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.     

Binding of isolectins from red kidney bean (Phaseolus vulgaris) to purified rat brush-border membranes

Ingestion of red kidney bean phytohemagglutinin causes impaired growth and intestinal malabsorption, and facilitates bacterial colonization in the small intestine of weanling rats. We have studied interactions of the highly purified phytohemagglutinin erythroagglutinating (E4) and mitogenic (L4) isolectins with microvillous membrane vesicles prepared from rat small intestines. E4 and L4 were radioiodinated with 125I by the chloramine-T technique. E4 and L4 isolectins both bound to microvillous membrane vesicles. Binding was saturable and reversible. Each mg of membrane protein bound 744 +/- 86 micrograms E4 and 213 +/- 21 micrograms L4. The apparent Ka for E4 and L4 binding was 2.5 x 10(-6) and 13.0 x 10(-6) M-1, respectively. Binding of each 125I-labelled isolectin was abolished by 100-fold excess of unlabelled isolectin. In each case binding also was inhibited by appropriate oligosaccharide inhibitors, indicating that isolectin-microvillous membrane interactions were mediated by carbohydrate recognition. Patterns of saccharide inhibition of isolectin binding were different for E4 and L4. Competitive binding experiments demonstrated mutual noncompetitive inhibition of E4 and L4 binding consistent with steric hindrance. Therefore, E4 and L4 each bound to its own set of receptors. Based on the known saccharide specificities of E4 and L4, these data indicate that there are differences in expression of complex asparagine-linked biantennary and tri- or tetraantennary oligosaccharides at the microvillous surface. The data also provide the possibility that direct interactions of one or more phytohemagglutinin isolectins with intestinal mucosa in vivo may contribute to the antinutritional effects associated with ingestion of crude red kidney beans.     

Regulation of glycan processing by Golgi enzymes from red kidney bean (Phaseolus vulgaris) seedlings.

The effect of a protein matrix on the processing of glycoprotein glycans by Golgi enzymes from plant seedlings has been determined with an artificial glycoprotein system, comparing the processing rates of glycan-(biotinyl)Asn (or glycan-(biotinamidohexanoyl)Asn) substrates either free or bound to avidin. An analysis of the pooled glycoproteins from the seedlings suggested that the most common glycan structure is a complex one (GlcNAc-Man3GlycNAc2-protein), and consistent with this processing end-product, mannosidases I and II and GlcNAc transferases I and II were all found to be present in the seedling Golgi membrane preparations. The effect of the avidin matrix either in a proximal (biotinyl substrates) or distal (N-(biotinamido)hexonoyl substrates) association with the appropriate glycan substrate for these four enzymes was assessed from the direct comparison of the apparent first-order rate constants for the free and avidin-bound substrate-product conversions. All four plant enzymes were inhibited by the association of the glycan substrates with avidin, but the inhibition was much less pronounced than that observed with the corresponding enzymes from rat liver and hen oviduct. The rate effect shows a progression from 3- to 10-fold rate decreases in the proximal complexes and 2- to 3-fold in the distal complexes in going from the first (mannosidase I) to the fourth (GlcNAc transferase II) enzyme; with the mammalian and avian enzymes the largest effects were for the first ones and much larger absolute rate effects were observed. The results suggest that the nature of the processing enzymes in terms of this response to the avidin glycan substrates may differ in different organisms.     

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.     

Conidiogenic effects of mannose-binding lectins isolated from cotyledons of red kidney bean (Phaseolus vulgaris) on Alternaria alternata

Effect of proteinaceous extracts from red kidney bean cotyledons on mycelium of Alternaria alternata growing on potato dextrose agar (PDA) plates was investigated. Unexpectedly, conidia formation was induced in response to applied crude extracts. A PDA disc method was developed to quantify conidia formed. A purified fraction retaining conidiation inducing effect (CIE) was obtained following several protein purification procedures including the last step of eluting bound proteins from an Affi-gel blue gel column. Based on MALDI (matrix assisted laser desorption/ionization) mass spectrometric analysis, a previously identified mannose-binding lectin (MBL) called PvFRIL (Phaseolus vulgaris fetal liver tyrosine kinase 3-receptor interacting lectin) was present in this conidiation inducing fraction. The PvFRIL was subsequently purified using a single step mannose-agarose affinity column chromatography. When the lectin was applied exogenously to A. alternata, increased conidiation resulted. The conidia produced in response to the MBL were similar to those induced by other methods and their germ tubes were longer after 12 h growth than those induced under white light. To our knowledge this is the first report of exogenous application of a PvFRIL or another purified protein from a plant inducing conidia formation in a fungus.     

