Con A-Sepharose 4B Research Articles

Low molecular weight α-galactosidase from black gram (Vigna mungo): Purification and insights towards biochemical and biophysical properties

A hitherto unknown low molecular weight form of α-galactosidase (VM-αGal-P) from germinating black gram (Vigna mungo) seeds was purified (324 U/mg specific activity, 1157-fold purification, ~45 kDa) using ion-exchange (DEAE-cellulose, CM-sepharose), gel filtration (Sephadex G-75) and affinity (Con-A Sepharose 4B) chromatography but with poor yield (0.75%). Partially purified enzyme (VM-αGal) (146.3 U/mg specific activity, 522.5-fold purification) was used for further studies. VM-αGal showed optimal activity at pH 5 and 55 °C. Hg2+ and SDS completely inhibited VM-αGal activity. The Km, Vmax and catalytic efficiency (kcat/Km) of VM-αGal for pNPG and raffinose was 0.99, 17.23 mM, 1.66, 0.146 μmol ml−1 min−1, and 0.413, 0.0026 s−1 mM−1, respectively. VM-αGal was competitively inhibited by galactose (Ki 7.70 mM). Thermodynamic parameters [activation enthalpy (ΔH), activation entropy (ΔS) and free energy (ΔG)] of VM-αGal at 45-51 °C showed that VM-αGal was in a less energetic state and had susceptibility towards denaturation. Temperature-induced structural unfolding studies of VM-αGal probed by fluorescence, and far-UV CD spectroscopy revealed significant loss in tertiary structure and a steep decline in β-sheet content at 45–65 °C, and above 55 °C, respectively. VM-αGal improved the nutritional quality of soymilk by hydrolyzing raffinose family oligosaccharides (26.5% and 18.45% decrease in stachyose and raffinose, respectively).

Study on isolation and determination of glycoprotein from tea plant leaves

Tea is a healthy food, because it has various bioactive components. Recent research indicated that tea glycoprotein might be a bioactive component in tea. In this report, total proteins were extracted from tea fresh leaves, and coarse glycoproteins were isolated through the methods of ammonium sulfate fractions (45%--70%) and Sephadex G-100 chromatography. According to control staining of glycan and polypeptide, glycoproteins were determinated by using Pierce GelCode Glycoprotein Staining Kit and Coomassie Brilliant Blue R250, and three tea glycoproteins were fast purified from the SDS-gels. Sixteen native bioactive glycoproteins were gained from tea total proteins by ConA-Sepharose 4B affinity chromatography.

A glucose/mannose binding lectin from litchi (Litchi chinensis) seeds: Biochemical and biophysical characterizations

BackgroundLectins are highly important biomolecules to study several biological processes. A novel α-D-glucose/mannose specific lectin was isolated from the seeds of litchi fruits (Litchi chinensis) and its various biophysical and biochemical properties were studied. MethodsPurification was done by successive Sephadex G 100 and Con A-Sepharose 4B affinity chromatography. SDS-PAGE, Surface Plasmon Resonance (SPR), steady state absorbance, fluorescence, time-correlated single-photon counting, circular dichroism and antibiofilm activity by measuring total protein estimation and azocasein degradation assay have been performed. ResultsThe purified lectin is a homodimer of molecular mass ~ 54kDa. The amount of lectin required for hemagglutination of normal human O erythrocytes was 6.72µg/ml. Among the saccharides tested, Man-α-(1,6)-Man was found to be the most potent inhibitor (0.01mM) determined by hemagglutination inhibition assay. Steady state and time resolved fluorescence measurements revealed that litchi lectin formed ground state complex with maltose (Ka=4.9 (±0.2)×104M−1), which indicated static quenching (Stern-Volmer (SV) constant Ksv=4.6 (±0.2)×104M−1). CD measurements demonstrated that litchi lectin showed no overall conformational change during the binding process with maltose. The lectin showed antibiofilm activity against Pseudomonus aeruginosa. ConclusionsA novel homodimeric lectin has been purified from the seeds of litchi fruits (Litchi chinensis) having specificity for α-d-glucose/mannose. The thermodynamics and conformational aspects of its interaction with maltose have been studied in detail. The antibiofilm activity of this lectin towards Pseudomonus aeruginosa has been explored. General significanceThe newly identified litchi lectin is highly specific for α-d-glucose/mannose with an important antibiofilm activity towards Pseudomonus aeruginosa.

