A tuber lectin from Arisaema helleborifolium Schott with anti-insect activity against melon fruit fly, Bactrocera cucurbitae (Coquillett) and anti-cancer effect on human cancer cell lines
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Chemicals and reagents
Fetal calf serum from Sera Lab (GB) and RPMI 1640 from Gibco-BRL, New York, USA, were procured and stored at 4 °C. All sugars/derivatives, glycoproteins, bovine serum albumin, Freund’s complete adjuvant, adriamycin, 5-fluorouracil, and paclitaxel were obtained from Sigma Chemical, St. Louis, USA. Standard molecular weight markers, gel filtration markers and ampholine of pI range 3.0–9.5 were from Amersham Pharmacia, New Jersey, USA. Amino activated silica beads used were from Clifmar, UK. Biogel
Protein and neutral sugar content analyses
Protein concentration in the crude and purified lectin samples was determined by the method of Lowry et al. [13] using bovine serum albumin as standard while neutral sugar content of the purified lectin preparation was estimated by anthrone method [14] using d-glucose as standard.
Polyacrylamide gel electrophoresis of native and denatured lectin
Native polyacrylamide gel electrophoresis (PAGE) was carried out using 7.5% (w/v) gel at pH 4.5 [15] and 10% (w/v) gel at pH 8.3 [16]. SDS–PAGE was performed according to Laemmli [17] using 11% (w/v) separating gel.
Anti-insect activity
Effect of AHL on development of melon fruit fly, B. cucurbitae (Coquillett) along with its effect on the activity of some of the hydrolytic enzymes was studied. Cultures of B. cucurbitae were maintained at 25 ± 2 °C, photoperiod (L10:D14) and 70–80% relative humidity. Diet was prepared according to the method described by Gupta et al. [24].
AHL at 10, 20, 40, 80, and 160 μg/ml was incorporated in artificial diet prepared according to the method described by Srivastava [25]. Diet was poured into
Results and discussion
The present study describes the purification and characterization of an anti-cancer and anti-insect lectin from the tubers of A. helleborifolium. The lectin was purified by affinity chromatography and asialofetuin, which was one of the hemagglutination inhibitor, was used as a ligand. The results of isolation procedure are summarized in Table 1. The unbound fractions in PBS were devoid of any lectin activity indicating that there was complete adsorption of AHL to the column. The lectin was
Acknowledgments
This work was supported by a research grant from the CSIR. First and second authors are recipients of Senior Research Fellowship from CSIR, New Delhi, India. We are also thankful to sophisticated instrumentation research center, Punjab University, Chandigarh to carry out metal ion analysis.
References (35)
- et al.
Toxicon
(2002) - et al.
Biochim. Biophys. Acta
(2002) - et al.
Phytochemistry
(1995) - et al.
Biochem. Biophys. Res. Commun.
(2004) - et al.
J. Biol. Chem.
(1951) Methods Enzymol.
(1966)- et al.
Ann. Biochem.
(1987) - et al.
Biochem. Biophys. Acta
(1971) - et al.
Arch. Biochem. Biophys.
(2004) - et al.
J. Insect Physiol.
(1971)