An antifungal peptide from Phaseolus vulgaris cv. brown kidney bean

A 5.4-kDa antifungal peptide, with an N-terminal sequence highly homologous to defensins and inhibitory activity against Mycosphaerella arachidicola (IC(50)= 3 μM), Setospaeria turcica and Bipolaris maydis, was isolated from the seeds of Phaseolus vulgaris cv. brown kidney bean. The peptide was purified by employing a protocol that entailed adsorption on Affi-gel blue gel and Mono S and finally gel filtration on Superdex 75. The antifungal activity of the peptide against M. arachidicola was stable in the pH range 3-12 and in the temperature range 0°C to 80°C. There was a slight reduction of the antifungal activity at pH 2 and 13, and the activity was indiscernible at pH 0, 1, and 14. The activity at 90°C and 100°C was slightly diminished. Deposition of Congo red at the hyphal tips of M. arachidicola was induced by the peptide indicating inhibition of hyphal growth. The lack of antiproliferative activity of brown kidney bean antifungal peptide toward tumor cells, in contrast to the presence of such activity of other antifungal peptides, indicates that different domains are responsible for the antifungal and antiproliferative activities.     

Fourier-transform infrared spectroscopic study of globulin from Phaseolus angularis (red bean).

The conformation of red bean globulin dispersions (≈10% in D₂O or deuterated phosphate buffer pD 7.4) under the influence of pH, chaotropic salts, protein structure perturbants, and heating conditions was studied by Fourier-transform infrared (FTIR) spectroscopy. The FTIR spectrum of red bean globulin showed major bands from 1682 to 1637 cm⁻¹ in the amide I′ region, corresponding to the four types of secondary structures, i.e. β-turns, β-sheets, -helix and random coils. At extreme pH conditions, there were changes in intensity in bands attributed to β-sheet (1637 and 1618 cm⁻¹) and random coil (1644 cm⁻¹) structures, and shifts of these bands to lower or higher wavenumbers, indicating changes in protein conformation. Chaotropic salts caused progressive increases in random coil structures and concomitant decreases in β-sheet bands, following the lyotrophic series of anions. In the presence of sodium dodecyl sulfate and ethylene glycol, pronounced increases in the random coil band were observed, accompanied by slight shifts of the β-sheet band. Addition of dithiothreitol and N-ethylmaleimide did not cause marked changes in the FTIR spectra. Heating at increasing temperature led to progressive decreases in the intensity of the -helix and β-sheet bands and increases in random coil band intensity, leveling off at around 60 °C. The data suggest that re-organization of protein structure occurred at temperatures well below the denaturation temperature of red bean globulin (86 °C) as determined by differential scanning calorimetry. This was accompanied by pronounced increases in the intensity of the two intermolecular β-sheet bands (1682 and 1619–1620 cm⁻¹) associated with the formation of aggregated strands at higher temperatures (80–90 °C). Increases in intensity of the aggregation bands were also observed in the heat-induced buffer-soluble and insoluble aggregates.     

Enzymatic characterization of starch synthase III from kidney bean (Phaseolus vulgaris L.)

In plants and green algae, several starch synthase isozymes are responsible for the elongation of glucan chains in the biosynthesis of amylose and amylopectin. Multiple starch synthase isozymes, which are classified into five major classes (granule-bound starch synthases, SSI, SSII, SSIII, and SSIV) according to their primary sequences, have distinct enzymatic properties. All the starch synthase isozymes consist of a transit peptide, an N-terminal noncatalytic region (N-domain), and a C-terminal catalytic region (C-domain). To elucidate the enzymatic properties of kidney bean (Phaseolus vulgaris L.) SSIII and the function of the N-domain of kidney bean SSIII, three recombinant proteins were constructed: putative mature recombinant SSIII, recombinant kidney bean SSIII N-domain, and recombinant kidney bean SSIII C-domain. Purified recombinant kidney bean SSIII displayed high specific activities for primers as compared to the other starch synthase isozymes from kidney bean. Kinetic analysis showed that the high specific activities of recombinant kidney bean SSIII are attributable to the high k(cat) values, and that the K(m) values of recombinant kidney bean SSIII C-domain for primers were much higher than those of recombinant kidney bean recombinant SSIII. Recombinant kidney bean SSIII and recombinant kidney bean SSIII C-domain had similar chain-length specificities for the extension of glucan chains, indicating that the N-domain of kidney bean SSIII does not affect the chain-length specificity. Affinity gel electrophoresis indicated that recombinant kidney bean SSIII and recombinant kidney bean SSIII N-domain have high affinities for amylose and amylopectin. The data presented in this study provide direct evidence for the function of the N-domain of kidney bean SSIII as a carbohydrate-binding module.     

Properties of a glycopeptide isolated from human Tamm-Horsfall glycoprotein. Interaction with leucoagglutinin and anti-(human Tamm-Horsfall glycoprotein) antibodies.

A sialylated glycopeptide isolated after Pronase digestion of human Tamm-Horsfall glycoprotein behaves as a powerful monovalent hapten in the precipitin reaction between human Tamm-Horsfall glycoprotein and leucoagglutinin, but fails to inhibit the interaction of the glycoprotein with rabbit anti-(human Tamm-Horsfall glycoprotein) antibodies. The glycopeptide is much less active than the intact glycoprotein as an inhibitor of lymphocyte transformation induced by leucoagglutinin.     

