St. John’s Wort p27 SJ, a suppressor of HIV-1 expression, are ubiquitous in plants

Proteins belonging to the family of DING proteins are ubiquitous in animals and several of them are associated with various diseases. Their presence in a few plant species has previously been reported and the St John’s Wort DING protein was recently described as an inhibitor of HIV replication and transcription. However, data about DING protein occurrence in plants and their biochemical properties remain almost nonexistent. We describe methods for the purification of DING proteins from plants that may have general applicability since they are not dependent upon specific affinity ligands, contrary to previously described protocols. Cibacron Blue chromatography, sometimes preceded by an ion-exchange chromatographic step, is suitable for most plant extracts. DING proteins were purified from various species and cell types and their identity was confirmed immunologically and, in some cases, by N-terminal sequence analysis, indicating that they are ubiquitous in the plant kingdom. They are associated with the cell wall and sometimes secreted in the medium for in vitro grown cells. High-molecular-weight DING precursors were often observed. Internal peptides were also sequenced, as a prelude to gene cloning experiments.

Immunolocalization of the Ecto-ATPase and Ecto-apyrase in Chicken Gizzard and Stomach: PURIFICATION AND N-TERMINAL SEQUENCE OF THE STOMACH ECTO-APYRASE

We have examined the in vivo localization of extracellular ecto-ATPase and ecto-apyrase (ATPDase) in adult chicken gizzard and stomach by immunofluorescence and laser scanning confocal microscopy. In chicken gizzard, the ecto-ATPase was distributed in discrete clusters restricted to the sarcolemma of the smooth muscle cells. Anti-ecto-apyrase antibody detected a single 80-kDa band (putative apyrase) in Western blots of both chicken gizzard membrane extracts and partially purified anion exchange fractions, but the antibody did not detect ecto-apyrase in immunolabeled gizzard cryosections. In adult chicken stomach, the ecto-apyrase was observed at the apical membrane of the glandular oxyntico-peptic cells as described in previous immunoperoxidase studies (Stout, J. G., R. S. Strobel, and T. L. Kirley (1995) Biochem. Mol. Biol. Int. 36, 529-535). However, ecto-ATPase was clustered in the sarcolemma of the organized layer of circular smooth muscle and in smooth muscle cells of the septa surrounding the glandular tissue, but not in the glandular cells containing the ecto-apyrase. The findings indicate compartmentalization of the two related extracellular nucleotide hydrolyzing enzymes and suggest differential functions that are specialized for different regions of the chicken stomach. We also partially purified the ecto-apyrase of chicken stomach, an 80-kDa membrane glycoprotein. Chicken stomach membranes were solubilized in digitonin, glycoproteins were separated from solubilized proteins by lectin chromatography, and nucleotide-binding glycoproteins were selected by immobilized Cibacron blue chromatography. Further purification by size exclusion and anion exchange chromatography yielded purification of 94-fold. The ATPase specific activity of the purified stomach ecto-apyrase was 75,000 micromol of Pi/mg of protein/h, and the purified preparation consisted of a major band (55% of total protein) at 80 kDa. The purified enzyme could be deglycosylated with peptide N-glycosidase-F to a core molecular mass of 54 kDa. The N-terminal sequence of the 80-kDa stomach ecto-apyrase band (which reacted with anti-ecto-ATPDase antibodies) was determined to be: MEYKGKVVAGLLTATWV. Immunological cross-reactivity data indicate that the stomach 80-kDa protein isolated is an ecto-apyrase and is related to both the chicken liver and oviduct ecto-ATPDase enzymes characterized earlier, as well as to the human lymphoid cell activation antigen, CD39.

