Indian Journal of Biochemistry & Biophysics

Vol. 42, February 2005, pp. 34-40

Isolation and characterization of two N-acetyl-D-lactosamine specific lectins from

tubers of Arisaema intermedium Blume and A. wallichianum Hook f.

Manpreet Kaur, Jatinder Singh*, Sukhdev Singh Kamboj, Jagmohan Singh, Amandeep Kaur, S K Sooda and A K Saxena

b

Department of Molecular Biology and Biochemistry, Guru Nanak Dev University, Amritsar 1430 05, India aDepartment of Biosciences, Himachal Pradesh University, Shimla, India

bDepartment of Pharmacology, Regional Research Laboratory, Jammu-Tawi 180 001, India

Received 16 June 2004; revised 14 December 2004

Two new lectins were purified from the tubers of Arisaema intermedium Blume and A. wallichianum Hook. f. (family:

Araceae) by affinity chromatography on asialofetuin-linked amino activated silica beads. The bound lectins were eluted with

0.1 M glycine-HCl, pH 2.5. They gave a single band corresponding to subunit Mr 13.4 kDa in SDS-PAGE, pH 8.3. On gel

filtration chromatography, the lectins showed a Mr of 51.2 kDa, suggesting a homotetrameric structure. Both the lectins gave

a single peak on size exclusion HPLC and cation-exchange columns and a single band on PAGE, pH 4.5. However, like

other monocot lectins, they gave multiple bands in isoelectric focusing and at PAGE 8.3. The lectins were inhibited by

N-acetyl-D-lactosamine (LacNAc), a disaccharide and asialofetuin, a complex desialylated serum glycoprotein. They had no

requirement for divalent metal ions i.e., Ca2+ and Mn2+ for their activity and were found to be mitogenic towards human

lymphocytes. A. intermedium showed antiproliferative effect against various human cancer cell lines in vitro.

Keywords: Araceae, Arisaema, tuber, N-acetyl-D-lactosamine, asialofetuin, lectin, mitogenic, antiproliferative effect,

human lymphocytes, human cancer cell lines, carbohydrate specificity, denaturing agents.

IPC Code: C 07K 14/42, G 01N 33/53

Plant lectins, a heterogeneous group of proteins or

glycoproteins are classified on the basis of their ability

to recognize and specifically bind to carbohydrate

ligands1. They are widely distributed in dicotyledonous

plants and in a large number of other plant families.

Among monocotyledonous plants, several lectins with

interesting properties have been isolated from species,

belonging to Poaceae2, Alliaceae

3, Amaryllidaceae

4,

Liliaceae5 and Orchidaceae

6. These lectins differ from

the dicotyledonous lectins, especially with respect to

their carbohydrate-binding specificity, and other

biochemical and biophysical properties. For instance,

the lectins from several Amaryllidaceae, Alliaceae and

Orchidaceae species7 have shown exclusive specificity

towards mannose and differ from the

mannose/glucose/N-acetyl-D-glucosamine binding con

A and other related legume lectins, and this property

has been exploited for the analysis and isolation of

mannose-containing glycoconjugates8.

Some monocot mannose-binding lectins have also

found applications in biomedical field, due to their

potential inhibitory effect against human and animal

retroviruses, including HIV9,10

and their ability to block

the adhesion receptors of mannose-fimbriated

Escherchia coli in the small intestine of rat11

. In

addition, they are also finding use as an important tool

in the plant protection and plant biotechnology, as their

genes confer resistance against sucking insects12,13

and

nematodes14

. Araceae is a lectin-rich monocot family,

in which lectins comprise 70-80% of storage

protein15,16

. In the present communication, the

purification and biochemical and biophysical

characterization of two new lectins (from the tubers)

from the genus Arisaema i.e., A. intermedium and

A. wallichianum (family Araceae) have been described.

A preliminary study about their mitogenic potential and

inhibition against some cancer lines is also reported.

Materials and Methods

Chemicals

Amino activated silica beads were purchased from

Clifmar Associates, U.K. All sugars/derivatives,

_________________

*Corresponding author

Tel: (+91) 183-2258802-09 Ext. 3313; (+91) 183-2450601-14

Fax: (+91)-183-2258819, 2258820

E-mail: jst2002@rediffmail.com

Abbreviations: AIL, Arisaema intermedium lectin; AWL,

Arisaema wallichianum lectin; LacNAc, N-acetyl-D-lactosamine;

PBMC, peripheral blood mononuclear cells; MEAPC, minimal

erythrocyte agglutinating protein concentration; RBCs, red blood

cells; IEF, isoelectric focusing, SRB, sulphorhodamine B.