Use of glycosylated ferritin assay to aid the diagnosis of adult-onset Still’s disease: a local laboratory experience in Hong Kong

Adult-onset Still’s disease (AOSD) is a multi-systemic inXammatory disorder aVecting all ethnic groups. Even though there are several published diagnostic criteria, AOSD remains diYcult to diagnose clinically due to the overlapping features with infective, neoplastic and other rheumatological conditions [1, 2]. The problem is further complicated by the fact that treatments for AOSD, including steroid and immunosuppressive agents, can worsen the clinical course of the above conditions. Regarding laboratory markers, a markedly increased serum ferritin concentration is a common diagnostic marker among AOSD patients in active disease [3–5]. Generally speaking, serum ferritin concentration above 2,000 g/L is considered to be consistent with AOSD. Fautrel et al. [6] reported that a Wvefold increase in serum ferritin over upper limit of normal had 80% sensitivity and 41% speciWcity for AOSD. As marked elevation of serum ferritin concentration can also be seen in other disorders, the low speciWcity limits its diagnostic use. The percentage of glycosylated ferritin (GF), an isoform of ferritin, over total ferritin oVers additional diagnostic value. In healthy participants, 50–80% of ferritin is glycosylated. In inXammatory diseases, the glycosylated portion drops to 20–50%, while in AOSD, it is often ·20%. Using ·20% as the cut-oV, the sensitivity and speciWcity of percentage GF for AOSD were 80 and 66%, respectively. The combination of a percentage GF · 20% with ferritin over Wve times of normal produced a sensitivity of 43% and speciWcity of 93% [6]. We decided to develop an assay for the measurement of serum GF. Based on method described by Vignes et al. [7], GF could be easily separated from non-glycosylated ferritin by dividing the target serum sample into two aliquots, adding concanavalin A (Con-A) Sepharose 4B to one aliquot and only Sepharose 4B to the other aliquot and incubating them in a water bath for 2 h at room temperature. GF bound to Con-A, whereas non-glycosylated ferritin did not. The two aliquots were then centrifuged at 3,000 rpm for 15 min. The non-glycosylated ferritin unbound to Con-A was recovered in the supernatant and measured by immunometric assay. Accordingly, the non-glycosylated ferritin and total ferritin concentrations could be determined in the aliquots with Con-A Sepharose 4B and Sepharose 4B, respectively. The percentage GF was calculated from the diVerence between total ferritin and non-glycosylated ferritin divided by total ferritin. We had retrospectively analysed archived serum samples taken on admission from 5 newly diagnosed AOSD patients referred to our laboratory, in whom diagnosis was made clinically according to the Yamaguchi’s criteria. These 5 patients had serum ferritin concentrations of 8,047–41,479 g/L and percentage GF of 1.9–16.2%. The combined results were compatible with the diagnosis of AOSD. We had also randomly selected 15 patient samples with ferritin level over 2,000 g/L. These were patients with chronic anaemia requiring regular transfusion and patients with fulminant sepsis. Their serum ferritin concentrations ranged from 2,092 to 13,773 g/L and percentage GF varies from 28.6 to 80.6%. J. S.-S. Kwok (&) · M. H.-M. Chan Department of Chemical Pathology, The Chinese University of Hong Kong, Hong Kong SAR, China e-mail: jeffreykwok@cuhk.edu.hk

Hydrophobicity as a driver of MHC class I antigen processing

The forces that drive conversion of nascent protein to major histocompatibility complex (MHC) class I-restricted peptides remain unknown. We explored the fundamental property of overt hydrophobicity as such a driver. Relocation of a membrane glycoprotein to the cytosol via signal sequence ablation resulted in rapid processing of nascent protein not because of the misfolded luminal domain but because of the unembedded transmembrane (TM) domain, which serves as a dose-dependent degradation motif. Dislocation of the TM domain during the natural process of endoplasmic reticulum-associated degradation (ERAD) similarly accelerated peptide production, but in the context of markedly prolonged processing that included nonnascent species. These insights into intracellular proteolytic pathways and their selective contributions to MHC class I-restricted peptide supply, may point to new approaches in rational vaccine design.

