Purification and characterization of Tora-bean (Phaseolus vulgaris) lectin

A lectin purified from the Tora-bean (Phaseolus vulgaris) by affinity chromatography with Con-A Sepharose was shown to be a glycoprotein containing 7.8% neutral sugars (D-mannose, N-acetyl-D-glucosamine, L-fucose, and D-xylose, in a molar ratio of 9.6 : 2.0 : 0.6 : 0.7). Its molecular weight was 130,000, as estimated by exclusion gel chromatography, and SDS gel electrophoresis showed that it consists of four subunits of molecular weight 32,000. The lectin reacts with various glycoproteins, i.e., blood group substances, human parotid salivary glycoprotein, fetuin, and bovine submaxillary mucin. Divalent cations, such as Ca2+, Mn2+, and Mg2+, appear to stimulate its reactivity. Inhibition tests using the glycopeptide fragment from fetuin and some oligosaccharides, as well as the binding test with 14C-N-acetyl-lactosamine suggest that the sequence of D-galactose, N-acetyl-D-glucosamine, and D-mannose residues in the carbohydrate chain of fetuin is essential for binding.     

Isolation and characterization of lectins from kidney beans (Phaseolus vulgaris)

The bioactive properties of lectins obtained from raw and canned red kidney bean (Phaseolus vulgaris) were studied to determine the changes in their bioactivity during the canning process. Phytohaemagglutinin (PHA) was extracted using Affi-gel Blue gel and thyroglobulin-Sepharose and had a molecular weight of 32 kDa. Both the raw and the canned kidney beans possessed the ability to agglutinate red blood cells and inhibit α-glucosidase. The activity found in the canned beans was similar to that from the in the raw kidney beans. However, the amount of lectin that could be extracted from thyroglobulin-Sepharose was much less in the canned samples than in the raw kidney bean samples. The extracted lectin from the raw kidney beans was also subjected to a heating and cooling treatment using a differential scanning calorimeter. The lectin had a nonset denaturation temperature of 77.76 °C and it did not renature upon cooling. In this study, we demonstrated that extracts from raw red kidney bean and canned red kidney bean contain bioactive compounds capable of inhibiting HIV-1 RT in vitro.     

Isolation and characterization of a specific enterokinase inhibitor from kidney bean (Phaseolus vulgaris)

A specific enterokinase inhibitor from kidney bean (Phaseolus vulgaris) was purified to homogeneity. It showed a single protein band on sodium dodecyl sulphate/polyacryl-amide-gel electrophoresis in the presence of mercaptoethanol, and the Mr was 31000. Aspartic acid was identified as the N-terminus of the inhibitor. The Mr by gel chromatography on Sephadex G-200 was found to be 60000, indicating the dimeric nature of the inhibitor. The inhibitor was found to be a glycoprotein. The monosaccharide moieties were glucose, mannose, glucuronic acid and glucosamine in the proportions 3.15%, 5.0%, 0.85% and 1.3% respectively. The inhibitor was most active on pig enterokinase, followed by bovine and human enterokinases. Maximal inhibitory activity was elicited by preincubation of the inhibitor with the enzyme for 15 min. Digestion with pepsin resulted in loss of inhibitory activity. The inhibitor was stable to exposure to a wide range of pH values (2-10), and exposure to pH above 10 resulted in loss of inhibitory activity. Modification of arginine residues by cyclohexane 1,2-dione and ninhydrin led to complete loss of enterokinase-inhibitory activity.     

Polysaccharide from Dry Navy Beans, Phaseolus vulgaris: Its Isolation and Stimulation of Clostridium perfringens

A microbiological assay is described for determining gas produced by Clostridium perfringens (Veilon and Zuber) Holland from materials believed to be flatulent. A rationale for its use is given and analyzed. The fractionation and the results of the assay for several components of lima beans, Phaseolus lunatus L., and navy beans, P. vulgaris L., are given. In particular, a polysaccharide was isolated from dried P. vulgaris seeds. Only about one-seventh as much could be isolated from green P. lunatus seeds as from P. vulgaris seeds. The polysaccharide stimulates voluminous gas production by C. perfringens and meets all criteria of the rationale for flatulent sus pects. It has not been tested on humans; however, data from the microbiological assay correlate well with human results for green and dry lima beans and navy beans.     

Isolation and characterization of two mouse L cell lines resistant to the toxic lectin ricin.