KAUR et al.: N-ACETYL-D-LACTOSAMINE SPECIFIC LECTINS FROM ARISAEMA

35

glycoproteins, molecular weight markers for SDS-

PAGE and gel filtration chromatography, pI markers

and ampholines (pH 3-10) for isoelectric focusing

were the products of Sigma Chemical, Co. USA.

Biogel P-200 for gel filtration chromatography was

supplied by Bio-Rad U.S.A. All other chemicals were

of analytical grade.

Plant material

The tubers of Arisaema intermedium Blume and A.

wallichianum Hook. f. were collected from Shimla

during the month of September.

Isolation and purification

A. intermedium lectin (AIL) and A. wallichianum

lectin (AWL) were purified by affinity

chromatography using asialofetuin-linked porous

amino-activated silica beads15

(pore size 1000 Ǻ,

diameter 100 µ). The extract of fresh tubers was

prepared in 0.01 M PBS, pH 7.2 (1:5 w/v). The homogenate was filtered and then centrifuged at

20,000 × g for 30 min at 4°C. The clear supernatant was extensively dialyzed against 0.01 M PBS, pH 7.2

at 4°C to remove any low molecular weight substances, which may interfere in lectin activity. The

crude dialyzed extract was applied to affinity matrix,

packed in polypropylene column (Bio-Rad) and after

recirculating 3-4 times, the unbound proteins were

washed off with 0.01 M PBS. The bound lectins were

eluted with 0.1 M glycine-HCl buffer, pH 2.5 and the

eluted fractions were neutralized immediately with

2 M Tris-HCl buffer, pH 8.8. The active fractions

were pooled and extensively dialyzed against 0.01 M

PBS at 4°C to bring the purified lectin in physiological buffer and to remove tris ions which

interfere in protein estimation.

Electrophoretic analysis

Polyacrylamide gel electrophoresis (PAGE) using a

discontinuous buffer system was carried out using

7.5% (w/v) gel at pH 4.517

and 10% (w/v) gel at pH

8.318

. SDS-PAGE at pH 8.3 was carried out on 11%

gel, both under reducing and non-reducing

conditions19

. Isoelectric focusing of purified lectins

was carried out in 5% polyacrylamide tube gels using

carrier ampholine of pH range 3.0-10.020

.

Gel-exclusion chromatography

Native molecular mass of the lectins was

determined by gel filtration chromatography on a

calibrated Biogel P-200 column (1.6 cm × 67 cm),

equilibrated and eluted with 0.01 M PBS, pH 7.2. The

following standard markers were employed:

cytochrome c (12.4 kDa), carbonic anhydrase

(29 kDa), glyceraldehyde-3-phosphate dehydrogenase

(36 kDa), ovalbumin (45 kDa) and bovine albumin

(66 kDa). Gel-exclusion chromatography using 300-

SW, HPLC column (Waters) was performed with

affinity purified lectins to check the purity.

Ion-exchange chromatography

Ion-exchange chromatography was carried out on

DEAE-5PW and SP-5PW HPLC columns (Waters).

Lectins dialyzed against running buffers i.e., 0.01 M

Tris-HCl, pH 8.3 and 0.05 M acetate-acetic acid, pH

4.5 were applied to the DEAE-5PW and SP-5PW

columns, respectively. The adsorbed lectin was eluted

using a linear gradient of increasing NaCl

concentration (0-2 M) in running buffer.

Protein and carbohydrate analyses

Protein concentration in both crude and purified

agglutinins was determined by the method of Lowry

et al21

. Total neutral sugar content of the purified

lectin preparations was estimated by anthrone method

using D-glucose as standard22

.

Thermal stability

To determine the thermal stability, 30 µl of the

purified lectin (2 mg/ml) was incubated at

temperatures ranging between 40-95ºC with 5ºC

interval for 15 min at each temperature in a water

bath. The samples were cooled to room temperature,

centrifuged to eliminate precipitated material and

evaluated for hemagglutination activity.