A Novel Monoclonal Antibody Against A60 Antigen ofMycobacterium bovisBacillus Calmette-Guerin

Mycobacterium contains several immunologically active substances, which play a principal role in mycobacterial diseases. The majority of the highly antigenic proteins present in mycobacterial homogenates are components of the A60 complex. In this study, A60 antigen was prepared from cytoplasm of Mycobacterium bovis Bacillus Calmette-Guerin (BCG). Cytoplasm was fractionated by passage through the column of sepharose 6B and ConA-sepharose 4B. After purification of spleen cells of the immunized mice, the cells were fused with SP2/0 myeloma cells. Four clone cell lines producing antibody against A60 antigens were established and each clone was tested for immunoreactivity against purified A60 by ELISA and immunoblotting. The clone designated DEB7 reacted strongly with A60. Immunoblotting using MAb DEB7 showed that this MAb binds to a single protein of A60 subunit with a molecular weight of 65 kDa. This subunit of A60 M. bovis recognized by DEB7 MAb could be used to increase the sensitivity and specificity of immunoassay or other potential roles in mycobacterium infection.

Purification and Characterization of an α-Mannosidase from the Tropical Fruit Babaco (Vasconcellea × Heilbornii Cv. Babaco)

An alpha-mannosidase (EC 3.2.1.24) present in the lyophilized latex of babaco ( Vasconcellea heilbornii ) has been purified to apparent homogeneity by native PAGE. The purification involves a three-step procedure with successive anion exchange with Q Sepharose HP, lectin affinity chromatography using ConA Sepharose 4B, and gel filtration using Superdex 200 prep grade. The molecular mass was determined to be in the range of 260-280 kDa by Superdex 200 prep grade gel filtration, and isoelectric focusing showed a pI range between 5.85 and 6.55, suggesting different glycosylated isoforms. The optimal temperature for the alpha-mannosidase was determined to lie between 50 and 60 degrees C, and the optimal pH was 4.5 at 50 degrees C. The K(m) value for p-nitrophenyl alpha-mannopyranoside (pNPM) was found to be 1.25 mM and the V(max), 2.4 microkat mg(-1) at 50 degrees C and 1.94 microkat mg(-1) at 40 degrees C. The pure alpha-mannosidase was specific for mannose and did not display activity for any other tested synthetic substrates.

TbVps34, the Trypanosome Orthologue of Vps34, Is Required for Golgi Complex Segregation

Phosphoinositides are important regulators of numerous cellular functions. The yeast class III phosphatidylinositol 3-kinase Vps34p, and its human orthologue hVPS34, are implicated in control of several key pathways, including endosome to lysosome transport, retrograde endosome to Golgi traffic, multivesicular body formation, and autophagy. We have identified the Vps34p orthologue in the African trypanosome, TbVps34. Knockdown of TbVps34 expression by RNA interference induces a severe growth defect, with a post-mitotic block to cytokinesis accompanied by a variety of morphological abnormalities. GFP2xFYVE, a chimeric protein that specifically binds phosphatidylinositol 3-phosphate, localizes to the trypanosome endosomal system and is delocalized under TbVps34 RNA interference (RNAi), confirming that TbVps34 is an authentic phosphatidylinositol 3-kinase. Expression of GFP2xFYVE enhances the TbVps34 RNAi-associated growth defect, suggesting a synthetic interaction via competition for phosphatidylinositol 3-phosphate-binding sites with endogenous FYVE domain proteins. Endocytosis of a fluid phase marker is unaffected by TbVps34 RNAi, but receptor-mediated endocytosis of transferrin and transport of concanavalin A to the lysosome are both impaired, confirming a role in membranous endocytic trafficking for TbVps34. TbVps34 knockdown inhibits export of variant surface glycoprotein, indicating a function in exocytic transport. Ultrastructural analysis revealed a highly extended Golgi apparatus following TbVps34 RNAi, whereas expression of the Golgi marker red fluorescent protein-GRASP (Grp1 (general receptor for phosphoinositides-1)-associated scaffold protein) demonstrated that trypanosomes are able to duplicate the Golgi complex but failed to complete segregation during mitosis, despite faithful replication and segregation of basal bodies and the kinetoplast. These observations implicate TbVps34 as having a role in coordinating segregation of the Golgi complex at cell division.