Two variant mouse L cell lines (termed CL 3 and CL 6) have been selected for resistant to ricin, a galactose-binding lectin with potent cytotoxic activity. The resistant lines exhibit a 50 to 70% decrease in ricin binding and a 300- to 500-fold increase in resistance to the toxic effects of ricin. Crude membrane preparations of CL 3 cells have increased sialic acid content (200% of control), while the galactose, mannose, and hexosamine content is within normal limits. Both the glycoproteins and glycolipids of CL 3 cells have increased sialic acid, with the GM3:lactosylceramide ratios for parent L and CL 3 cells being 0.29 and 1.5, respectively. In contrast, the membranes of CL 6 cells have a decrease in sialic acid, galactose, and hexosamine content with mannose being normal. Both cell lines have specific alterations in glycosyltransferase activities which can account for the observed membrane sugar changes. CL 3 cells have increased CMP-sialic acid:glycoprotein sialyltransferase and GM3 synthetase activities, while CL 6 cells have decrease UDP-GlcNAc:glycoproteinN-acetylglucosaminyltransferase and DPU-galactose:glycoprotein galactosyltransferase activities. The increased sialic acid content of CL 3 cells serves to mask ricin binding sites, since neuraminidase treatment of this cell line restores ricin binding to essentially normal levels. However, the fact that neuraminidase-treated CL 3 cells are still 45-fold resistant to ricin indicates that either a special class of productive ricin binding sites is not being exposed or that the cell line has a second mechanism for ricin resistance.     

Purification and characterization of a lectin from Phaseolus acutifolius var. latifolius

The lectin from Phaseolus acutifolius var. latifolius has been purified to homogeneity by affinity chromatography using stroma-sephadex, gel filtration and electrofocusing. The purified lectin consists of four subunits of 21 Kd molecular mass each one with 7.4% w/w total carbohydrate. Red cells of human and animal species, are agglutinated but are not inhibited by a series of sugars. Mitogenicity and immunosuppressive activities were demonstrated. Agglutination requires magnesium and calcium ions.     

Isolation and characterization of the subunits of Phaseolus vulgaris alpha-amylase inhibitor.

An alpha-amylase inhibitor (PHA-I) of the white kidney bean (Phaseolus vulgaris) was found to be composed of two kinds of subunits and they were isolated on a size-exclusion column by HPLC under denaturing conditions. The alpha-subunit was free from tryptophan and cysteine and the beta-subunit contained no methionine or cysteine. There was no marked resemblance in tryptic peptide map between these subunit polypeptides. The alpha-subunit contained 28% by weight of carbohydrate, mainly made up of high mannose-type oligosacharides, whereas the sugar moiety of the beta-subunit amounted to 7% by weight and seemed to be predominantly composed of xylomannose-type oligosaccharides. By SDS-PAGE following deglycosylation, the molecular weights of the polypeptides of alpha- and beta-subunits were shown to be 7,800 and 14,000, respectively. These values were consistent with molecular sizes obtained for alpha- and beta-subunits by gel permeation HPLC in 6 M guanidine hydrochloride. The molecular weight of the native PHA-I, 28,800, obtained by gel permeation HPLC under non-denaturing conditions, suggested a heterodimeric structure for PHA-I.     

Isolation and characterization of a lectin from peanut roots.

A glucose-specific lectin has been purified to apparent homogeneity from 7-day-old peanut (Arachis hypogaea) roots by affinity chromatography on a Sephadex G-50. The lectin has a 66 kDa native molecular mass and a 33 kDa subunit molecular mass as revealed by native and denaturing sodium dodecyl sulphate-polyacrylamide gel electrophoresis, respectively. The purified lectin, gives a single precipitin line with the antiserum produced against 7-day-old root extract and shows 5 bands in the pH range of 4.4-5.4 in the isoelectric focusing gel. The glucose-specific lectin activity in the peanut roots appears from the fourth day onwards. Lipopolysaccharides isolated from the host specific Rhizobium strain are a 68-fold more potent inhibitor of the lectin as compared to glucose.     

Distribution of a lectin in tissues of Phaseolus vulgaris

The distribution of lectin in various tissues of Phaseolus vulgaris L. (var. red) has been investigated with the use of a sensitive solid phase enzyme immunoassay. Plants were divided into roots, stems and leaves with a further dissection of the stem into internodes. Isolated tissues, from plants 3-10 weeks old, were screened for their lectin content, and the development of the lectin over this period determined. Stems and roots contained significant amounts of lectin, whereas the leaves only exhibited very low levels. Furthermore, a pronounced and differential increase in the biosynthesis of the lectin was observed in stem sections between 6 and 10 weeks, where the third internode displayed the highest lectin level.     

Tissue abundance and characterization of two purified proteins with allantoinase activity from French bean (Phaseolus vulgaris)