Purification and characterization of a plasma membrane ferricyanide-utilizing NADH dehydrogenase from Ehrlich tumour cells

A ferricyanide-utilizing NADH dehydrogenase (NADH-ferricyanide oxidoreductase) from the plasma membrane of Ehrlich ascites tumour cells has been purified about 1500-fold to apparent homogeneity. The method comprises the isolation of an enriched plasma membrane fraction, solubilization with Triton X-100, ion-exchange chromatography, ammonium sulphate precipitation, Cibacron Blue chromatography and fast-protein liquid chromatography with a Superose-6 gel filtration column. The specific activity of the final pool was more than 61 units/mg protein. The pure enzyme examined by SDS/PAGE displayed only one type of subunit with an apparent molecular mass of 32.0 kDa. The molecular mass of the native protein (117.0 kDa) was estimated by gel filtration; these results suggest a protein composed of four subunits of identical molecular mass. The enzyme was stable in the pH interval between 6 and 9, with maximum activity at pH values from 7.5 to 8.5. The purified enzyme showed Michaelis-Menten kinetics for the substrates, with apparent K(m) values of 4.3 X 10(-5) M and 6.7 X 10(-5) M for NADH and ferricyanide respectively. The isolated protein was strongly inhibited by Zn2+ and the thio-specific reagents mersalyl and p-chloromercuribenzenesulphonic acid.

Preparation of fatty-acylated derivatives of acyl carrier protein using Vibrio harveyi acyl-ACP synthetase

A simple two-step purification of Vibrio harveyi fatty acyl—acyl carrier protein (acyl-ACP) synthetase, which is useful for the quantitative preparation and analysis of fatty-acylated derivatives of ACP, is described. Acyl-ACP synthetase can be partially purified from extracts of this bioluminescent bacterium by Cibacron blue chromatography and Sephacryl S-300 gel filtration and is stable for months at −20δC in the presence of glycerol. Incubation of ACP from Escherichia coli with ATP and radiolabeled fatty acids (6 to 16 carbons in length) in the presence of the enzyme resulted in quantitative conversion to biologically active acylated derivatives. The enzyme reaction can be monitored by a filter disk assay to quantitate levels of ACP or by sodium dodecyl sulfate—polyacrylamide gel electrophoresis and fluorography to detect ACP in cell extracts. With its broad fatty acid chain length specificity and optimal activity in mild nondenaturing buffers, the soluble V. harveyi acyl-ACP synthetase provides an attractive alternative to current chemical and enzymatic methods of acyl-ACP preparation and analysis.

Purification and properties of xylitol dehydrogenase from the xylose-fermenting yeastCandida shehatae

Xylitol dehydrogenase (EC1.1.1.9) from xylose-grown cells ofCandida shehatae was purified 215-fold by sequential chromatography on NAD-C8 affinity, Superose-12, and Cibacron blue columns, and a single band was observed by SDS gel electrophoresis. The purified enzyme had a native molecular weight of 82 kDa and a denatured molecular weight of 40 kDa following SDS gel electrophoresis, indicating that it was composed of two subunits. Alcohol dehydrogenase copurified on the NAD-C8 but was substantially removed by Superose-12 and was not detected following Cibacron blue chromatography. The kinetic properties of the C.shehatae xylitol dehydrogenase differed considerably from those described previously for thePachysolen tannophilus enzyme. The Km of the C.shehatae enzyme for xylitol was 3.8 times smaller, whereas the Km for xylulose was 1.7-fold bigger. These factors could account for the lower xylitol production by C.shehatae.