Purification and identification of rice soluble glycoproteins

The total proteins was extracted from rice leaves, and quantified by Coomassie Brilliant Blue G-250. We got four classifications from rice total proteins by using Ammonium Sulfate Precipitation, which was very important because we got easy way to separate and classify the total proteins. Also, it was useful for the purification of glycop roteins. The four classifications then purified by using ConA-Sepharose 4B Affinity Chromatography, the liquid of glycoproteins was collected by tubes in the same time of the appearances of absorption peaks. We even got some dry samples by using vacuum freezing and drying technology. Next, we done SDS-PAGE and determination respectively by Periodic AcidSchiff (PAS) staining and Coomassie Brilliant Blue (CBB) R-250 staining. In the gel of CBB staining, there are near 30 strips of different glycoproteins (some are glycopeptides) in the last three classifications; but the gel of PAS staining only showed 7 strips of different glycoproteins (some are glycopeptides) only because of the sensitivities of PAS much lower than CBB staining. The last step, we choose three glycoproteins showed on the gel of 50%--80% glycoproteins classification, all of them have higher contents than others. We further purified each of the three glycoproteins directly from the SDS-PAGE gels. They are proved purified glycoproteins or glycopeptides by CBB and PAS staining again. The three glycoproteins are named respectively as RG1, RG2 and RG3.

Purification and partial characterization of two acid phosphatase forms from pearl oyster ( Pinctada fucata)

The present study describes the details about the acid phosphatase forms in the pearl oyster, Pinctada fucata. Two isoenzymes (AcPase I and II) of acid phosphatase were separated and purified from viscera of pearl oyster, P. fucata to homogeneity by chromatography on DEAE-Sepharose Fast Flow, Sephadex G-200 superfine and ConA Sepharose 4B, and partial biochemical properties of AcPase I and II were studied. AcPase I and AcPase II had molecular weights of 208.8 and 64.3 kDa, respectively. AcPase I was a single polypeptide chain, while AcPase II was a dimeric enzyme composed of two equivalent subunits. AcPase I and II showed optimal pHs at 4.6 and 3.2 with p-nitrophenylphosphate as substrate. The optimal catalytic reaction temperature was 47 °C for AcPase I and 57 °C for AcPase II. Both enzyme forms were stable when incubated at 50 °C for 40 min. Tartrate and fluoride were the most effective inhibitors of the enzymes. Fe 3+, Zn 2+, Cu 2+ and Pb 2+ inhibited the activity of AcPase I and II to differing extents. AcPase I and II were apparently nonspecific and hydrolyzed various phosphoric esters. The different properties of AcPase I and II suggested that the two enzymes may play different roles in the pearl oyster.

Purification and characterization of extracellular invertase from the hypocotyls of mung bean (Phaseolus radiatus L.)