Allantoinase (allantoin amidohydrolase, EC 3.5.2.5) catalyses the hydrolysis of allantoin to allantoic acid, a key reaction in the biosynthesis and degradation of ureides. This activity was determined in different tissues of French bean plants (Phaseolus vulgaris L.) which were grown under nitrogen-fixing conditions. Allantoinase activity was detected in all tissues analysed, but the highest levels of specific activity were found in developing fruits, from which allantoinase has been purified to electrophoretic homogeneity and further characterized. After diethylaminoethyl (DEAE)-Sephacel chromatography, two peaks showing allantoinase activity were obtained in the chromatographic profile and the corresponding proteins were independently purified. Total allantoinase activity was purified 200-fold, indicating the relevance of this enzymatic activity in French bean developing fruits, with allantoinase representing 0.5% of total soluble protein. Both proteins with allantoinase activity are monomeric with molecular masses of 45 and 42 kDa. The specific activities of the purified proteins were 560 and 295 units mg(-1), which correspond to turnover numbers of 25,200 and 12,100 min(-1), respectively. The two proteins have very similar biochemical properties showing Michaelis-Menten kinetics for allantoin with K(m) values of about 60 mM, with high optimal temperatures; are metalloenzymes; are inhibited by compounds reacting with sulphydryl groups; and are unaffected by reducing agents. All analysed tissues exhibited the two activities responsible for allantoin degradation, although one of them was the main form in leaves (the most photosynthetic tissue) and the other protein was the main form in roots (non-photosynthetic tissue). The allantoinase activity and distribution of both proteins have been analysed during fruit development. For both proteins, the allantoinase activity and distribution pattern were the same in plants growing either under nitrogen-fixing conditions or fertilized with nitrate.     

Isolation, characterization and cloning of a cDNA encoding a new antifungal defensin from Phaseolus vulgaris L. seeds

The PvD1 defensin was purified from Phaseolus vulgaris (cv. Pérola) seeds, basically as described by Terras et al. [Terras FRG, Schoofs HME, De Bolle MFC, Van Leuven F, Ress SB, Vanderleyden J, Cammue BPA, Broekaer TWF. Analysis of two novel classes of plant antifungal proteins from radish (Raphanus sativus L.) seeds. J Biol Chem 1992;267(22):15301–9], with some modifications. A DEAE-Sepharose, equilibrated with 20 mM Tris–HCl, pH 8.0, was initially utilized for the separation of peptides after ammonium sulfate fractionation. The basic fraction (the non-retained peak) obtained showed the presence of one unique band in SDS–Tricine gel electrophoresis with a molecular mass of approximately 6 kDa. The purification of this peptide was confirmed after a reverse-phase chromatography in a C2/C18 column by HPLC, where once again only one peak was observed and denominated H1. H1 was submitted to N-terminal sequencing and the comparative analysis in databanks revealed high similarity with sequences of different defensins isolated from other plants species. The N-terminal sequence of the mature defensin isolated was used to produce a degenerated primer. This primer allowed the amplification of the defensin cDNA by RT-PCR from mRNA of P. vulgaris seeds. The sequence analysis of the cloned cDNA, named PVD1, demonstrated 314 bp encoding a polypeptide of 47 amino acids. The deduced peptide presented high similarity with plant defensins of Vigna unguiculata (93%), Cicer arietinum (95%) and Pachyrhizus erosus (87%). PvD1 inhibited the growth of the yeasts, Candida albicans, Candida parapsilosis, Candida tropicalis, Candida guilliermondii, Kluyveromyces marxiannus and Saccharomyces cerevisiae. PvD1 also presented an inhibitory activity against the growth of phytopathogenic fungi including Fusarium oxysporum, Fusarium solani, Fusarium lateritium and Rizoctonia solani.     

Extraction and purification of a lectin from small black kidney bean (Phaseolus vulgaris) using a reversed micellar system

Lectin from crude extract of small black kidney bean (Phaseolus vulgaris) was successfully extracted using the reversed micellar extraction (RME). The effects of water content of organic phase (W_o), ionic strength, pH, Aerosol-OT (AOT) concentration and extraction time on the forward extraction and the pH and ionic strength in the backward extraction were studied to optimize the extraction efficiency and purification factor. Forward extraction of lectin was found to be maximum after 15 min of contact using 50 mM AOT in organic phase with W_o 27 and 10 mM citrate-phosphate buffer at pH 5.5 containing 100 mM NaCl in the aqueous phase. Lectin was backward extracted into a fresh aqueous phase using sodium-phosphate buffer (10 mM, pH 7.0) containing 500 mM KCl. The overall yield of the process was 53.28% for protein recovery and 8.2-fold for purification factor. The efficiency of the process was confirmed by gel electrophoresis analysis.     

Unfolding and refolding of leucoagglutinin (PHA-L), an oligomeric lectin from kidney beans (Phaseolus vulgaris)

The unfolding and refolding of Phaseolus vulgaris Leucoagglutinin, a homotetrameric legume lectin, was studied at pH 2.5 and 7.2 using fluorescence, far- and near-UV circular dichroism (CD) spectroscopy, 8-anilino-1-naphthalene sulfonate (ANS) binding and FPLC techniques. This protein was found to refold even at pH 2.5 and also exhibited high refolding yield around 60% at pH 2.5 and 85% at pH 7.2. The refolding at pH 2.5 takes place with the formation of a dimeric intermediate. Although the hydrodynamic radius of the completely renatured protein and the dimer at pH 2.5 was found to be same, the ANS binding as well as far-UV CD spectra of the two were different. The denaturation kinetics at pH 2.5 followed single exponential pattern with the rate of denaturation being independent of protein concentration. The renaturation kinetics on the other hand was dependent on the protein concentration providing further evidence of an intermediate state during refolding. From these experiments the folding pathway of the protein at pH 2.5 was proposed.