The purification and properties of two soluble reduced nicotinamide: acceptor oxidoreductases from Trichomonas vaginalis

The occurrence of soluble reduced nicotinamide nucleotide:acceptor oxidoreductases has been reported in a number of strains of the oxygen-tolerant anaerobe Trichomonas vaginalis and other trichomonad species. The quantitatively more important enzyme in most strains of T. vaginalis is an NADH oxidase which produces water from the reduction of oxygen. This enzyme has been purified by a combination of gel filtration, chromatofocusing, Cibacron Blue chromatography and high pressure gel permeation chromatography. It is a monomeric protein with an estimated molecular mass from sodium dodecyl sulphate gel electrophoresis of 98 kDa; an isoelectric point of approximately pH 5.5 and a K m for NADH of 5.4 μM. The purified NADH oxidase is significantly inactivated during turnover under air ( t 1 2 3.65 min ) and rapidly inactivated by μM levels of hydrogen peroxide. The NADPH-dependent minor activity requires a flavin. It has been partially purified by gel filtration and chromatofocusing. The apparent molecular mass of this enzyme is 36 kDa by gel filtration; it has an isoelectric point of approximately pH 5.2 and K m values for NADPH and FMN of 16.6 μM and 6.1 μM respectively. The product of oxygen reduction by this enzyme, using FMN as acceptor is hydrogen peroxide. The possible role of these two enzymes in the cell and their affinity with related enzymes from other organisms is discussed.

Study of anti-DNA antibodies prepared by DNA cellulose or Cibacron blue chromatography

We prepared anti-DNA antibodies from sera of lupus patients by either DNA cellulose or by Cibacron blue chromatography. Eluates from both columns were studied with respect to recovery of IgG, recovery, purification and specificity of anti-DNA activity. An attempt was made to raise rabbit anti-idiotypic antibodies against both eluates. DNA cellulose chromatography — if DNA leakage was prevented — yielded 58% recovery and 58-fold purification of the anti-DNA activity present in the original purified IgG ample. 1% of loaded IgG was recovered. Cibacron blue chromatography yielded 32% recovery and 1.1-fold purification of the anti-DNA activity. 29% of loaded IgG was recovered. Eluates of Cibacron blue were not pure as shown by their high binding activity against an unrelated antigen, tetanus toxoid. Eluates from DNA cellulose were pure and did not show anti-tetanus toxoid activity. Rabbit anti-idiotypic antibodies could be raised only against eluates of DNA cellulose suggesting that the eluates of Cibacron blue did not contain enough idiotypes to induce rabbit anti-idiotypic antibodies. The characterization of the rabbit anti-idiotypic antibodies showed that it contained two populations, one against site-specific idiotypes and the other against framework idiotypes. Anti-DNA antibodies prepared by Cibacron blue had idiotypes similar to those prepared by DNA cellulose. The present study demonstrates that DNA cellulose chromatography — if leakage of DNA is prevented — can yield excellent recovery and purification of anti-DNA activity. Anti-DNA antibodies prepared by DNA cellulose were enriched and could induce anti-idiotypic antibodies in rabbits. Tube chromatography on Cibacron blue yielded poor recovery and minimal enrichment of anti-DNA activity.

Purification of DNA antibodies using cibarcon blue F3GA affinity chromatography

Isolation of DNA antibodies by conventional affinity chromatography has been difficult, often requiring harsh eluting conditions and giving low yields. The triazene dye cibacron blue F3GA is a nucleotide analogue which, when bound to an agarose,matrix, has been used to isolate numerous DNA-binding proteins. In this study, we have used cibacron blue chromatography to bind and purify another group of DNA-binding proteins, DNA antibodies. Greater than 90% of both ssDNA and dsDNA antibody activity from 4 SLE plasmas bound to the cibacron blue matrix. Between 30 and 65 of this antibody activity could be eluted from the column with 1.0 M NaCl, with a net 50–60-fold antibody purification from plasma. Studies with 2 monoclonal antibodies showed that a DNA antibody directed against the DNA phosphate backbone bound to cibacron blue, but a monoclonal antibody directed primarily against bases did not bind. Inhibition studies showed that DNA antibodies bound to the cibacron blue matrix at the antigen binding site, suggesting that cibacron blue does act as an antigen analogue. Although cibacron blue chromatography yields only partially purified DNA antibodies, this method should be useful in producing enriched DNA antibody preparations from ascites fluid, tissue culture supernatants, or serum.