The extracellular invertase (β-D-fructofuranoside fructohydrolase, EC 3.2.1.26) was isolated and characterized from the hypocotyls of mung bean (Phaseolus radiatus L.). The enzyme was purified to apparent homogeneity by ammonium sulfate fractionation and sequential chromatography over diethylaminoethyl (DEAE)-cellulose anion exchange, Concanavalin (Con) A-Sepharose 4B affinity and Sephadex G-200. The overall purification was about 77-fold with a recovery of about 11%. The finally purified enzyme exhibited a specific activity of about 113 μmol of glucose produced mg-1 protein min-1 at pH 5.0 and appeared to be a single protein by nondenaturing polyacrylamide gel electrophoresis (PAGE) and sodium dodecyl sulfate (SDS)-PAGE. The enzyme had the native molecular mass of 134 kD and subunit molecular weight of 67 kD as estimated by Sephadex G-200 chromatography and SDS-PAGE, respectively, suggesting that the enzyme was composed of homodimeric proteins. On the other hand, the enzyme appeared to be a glycoprotein containing mannosyl residues on the basis of its ability to interact specifically with the immobilized Con A and the separability of invertase-Con A complex by methyl-α-D-mannopyranoside. The enzyme had a Km for sucrose of 3.4 mM and its pH optimum of 4.0. The enzyme showed highest enzyme activity with sucrose as substrate. Raffinose and cellobiose were hydrolyzed at a low rate, maltose and lactose were not cleaved by the enzyme. These results indicate the extracellular invertase is a β-fructofuranosidase.

Evidence for glycosylation of the juvenile-hormone-binding protein from Galleria mellonella hemolymph.

The juvenile-hormone-binding protein (JHBP) from Galleria mellonella hemolymph, which is a member of the high-affinity/low-molecular-mass group of JHBP proteins, was found to be glycosylated. Glycosylation was confirmed by the following evidence. Carbohydrate gas-liquid chromatography analysis of the purified JHBP preparations showed the presence of a low amount of sugars (Man and GlcNAc were the major components). The JHBP electrophoretic band blotted onto nitrocellulose was stained with GlycoTrack (a reagent kit used for the detection of protein glycosylation) and showed strong binding of concanavalin A (ConA). JHBP was fractionated on a ConA-Sepharose 4B column into ConA-bound (strongly stained with ConA) and ConA-unbound (hardly stained with ConA) portions. Both fractions showed juvenile-hormone-binding activity and were glycosylated, as revealed by staining both of them with GlycoTrack. Electrospray-ionization mass spectrometry of JHBP suggested the presence of a small amount of presumably nonglycosylated protein (24988 Da) and five glycoforms, two of which (containing Man2GlcNAc, or Man2Fuc1GlcNAc2 chain) were not bound or were weakly bound to ConA, and three (with Man3GlcNAc2, Man5Fuc1GlcNAc2, or Man5GlcNAc2, chain) were present in the fraction strongly bound to ConA. In conclusion, the monosugar composition, GlycoTrack staining, ConA-binding properties and molecular mass analyses of JHBP supplied convincing evidence for its glycosylation and some information on the character of the oligosaccharide chains.

N-Acetyl-β- d-glucosaminidase from ‘golden delicious’ apples

N-Acetyl-β- d-glucosaminidase (EC 3.2.1.30) was purified to homogeneity and characterized from climacteric ‘Golden Delicious’ apple fruit cortical tissue. Ammonium sulphate fractionation (30–70%), Mono-Q anion-exchange and Con A-Sepharose 4B chromatography resulted in a 4.7% yield and 763-fold purification. The M r of the native enzyme was ca 236 000 using Superose 12 gel filtration, suggesting it was composed of eight similar subunits since only a single M r 29 000 silver-stained band was observed upon SDS-PAGE. The enzyme had a K m and V max for p-nitrophenyl- N-acetyl-β- d-glucosaminide of 0.23 mM and 8.48 nmol min −1, respectively, and for p-nitrophenyl- N-acetyl-β- d-galactosaminide of 1.49 mM and 10.58 nmol min −1, respectively. Estimates of V max/ K m for both substrates indicated that p-nitrophenyl- N-acetyl-β- d-glucosaminide was the most efficient and preferred substrate. The kinetics of mixed-substrate reactions and N-acetyl-β- d-glucosamine inhibition indicated the enzyme has only one active site which binds both substrates.