Effect of pH

Appropriate pH of eluent (glycine-HCl) used in

affinity purification of lectins was ascertained by

incubating each lectin with 0.1 M glycine-HCl buffers

at pH 3.5, 3.0, 2.5 and 1.5 for 30 min. The contents

were neutralized with 2.0 M Tris-HCl buffer, pH 8.8

and hemagglutination activity of the control and

treated samples was evaluated.

Hemagglutination and hapten inhibition assay

Lectin-mediated agglutination of human and

animal red blood cells was performed in 96-well

polystyrene ‘U’ shaped plates both for crude and

purified lectins and after incubation for 1 hr at 37ºC,

the agglutination was observed visually23

. The titre

was expressed as the reciprocal of the highest dilution

of the lectin, showing visible agglutination and this

INDIAN J. BIOCHEM. BIOPHYS., VOL. 42, FEBRUARY 2005

36

concentration was denoted as one hemagglutination

unit (HU)23

.

To ascertain the carbohydrate specificity of the

lectins, hapten inhibition assay23

was performed with a

series of 39 sugars which included 4 pentoses:

D-arabinose, L-arabinose, D-ribose and D-xylose; 19

hexoses or their derivatives: D-fructose, D-galactose,

D-glucose, D-mannose, L-sorbose, L-fucose,

L-rhamnose, N-acetyl-D-galactosamine, N-acetyl-D-

glucosamine, N-acetyl-D-mannosamine, methyl-α- and

β-D-glucopyranosides, methyl-α-D-mannopyranoside,

methyl-α- and β-D-galactopyranosides, β-phenyl-D-glucopyranoside, sialic acid, adonitol and myo-inositol;

7 disaccharides: β-gentiobiose, D-lactose, D-maltose, D-

melibiose, D-trehalose, T-disaccharide and N-acetyl-D-

lactosamine; 3 trisaccharides: D-melizitose, D-raffinose;

and N,N’,N”-triacetylchitotriose, 3 polysaccharides i.e.,

chitin, glycogen and inulin. Three glycoproteins i.e.,

fetuin, asialofetuin and porcine mucin were also used.

The various sugars or their derivatives were tested at a

conc. of 100 mM while polysaccharides and

glycoproteins at a conc. of 4 mg/ml. Each lectin was

used at twice the lowest concentration causing

agglutination of rabbit RBCs as determined through

double dilution technique. The lowest concentration of

sugar that inhibited haemagglution was recorded and

used to define the inhibitory activity23

.

Effect of denaturing agents

The effect of denaturing agents i.e., urea, thiourea,

and guanidine hydrochloride was examined on lectin

activity. For this, wide ranges of concentrations of

denaturants from 0.5-8.0 M at 0.5 M interval were

used. The 50 µl of each solution was incubated with

50 µl of each lectin solution (2 mg/ml) in a microtitre

plate at 37ºC for 1 hr and the hemagglutination

activity was checked for untreated and treated

samples.

Metal ion requirement

Lectins were demetallized by dialysis against 0.1 M

EDTA for 72 hr, followed by remetallization with

0.1 M CaCl2 and MnCl2. The metal ion requirement

was determined by comparing the hemagglutination

titre after each step.

Mitogenic potential

Mitogenic potential of purified lectins was studied

by 3,4,5-dimethylthiazol-2-yl-2,5-diphenyltetrazolium

bromide (MTT) assay24

towards human peripheral

blood mononuclear cells (PBMC) using conA as

standard. The PBMC were isolated by the method as

described.25

Inhibition of cancer cell lines

Inhibitory potential of the lectins was checked at

doses ranging between 20-100 µg/ml against various human cancer cell lines i.e., PC3 (prostrate), SNB78

(central nervous system), A 549 (lung), and SiHa

(cervix), procured from Pune, using sulphorhodamine

B (SRB) assay26

. Anticancer drugs 5-flurouracil,

mytomycin C and paclitaxel at a concentration of

1 × 10-4 M were used as positive controls.