Dynamic of lectin activity during germination of bean seeds (Phaseolus vulgaris L.)

PHA quantity and activity dynamics during early germination of bean seed were investigated. Electrophoretic characteristics, subunits composition and carbohydrate-binding specificity of lectin extracted from white kidney bean cv. Bilozerna were studied. It was shown that investigated lectin consisted of 2 subunits E and L with molecular weight 34 and 36 kDa, respectively, analogously to purified PHA ("Serva", Germany), and specifically bound N-acetyl-D-galactosamin and galactose. During germination both quantity and activity of PHA were dramatically decreasing in embryonic axes and in cotyledons, possibly, as a result of the lectin release from seeds to the environment. It is very likely that one of the defence mechanisms of germinating seeds is related with the releasing of lectins that are able to bind components of the bacterial cell wall and to inhibit their growth.     

A specific inhibitor of Colletotrichum lindemuthianum protease from kidney bean (Phaseolus vulgaris) seeds

A specific protein—an inhibitor of Colletotrichum lindemuthianum protease—was isolated from kidney bean seeds in a homogeneous form. The purification procedure included gel filtration, isoelectric focusing and affinity chromatography on trypsin-Sepharose column. The latter was introduced to separate the fungal protease inhibitor from closely related trypsin and chymotrypsin inhibitors present in kidney bean seeds.     

Activities of some peptidases and proteinases in germinating kidney bean, Phaseolus vulgaris

The activities of aminopeptidase (EC 3.4.11), dipeptidase (EC 3.4.13), carboxypeptidase (EC 3.4.16), naphthylamidase (EC 3.4.11) and proteinases (EC 3.4.21) were assayed in extracts from the cotyledons and the axial tissues of resting and germinating kidney beans ( Phaseolus vulgaris L. cv. Processor).
The activities of the alkaline peptidases (aminopeptidase hydrolyzing Leu-Tyr at pH 9.2 and dipeptidase acting on Ala-Gly at pH 8.5) and naphthylamidases (hydrolyzing Leu-β-naphthylamide at pH 6.4) were high in the cotyledons of resting seeds, but decreased during germination. This decrease was faster than the loss of the total nitrogen. On the contrary, the activities of carboxypeptidase (hydrolyzing carbobenzoxy-Phe-Ala at pH 5.9) and proteinases (acting on haemoglobin at pH 3.7 and on casein at pH 5.4 and 7.0) were low in the resting seeds, but increased during germination reaching their maximal values when the mobilization of nitrogen was highest. It has been suggested that the breakdown of storage proteins is initiated inside the protein bodies by acid proteinases and carboxypeptidases. Although the activities of the alkaline peptidases and naphthylamidases decreased during germination, these were still relatively high and enough for the completion of the proteolytic breakdown. Thus, it is suggested that, as a final step in a chain of events, the main role for the alkaline peptidases in the cotyledons of germinating seeds is to provide amino acids for the growth of the seedling.     

Races of common bean (Phaseolus vulgaris,Fabaceae)

Evidence for genetic diversity in cultivated common bean (Phaseolus vulgaris) is reviewed. Multivariate statistical analyses of morphological, agronomic, and molecular data, as well as other available information on Latin American landraces representing various geographical and ecological regions of their primary centers of domestications in the Americas, reveal the existence of two major groups of germplasm: Middle American and Andean South American, which could be further divided into six races. Three races originated in Middle America (races Durango, Jalisco, and Mesoamerica) and three in Andean South America (races Chile, Nueva Granada, and Peru). Their distinctive characteristics and their relationships with previously reported gene pools are discussed.     

Thermal stability of Phaseolus vulgaris leucoagglutinin: a differential scanning calorimetry study

Phaseolus vulgaris phytohemagglutinin L is a homotetrameric-leucoagglutinating seed lectin. Its three-dimensional structure shows similarity with other members of the legume lectin family. The tetrameric form of this lectin is pH dependent. Gel filtration results showed that the protein exists in its dimeric state at pH 2.5 and as a tetramer at pH 7.2. Contrary to earlier reports on legume lectins that possess canonical dimers, thermal denaturation studies show that the refolding of phytohemagglutinin L at neutral pH is irreversible. Differential scanning calorimetry (DSC) was used to study the denaturation of this lectin as a function of pH that ranged from 2.0 to 3.0. The lectin was found to be extremely thermostable with a transition temperature around 82 degrees C and above 100 degrees C at pH 2.5 and 7.2, respectively. The ratio of calorimetric to vant Hoff enthalpy could not be calculated because of its irreversible-folding behavior. However, from the DSC data, it was discovered that the protein remains in its compact-folded state, even at pH 2.3, with the onset of denaturation occurring at 60 degrees C.