Purification and Characterization of Cathepsin E Type Acid Proteinase from Gastric Mucosa of Bullfrog, Rana catesbeiana

An acid proteinase different from pepsin was purified from bullfrog (Rana catesbeiana) gastric mucosa by chromatography on hydroxyapatite, Q-Sepharose, Con A-Sepharose 4B, and Mono Q columns. Its molecular weight after purification was estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis to be 45 kDa under reducing conditions and about 90 kDa under nonreducing conditions. Thus, it is a dimer of two identical subunits. On acid treatment, the molecular weight of the subunit decreased from 45 to 42 kDa, showing a similar change to that of pepsinogen in its activation under acidic conditions. Therefore, the enzyme was thought to have both a proform and a mature form. It preferred hemoglobin to other protein substrates examined and showed broad optimal activity in the range of pH 2.0 to 3.5 towards hemoglobin. Its proteolytic activity, like that of porcine pepsin, was strongly inhibited by pepstatin. Its amino acid composition was similar to those of other aspartic proteinases. From these results, the enzyme was identified as a cathepsin E type acid proteinase of bullfrog, and cathepsin E type enzyme was purified from anuran for the first time.

Effect of Phaseolus vulgaris lectins on glucose absorption, transport, and metabolism in rat everted intestinal sacs

Many legume seeds contain lectins. Some of these lectins are toxic when fed to humans or laboratory animals. Although the mechanism for such toxicity has not been clearly elucidated, lectin binding to the intestinal mucosa is an obligatory step. Therefore, the interaction of lectin and enterocytes may interfere with the digestive process. Lectins from toxic (red kidney bean) and non-toxic (mountaineer half runner) Phaseolus vulgaris varieties were tested for their effect on the intestinal absorption, transport, and metabolism of glucose. Both lectins were purified by affinity chromatography on Con A-Sepharose 4B. Everted sacs, 5 cm long, from the first third of the small intestine were incubated for 20 min in the presence of 10 mmol/L glucose in both the luminal and serosal media. Pre-incubation (15 min) of the everted sacs with increasing concentrations of the red kidney bean lectin (50–200 μg/mL mucosal solution) before glucose addition reduced both the absorption and transport of the sugar from the luminal to the serosal side. Glucose metabolism as judged by lactate formation was not affected. At a concentration of 100 μg/mL of red kidney bean lectin, absorption, and transport were inhibited by 40% and 72%, respectively. Lactate production was decreased by only 5%. Under the same conditions (100 μg/mL) mountaineer half runner lectin inhibited glucose absorption and transport by less than 10%. Interference of lectins with the absorption and transport of nutrients could partially explain the toxic effect of lectins from some Phaseolus vulgaris varieties.

PURIFICATION AND CHARACTERIZATION OF N-ACETYL-β-d-GLUCOSAMINIDASE FROM `GOLDEN DELICIOUS' APPLES.

The ripening-related biological activity (Priem and Gross, 1992, Plant Physiol 98:399-401) of free N-glycans in fruit was recently reported. Thus, enzymes involved in N-glycan metabolism may be important in understanding the regulation of ripening. N-acetyl-β-D-glucosaminidase (EC 3.2.1.30) was purified to homogeneity and characterized from climacteric `Golden Delicious' apple (Malus domestica Borkh.) fruit tissue. This enzyme cleaves terminal N-acetylglucosaminyl residues from the non-reducing end of glycans. The enzyme was extracted in 25 mM Na-acetate (pH 5.0) containing 2% PVP, 0.1 mM EDTA, and 0.5 mM DTT. Activity was 0.02 μmol/min/gfw in the crude extract. Ammonium sulfate fractionation (30 to 70%), Mono-Q FPLC and Con A-Sepharose 4B resulted in a 4.7% yield and 923-fold purification. The enzyme had a Km for P-nitrophenyl-N-acetyl-β-D-glucosaminide of 136 μM. Optimal pH and substrate concentration were 6.0 and 0.5 mM, respectively. Activity was inhibited at substrate concentrations above 0.5 mM. The molecular weight of the enzyme was estimated to be 29 kD using SDS-PAGE.