Results and Discussion Two new lectins from the tubers of Arisaema

intermedium (AIL) and A. wallichianum (AWL) have

been purified, and their physicochemical and

biological characterization is reported. The

purification protocol of AIL and AWL is summarized

in Table 1. The lectins were purified in a single step

Table 1Affinity purification of lectins from tubers of Araceous plants

Lectin Step Total protein

(mg)

Total activity

(HU)

Specific activity

(HU/mg)

Purification

fold

Recovery

(%)

MEAPC

(µg/ml)

Crude 444.5 16,000 36.0 1.0 100.0 27.8

Affinity chromatography

PBS fractions 257.0 ─ ─ ─ ─ ─

Arisaema intermedium

Glycine-HCl fractions 99.4 12,160 12.2 3.4 76.0 8.2

Crude 973.0 32,000 32.9 1.0 100.0 30.4

Affinity chromatography

PBS fractions 338.3 --- -- -- -- --

A. wallichianum

Glycine-HCl fractions 625.5 26,560 42.5 1.3 83.0 23.5

MEAPC, minimal erythrocyte agglutinating protein concentration; HU, hemagglutination unit

KAUR et al.: N-ACETYL-D-LACTOSAMINE SPECIFIC LECTINS FROM ARISAEMA

37

by affinity chromatography on asialofetuin-linked

amino-activated silica. The flows through fractions

(1-40) were devoid of hemagglutination activity,

indicating the complete adsorption of the lectins to the

matrix (Fig. 1). The overall recovery for AIL and

AWL was 76% and 83%, respectively. The

purification factor was low, indicating that lectins

comprise a major proportion of the total protein

content in crude tuber extracts. This is consistent with

earlier findings16

.

A single peak in gel filtration on 300-SW (Fig. 2)

and SP-5PW HPLC cation exchange column (Fig. 3

A, B), and a single band in PAGE 4.5 (Fig. 4A)

indicated the purity of the lectin preparations. To

Fig. 3Cation-exchange HPLC of Arisaema lectins. (A): AIL; and (B): AWL. 100 µg of purified lectins were chromatographed

on SP-5PW analytical column (0.8 × 7.5 cm). The running buffer was 0.05 M sodium acetate, pH 4.5. A, AIL; B, AWL. Anion-

exchange HPLC of lectins. (C): AIL; and (D): AWL .100 µg of

purified lectins were chromatographed on DEAE-5PW analytical

column (0.8 × 7.5 cm). The running buffer was 0.01 M Tris-HCl, pH 8.3.

Fig. 1Affinity purification of A. intermedium lectin (AIL) and A. wallichianum lectin (AWL) from tuber extracts on asialofetuin-linked

amino-activated silica beads. [Crude dialyzed extracts of AIL (32 mg) and AWL (28 mg) were applied to the column (0.8 × 6.0 cm), pre-equilibrated with 0.01 M PBS, pH, 7.2. Bound lectins were eluted with 0.1 M glycine-HCl, pH 2.5 at flow rate 30 ml/hr. (A): AIL; and

(B): AWL]

Fig. 2Size-exclusion chromatography of Arisaema lectins. [100 µg of purified lectins were chromatographed on 300-SW

analytical column (0.8 × 30 cm). The running buffer was 0.01 M PBS, pH 7.2 at a flow rate = 1 ml min-1. (A): AIL; and (B): AWL]

INDIAN J. BIOCHEM. BIOPHYS., VOL. 42, FEBRUARY 2005

38

determine the molecular mass, the purified lectins were

analyzed by SDS-PAGE and gel filtration

chromatography. Gel filtration chromatography gave a

single peak with an apparent molecular mass of 51.2

kDa for both the lectins, while the subunit molecular

mass as determined by SDS-PAGE was 13.4 kDa

(Fig. 4B). These results suggest that they are

homotetramers and their subunits are not held by

disulphide linkages. This finding is further supported

by the fact that most of the Araceous lectins are devoid

of cysteine residues15

. Both the lectins gave multiple

peaks in anion exchange DEAE-5PW HPLC column

(Fig. 3C, D), two bands in PAGE 8.3 (Fig. 4C) and

multiple bands in isoelectric focusing (Fig. 4D), which

may be due to the presence of charged isomers. Similar

results were reported earlier for many other purified

lectins15

. The charge microheterogeneity in the lectins

could be genetic4, as reported for lectins from

Amaryllidaceae

and Alliaceae or due to the

heterogeneity of the oligosaccharide chains27

.

