Isolation and properties of a lectin from the seeds of the Indian bean or lablab (Dolichos lablab L.)

Abstract

The lectin of the Indian bean or lablab (Dolichos lablab L.) was purified by affinity chromatography on two types of affinity carriers: O-a-D-mannopyranosyl-Separon and Separon-bound ovomucoid. The lectin is homogeneous in the ultracentrifuge: s2O, w= 6.14S, Mr= 110000; the molecule appears to comprise two pairs of two types of subunits (Mr 16000 and 40000), and contains 2% neutral sugar and 0.2 Mn and 0.5 Zn atom respectively. The lectin agglutinates human erythrocytes non-specifically with regard to ABO grouping at a limit concentration of 8,ug/ml, and this activity is inhibited most effectively by N-acetyl-D-glucosamine, methyl a-D-mannopyranoside and ovomucoid, but not by free D-mannose.The presence of a haemagglutinin in seeds of the Indian bean or lablab (Dolichos lablab L.) has been known for a long time (Tobis'ka, 1964). Extracts of D. lablab seeds were found also to be mitogenic (Aurich et al., 1971). To our knowledge, however, little attention, in comparison with other lectins, has been paid to the isolation and characterization ofthis lectin. Rao et al. (1976) used for its purification conventional methods of protein chemistry and described some of its properties.Though the erythroagglutinating activity of the D. lablab lectin was inhibited most effectively by methyl a-D-mannoside, in common with many other lectins specific for D-glucose and D-mannose [e.g. concanavalin A, Pisum sativum (pea) and Lens esculenta (lentil) lectins], further inhibition studies showed that the sugar specificity of the D. lablab lectin is quite different. Therefore it seemed worthwhile to seek a suitable affinity carrier for isolation of the lectin and to study its properties in more detail. Materials and methodsThe seeds of D. lablab L. were collected at Marmaris in the southern part of Turkey.Separon H-1000 was a product of Laboratorni Abbreviations used: SDS, sodium dodecyl sulphate; t.l.g.c., thin-layer gel chromatography. * This is paper 56 of the series 'Studies on lectins'. § To whom correspondence and requests for reprints should be sent. pristroje n.p., Praha, Czechoslovakia; it was glycosylated with free D-mannose or N-acetyl-D-glucosamine in dioxan with dry HCI as catalyst as described by Filka et al. (1978). Ovomucoid isolated from egg white (Lineweaver & Murray, 1947) or concanavalin A were bound to periodate-oxidized D-glucopyranosyl-Separon H-1000 containing 12.4% D-glucose (Filka et al., 1979). The watersoluble O-glycosyl-polyacrylamide copolymers used for affinity electrophoresis were prepared as described by Horejsi et al. (1978).

Purification and characterization of Dolichos lablab lectin

“…In the present study, the affinity purified DLL was eluted as a single symmetric peak on a Bio-Gel P150 column, with an apparent molecular weight of 67 kDa, which is in good agreement with the results obtained by Kumar and Rao (1986) and Gowda et al (1994). However, the subunit pattern of the present preparation is quite different from those reported previously (Guran et al, 1983;Gowda et al, 1994). Upon SDS-PAGE under both reducing and nonreducing condition, the purified lectin separated into five bands with apparent subunit masses ranging from 10 kDa to 22 kDa, of which band 3 and band 4 appear as doublets or triplets of closely migrating bands (Figure 3).…”

“…α-D-Glucans such as glycogen, and dextran 1355 S, which has numerous α1,3-glucosidic linkages, also failed to precipitate the DLL. Although DLL was found to be retained by ovalbumin-Sepharose and ovomucoid-Separon (Guran et al, 1983), neither ovalbumin nor ovomucoid gave a detectable precipitate with the lectin. Of many native and desialylated glycoproteins examined, only murine IgM gave a pronounced precipitation reaction with DLL.…”

“…Dolichos lablab lectin (DLL) has been isolated and characterized by several groups. However, reports on its molecular weight and carbohydrate-binding properties have varied, the discrepancy being attributed to the different varieties of Dolichos lablab used and/or the different climates in which they were grown (Salgarkar and Sohonie, 1965;Rao et al, 1976;Guran et al, 1983;Kumar and Rao, 1986;Favero et al, 1988;Silva-Lima et al, 1988;Mo et al, 1990).…”

Purification and Characterization of Lectins from Jute (Chorchorus olitorius) Leaves

“…Indian bean (Dolichos lablab L.), 18 Abrus precatorius 19 and Ricinus communis agglutinin 20 and very few contained four subunits of identical molecular weight viz. Pheseolus vulgaris.…”

Purification and characterization of three galactose specific lectins from Mulberry seeds (Morus sp.)

“…The lectins purified from plant sources contained mostly four subunits of two identical pairs, e.g. Indian bean (Dolichos lablab L.) [19], Arbus precatorius [20], and Ricinus comminis agglutinin [21] and very few contained four subunits of identical molecular mass, e.g. Phaseolus vulgaris [3].…”