INCREASE IN ACID INVERTASE ACTIVITY AND ACCUMULATION OF REDUCING SUGAR IN POTATOES STORED AT LOW TEMPERATURES AND PURIFICATION OF ACID INVERTASE

It is well known that storage of potato tubers at chilling temperatures results in an increase in the content of simple sugars, mainly sucrose, glucose, and fructose. We have observed that in Russet Burbank potatoes sucrose is the first sugar to accumulate, followed by a rise in glucose and fructose. The ratio of the increment of glucose to fructose is one, indicating that sucrose is the sole source for the reducing sugars. Our data show that there is a close temporal relationship between the accumulation of reducing sugars and rise in the activity of acid invertase (AI). Purification of combining Con A-Sepharose 4B affinity followed by DEAE-Sephacel ion exchange columns resulted in 144-fold purification of AI. SDS-PAGE indicating that the above procedure resulted in the concentration of 58 kDa polypeptide. IEF electrophoresis revealed the presence of five isoforms of AI with the following pI: 5.16, 5.25, 5.37, 5.55, 5.85. Two-D gel electrophoresis (native-to-denature) showed that a polypeptide of 49.48 kDa was associated with all isoforms of AI. There were also two addition polypeptides with 37.94 and 20.07 kDa which could be breakdown products of the 58 kDa protein or subunits of AI.

Leaching of concanavalin A during affinity chromatographic isolation of cell surface glycoproteins from human fetal neurons and glial cells

Preparation of surface glycoproteins from human fetal brain cells by affinity chromatography on Con A-Sepharose 4B was a problematic endeavor due to leaching of Con A from the matrix. Dissociation of Con A from the matrix took place irrespective of the presence of lipid and/or detergent and the buffer composition during chromatography and was apparently related to the nature of the protein under study. Pretreatment of Con A-Sepharose with 6 m guanidine or 8 m urea reduced Con A leaching. The Con A eluate also contained noncovalently associated glycolipid. Elution at 25°C rendered fractions containing a higher degree of Con A and glycolipid contamination compared to the negligible contamination by these two components when elution was carried out at 4°C. This phenomenon was attributed to the formation of heterogeneous mixed micelles of glycoprotein.

Immunological characterization of protective antigens prepared by alkaline treatment of whole cells and from the culture filtrate of Erysipelothrix rhusiopathiae

Culture filtrate and alkaline-extracted antigens from whole cells of an attenuated strain of Erysipelothrix rhusiopathiae (strain Koganei: serovar la) were fractionated with ammonium sulfate; both induced protective immunity in mice. Sephadex G-200 gel filtration revealed three protein fractions in the alkaline-extracted antigen and four protein fractions in the culture filtrate antigen. A fraction in the alkaline extract (NaOH P-2) and in the culture filtrate (CF P-2) induced protection in mice against challenge with a different serovar strain (strain Agata: serovar 5). Anti-NaOH P-2 and anti-CF P-2 mouse sera were protective against different serovars. Glycoprotein fraction derived from CF P-2 antigen by affinity chromatography with Con A-Sepharose 4B did not show protective activity. Western blotting between the antisera (anti-NaOH P-2, Anti-CF P-2 and anti-Koganei strain) and the antigens (NaOH P-2, and sonicated antigens of Agata, Fujisawa and Koganei strains) showed strong recognition of the same bands at 62, 42 and 41 kDa.

High molecular weight acid phosphatase from barley coleoptiles

The high M, acid phosphatase (EC 3.1.3.2) from barley ( Hordeum vulgare) coleoptiles was purified 57-fold by ammonium sulphate fractionation, DEAE-cellulose, Con A-Sepharose 4B and gel filtration chromatography. The molecular weight of the native enzyme was 104 000 and 106 000 measured by gel filtration on a Sephadex G-100 column and by density gradient ultracentrifugation, respectively. The enzyme is a heterodymer and consists of two subunits of 79 400 and 26 000 as determined by SDS-PAGE. The apparent K m value with p-nitrophenyl phosphate ( pNPP) as substrate was 0.10 mM. The enzyme activity was unaffected by phosphate acceptors such as glycerol, methanol, ethanol and glucose, but was strongly inhibited by Hg 2+, Cu 2+, molybdate ion and NaF. The acid phosphatase from barley coleoptiles hydrolysed a wide variety of phosphate esters and the highest activity was observed with ATP, inorganic pyrophosphate, pNPP and ADP.