Among the carbohydrates tested, N-acetyl-D-

lactosamine (LacNAc) and asialofetuin inhibited

lectin-induced haemagglutination. Earlier, Araceous

lectins, including those from A. curvatum and

A. consanguineum5 were inhibited by asialofetuin.

Interestingly, AIL and AWL have also shown

specificity towards LacNAc, which is an important

cancer marker28

, suggesting that they may prove to be

a useful tool for the detection of various types of

cancers. The minimal inhibitory concentration (MIC)

of AIL and AWL for LacNAc was 25 mM, while for

asialofetuin the values were 125 µg/ml and 62 µg/ml

respectively. The reactivity of the lectins towards

asialofetuin, but not with fetuin suggests that sialic

acid may hinder the binding of lectin to the

recognition sites in fetuin. Asialofetuin comprises of

80% Asn-linked oligosaccharides terminating in

LacNAc (Gal-β-1, 4 GlcNAc) and 20% Ser/Thr-

linked oligosaccharides having T-disaccharide (Gal-β-

1, 3-GalNAc)29

. The reactivity of AIL and AWL

towards asialofetuin may be attributed to the LacNAc

component, since these lectins could bind to LacNAc,

but not to T-disaccharide, when checked individually.

Furthermore, C-2 equatorial acetamido group appears

to be crucial in lectin binding, as these lectins showed

no specificity towards lactose. Similarly, they were

not inhibited by maltose (a glucose disaccharide),

indicating the importance of C-4 axial hydroxyl group

and β-1, 4-glycosidic linkage in their reactivity

towards LacNAc and asialofetuin.

The lectins were reactive towards erythrocytes

from rabbit, rat, goat, sheep and guinea pig, but were

Fig. 4(A): Discontinuous PAGE of Arisaema lectins at pH 4.5 using 7.5% gel (running time 8 hr at a constant 150 V). 80 µg protein

loaded in each lane; (B): SDS-PAGE of Arisaema lectins, pH 8.3 using 11% gel in the presence of 2% β-mercaptoethanol (running time 4

hr at a constant 150 V). 40 µg protein was loaded. Molecular mass markers (lane M) from top to bottom are: phosphorylase b (94 kDa);

albumin bovine (67 kDa); ovalbumin (45 kDa); carbonic anhydrase (30 kDa); trypsin inhibitor (20.1 kDa) and α-lactalbumin (14.4 kDa);

(C): Discontinuous PAGE, at pH 8.3 using 10% gel (running time 8 hr at a constant 100 V). 60 µg protein loaded in each lane; and (D): Isoelectric focusing of non-denatured lectins on 5% polyacrylamide gel using carrier ampholine of pH range 3.5-10.0 (running time 12 hr

at a constant 200 V). pI markers (lane M) used from top to bottom are: myoglobulin 1 (pI-7.2); myoglobulin 2 (pI-6.8); carbonic

anhydrase II (pI-5.5) and trypsin inhibitor (pI-4.6). Protein loaded was 40 µg. The gels were stained with Coomassie brilliant blue. 1, AIL; 2, AWL.

KAUR et al.: N-ACETYL-D-LACTOSAMINE SPECIFIC LECTINS FROM ARISAEMA

39

non-reactive towards human erythrocytes, irrespective

of their blood groups, even after neuraminidase

treatment (Table 2), as reported earlier for Araceous

lectins15

. Both lectins agglutinated normal and

neuraminidase-treated rabbit, rat, sheep and guinea

pig erythrocytes, but reacted with goat erythrocytes

only after neuraminidase treatment. No decrease in

minimal erythrocyte agglutinating concentration

(MEAPC) of the lectins was observed in the rabbit

RBCs after neuraminidase treatment, while

approximately 130 times and 64 times lower MEAPC

were recorded for RBCs from rat and sheep, when

desialylated erythrocytes were used. Thus, for

practical reasons i.e., better reactivity and easy

availability, rabbit RBCs were subsequently used in

hemagglutination and hemagglutination inhibition

assay.

Urea and guanidine-HCl at 3.0 M conc. decreased

the lectin activity to 50%, while a similar decrease

was observed at 4.0 M, in the case of thiourea. The

denaturation by these agents indicates the globular

nature of lectins, which is stabilized mainly by

hydrophobic interactions30

. Both AIL and AWL were

found to be glycoproteins containing 3.4% and 2.9%

carbohydrates respectively, unlike Amaryllidaceae

and Alliaceae lectins. These lectins were stable up to

55ºC for 15 min and thereafter, the activity starts

declining. However, 25% of the residual activity

remains, even after boiling in water-pan for 15 min.

Both the lectins were found to be stable at pH chosen

for elution i.e., pH 2.5 for 30 min. Demetallization

using EDTA as the chelating agent had no effect on

the hemagglutination activity of the lectins, indicating

that they don’t require metal ions for their activity.

The lectins showed the mitogenic potential towards

human peripheral blood mononuclear cells, which

was almost double, when compared with a well-

known mitogen con A (Fig. 5). The relative mitogenic

index of AIL and AWL was 258A.33 and 205.73,

respectively. Thus, these lectins can be applied as a

tool to study the lymphocytes transformation as a

model of antigen activation, initiation of cell division

and growth, and to know the immune status of an

individual31

, like other commercially popular

mitogenic lectins, such as concanavalin A (con A),

phytohemagglutinin (PHA) and pokeweed mitogen

(PWM)32

. The mitogenic potential of AIL and AWL,

as well as of earlier reported Araceous lectins

indicates that the family Araceae is rich in mitogenic

lectins. This is in contrast to the virtually non-

mitogenic lectins reported from the family

Amaryllidaceae32

. AIL showed anti-proliferative

activity in sulforhodamine B (SRB) assay for various

human cancer cell lines (Fig. 6). It showed 30.80%,

19.02% and 27.96% inhibition with PC 3, A549, and

SiHa cell lines, respectively, however, no such

activity was observed with AWL against these cell

lines. In view of the antiproliferative potential, AIL

needs to be screened further for other cell lines also.

The non-proliferative effect of AWL indicates that

though these lectins have apparently the same sugar

specificity, but they might differ in fine sugar

specificity33

.

In conclusion, both the lectins have almost similar

biochemical and biophysical properties. Thus, the

Araceous lectins seemed to be highly conserved

Table 2Biological specificity of affinity purified Arisaema lectins on the erythrocytes

MEAPC (µg/ml)

Lectin Human* Rabbit Guinea pig Rat Goat Sheep

UT NT UT NT UT NT UT NT UT NT UT NT

A. intermedium NA NA 8.2 8.2 16.4 3.28 131.2 1.0 NA 196.8 787.2 12.3

A. wallichianum NA NA 23.5 23.5 47.0 9.4 376.0 2.9 NA 564.0 952.4 14.9

*Erythrocytes from human blood groups A, B, AB and O were used; MEAPC, minimal erythrocyte agglutinating protein concentration;

UT, untreated cells; NT, neuraminidase-treated cells; NA, no agglutination

Fig. 5Histogram showing response of human peripheral blood mononuclear cells (PBMC) to the lectins using con A as positive

control [Relative mitogenic stimulation was calculated at optimum

dose i.e., 10 µg/ml and is an average of quadruplicate cell cultures.]

INDIAN J. BIOCHEM. BIOPHYS., VOL. 42, FEBRUARY 2005

40

proteins, having some important physiological role,

which needs further investigation. The specificity of

AIL towards LacNAc suggests that it could find use

as prognostic marker of cancer. Further, as some

monocot lectins exhibit insecticidal activity12,13

, AIL

and AWL might also show the insecticidal potential.

Acknowledgement One of us (MK) acknowledges of Council of

Scientific and Industrial Research, N. Delhi for award

of Junior Research Fellowship.

References 1 Goldstein I J & Poretz R D (1986) in The Lectins:

Properties, Functions and Applications in Biology and

Medicine (Liener I E, Sharon N, Goldstein I J eds), pp. 33-

248, Academic Press, New York

2 Stinissen H M & Peumans W J (1985) Biochem Physiol Plant 180, 85-106

3 Van Damme E J M, Goldstein I J & Peumans W J (1991) Phytochemistry 30, 509-514

4 Van Damme E J M, Goldstein I J, Vercammen G, Vuylsteke J & Peumans W J (1992) Physiol Plant 86, 245-252

5 Cammue B P A, Peeters B & Peumans W J (1986) Planta 169, 583-588

6 Van Damme E J M, Allen A K & Peumans W J (1987) Plant Physiol 85, 566-569

7 Saito K, Komae A, Kakuta A, Van Damme E J M & Peumans W J, Goldstein I J & Misaki A (1993) Eur J

Biochem 217, 677-681

8 Shibuya N, Berry J E & Goldstein I J (1998) Arch Biochem Biophys 267, 676-680

9 Heggelund L, Mollnes T E, Ureland T, Christophersen B, Aukrust P & Froland S S (2003) J Acqui Immune Defic

Syndr 32, 354-361

10 Charan R D, Munro M H G, O’Keefe B R, Sowder II R C, McKee T C, Currens M J, Pannell L K & Boyd M R (2000) J

Nat Prod 63, 1170-1174.

11 Pusztai A, Grant G, Spencer R J, Duruid T J, Brown D S, Ewen S W B, Peumans W J, Van Damme E J M & Bardoczs

(1993) J Bacteriol 75, 360-368

12 Gatehouse A M R, Powell K S, Peumans W J, Van Damme E J M & Gatehouse J A (1995) in Lectins:Biomedical

prespectives (A Pusztai & S Bardocz, eds.), pp. 35-37,

Taylor and francis, London

13 Hilder V A, Powell K S, Gatehouse A M R, Gatehouse J A, Gatehouse L N, Shi Y, Hamilton W D O, Merryweather A,

Newell C, Timans J C, Peumans W J, Van Damme E J M &

Boulter D (1995) Transgenic Res 4,18-25

14 Van Damme E J M, Peumans W J, Barre A & Rouge P (1998) Cri Rev Plant Sci 17, 575-692

15 Shangary S, Singh J, Kamboj S S, Kamboj S S, Kamboj K K & Sandhu R S (1995) Phytochemistry 40, 449-455

16 Van Damme E J M, Gossens K, Smeets K, Van L F, Verhaert P & Peumans W J (1995) Plant Physiol 107, 1147-

1158

17 Reisfeld R A, Lewis O J & Williams D E (1962) Nature 145, 281-283

18 Davis B J (1964) Ann N Y Acad Sci 121, 404-427 19 Laemmli U K (1970) Nature 277, 680-685 20 Giulian G G, Moss R L & Greaser M (1984) Anal Biochem

142, 421-436

21 Lowry O H, Rosebrough N J, Farr A L & Randall R J (1951) J Biol Chem 193, 265-275

22 Spiro R G (1996) Methods Enzymol 8, 3-26 23 Kaur N, Singh J & Kamboj S S (2002) Indian J Biochem

Biophys 39, 49-54

24 Mossmann T (1983) J Immunol Methods 65, 55-63 25 Boyum A (1968) Scand J Clin Lab Invest (Suppl 97) 21, 77-

89

26 Monks A, Scudiero D, Skehan P, Shoemaker R, Paull K, Vistica D, Hose C, Langley J, Cronise P, Wolff A V,

Goodrich M G, Campbell H, Mayo J & Boyd M (1991) J Nat

Cancer Inst 83, 757-766

27 Krickeberg H, Mauff G, Mertens T, Plum G & Heitmann K (1990) The ARC-IVIG. Study Group Vox Sang 59, 38-43

28 Green E D, Adelt G, Baenziger J U, Wilson S & Van H H (1998) J Biol Chem 263, 18253-18268

29 Hayes C E & Goldstein I J (1974) J Biol Chem 249, 1904-1914

30 Nelson D L & Cox M N (2001) in Lehninger Principles of Biochemistry, pp. 159-202, Macmillon Worth Publishers

31 Kilpatrick D C, Peumans W J & Van Damme E J M (1990) in Lectins Biology, Biochemistry and Clinical Biochemistry

(J Kocourek & J Freed, eds.), Vol. 7, pp. 259-263, Sigma

Chemicals Co., St. Louis

32 Kilpatrick D C (1991) in Lectin reviews (Kilpatrick D C, Van Driessche E & Bog T C, eds.), pp.69-80, Sigma Chemical

Co., St. Louis.

33 Leathem A J & Brooks S A (1998) in Lectin Methods and Protocols (Rhodes J M & Milton J D, eds.), pp. 3-20, Human

Press, Totowa, New Jersey

Fig. 6Histogram showing percentage inhibition of human cancer cell lines i.e., PC-3, SNB-78, A549, SiHa with AIL at a

conc. of 100 µg/ml [Pacitaxel, 5-fluorouracil and mytomycin C

were used as standards at a dose of 1 × 10-4 M.]