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Isolation of a glucosamine binding leguminous lectin with mitogenic activity towards splenocytes and anti-proliferative activity towards tumor cells.

Chan YS, Wong JH, Fang EF, Pan W, Ng TB - PLoS ONE (2012)

Bottom Line: However, drastic reduction of the activity occurred at temperatures above 65 °C.Flow cytometry with Annexin V and propidum iodide staining indicated apoptosis of MCF7 cells.Western blotting revealed that the lectin-induced apoptosis involved ER stress and unfolded protein response.

View Article: PubMed Central - PubMed

Affiliation: School of Biomedical Sciences, The Chinese University of Hong Kong, Hong Kong.

ABSTRACT
A dimeric 64-kDa glucosamine-specific lectin was purified from seeds of Phaseolus vulgaris cv. "brown kidney bean." The simple 2-step purification protocol involved affinity chromatography on Affi-gel blue gel and gel filtration by FPLC on Superdex 75. The lectin was absorbed on Affi-gel blue gel and desorbed using 1M NaCl in the starting buffer. Gel filtration on Superdex 75 yielded a major absorbance peak that gave a single 32-kDa band in SDS-PAGE. Hemagglutinating activity was completely preserved when the ambient temperature was in the range of 20 °C-60 °C. However, drastic reduction of the activity occurred at temperatures above 65 °C. Full hemagglutinating activity of the lectin was observed at an ambient pH of 3 to 12. About 50% activity remained at pH 0-2, and only residual activity was observed at pH 13-14. Hemagglutinating activity of the lectin was inhibited by glucosamine. The brown kidney bean lectin elicited maximum mitogenic activity toward murine splenocytes at 2.5 µM. The mitogenic activity was nearly completely eliminated in the presence of 250 mM glucosamine. The lectin also increased mRNA expression of the cytokines IL-2, TNF-α and IFN-γ. The lectin exhibited antiproliferative activity toward human breast cancer (MCF7) cells, hepatoma (HepG2) cells and nasopharyngeal carcinoma (CNE1 and CNE2) cells with IC(50) of 5.12 µM, 32.85 µM, 3.12 µM and 40.12 µM respectively after treatment for 24 hours. Flow cytometry with Annexin V and propidum iodide staining indicated apoptosis of MCF7 cells. Hoechst 33342 staining also indicated formation of apoptotic bodies in MCF7 cells after exposure to brown kidney bean lectin. Western blotting revealed that the lectin-induced apoptosis involved ER stress and unfolded protein response.

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Profile of elution of bean extract from (A) Affi-gel blue gel and (B) Superdex 75.
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pone-0038961-g001: Profile of elution of bean extract from (A) Affi-gel blue gel and (B) Superdex 75.

Mentions: A 2-step purification enabled acquisition of brown kidney bean lectin (BKBL) with satisfactory purity from a crude extract of the beans. This involved collection of the fraction (fraction II in Fig. 1A) adsorbed on Affi-gel blue gel, followed by the major peak (fraction III in Fig. 1B) from Superdex FPLC-gel filtration column. The first chromatographic step on Affi-gel blue gel helped to remove most of the impurities in the crude extract, allowing BKBL to remain as the major protein in the adsorbed fraction (Fig. 2B). This step led to purification of BKBL with about 13.3 folds (Table 2). The second step, FPLC-gel filtration on Superdex 75 10/300 GL column, helped to remove the remaining impurities to yield purified BKBL (Fig. 2C). The 2-step purification protocol facilitated simple and efficient purification of BKBL with a high yield and protein recovery.


Isolation of a glucosamine binding leguminous lectin with mitogenic activity towards splenocytes and anti-proliferative activity towards tumor cells.

Chan YS, Wong JH, Fang EF, Pan W, Ng TB - PLoS ONE (2012)

Profile of elution of bean extract from (A) Affi-gel blue gel and (B) Superdex 75.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3375228&req=5

pone-0038961-g001: Profile of elution of bean extract from (A) Affi-gel blue gel and (B) Superdex 75.
Mentions: A 2-step purification enabled acquisition of brown kidney bean lectin (BKBL) with satisfactory purity from a crude extract of the beans. This involved collection of the fraction (fraction II in Fig. 1A) adsorbed on Affi-gel blue gel, followed by the major peak (fraction III in Fig. 1B) from Superdex FPLC-gel filtration column. The first chromatographic step on Affi-gel blue gel helped to remove most of the impurities in the crude extract, allowing BKBL to remain as the major protein in the adsorbed fraction (Fig. 2B). This step led to purification of BKBL with about 13.3 folds (Table 2). The second step, FPLC-gel filtration on Superdex 75 10/300 GL column, helped to remove the remaining impurities to yield purified BKBL (Fig. 2C). The 2-step purification protocol facilitated simple and efficient purification of BKBL with a high yield and protein recovery.

Bottom Line: However, drastic reduction of the activity occurred at temperatures above 65 °C.Flow cytometry with Annexin V and propidum iodide staining indicated apoptosis of MCF7 cells.Western blotting revealed that the lectin-induced apoptosis involved ER stress and unfolded protein response.

View Article: PubMed Central - PubMed

Affiliation: School of Biomedical Sciences, The Chinese University of Hong Kong, Hong Kong.

ABSTRACT
A dimeric 64-kDa glucosamine-specific lectin was purified from seeds of Phaseolus vulgaris cv. "brown kidney bean." The simple 2-step purification protocol involved affinity chromatography on Affi-gel blue gel and gel filtration by FPLC on Superdex 75. The lectin was absorbed on Affi-gel blue gel and desorbed using 1M NaCl in the starting buffer. Gel filtration on Superdex 75 yielded a major absorbance peak that gave a single 32-kDa band in SDS-PAGE. Hemagglutinating activity was completely preserved when the ambient temperature was in the range of 20 °C-60 °C. However, drastic reduction of the activity occurred at temperatures above 65 °C. Full hemagglutinating activity of the lectin was observed at an ambient pH of 3 to 12. About 50% activity remained at pH 0-2, and only residual activity was observed at pH 13-14. Hemagglutinating activity of the lectin was inhibited by glucosamine. The brown kidney bean lectin elicited maximum mitogenic activity toward murine splenocytes at 2.5 µM. The mitogenic activity was nearly completely eliminated in the presence of 250 mM glucosamine. The lectin also increased mRNA expression of the cytokines IL-2, TNF-α and IFN-γ. The lectin exhibited antiproliferative activity toward human breast cancer (MCF7) cells, hepatoma (HepG2) cells and nasopharyngeal carcinoma (CNE1 and CNE2) cells with IC(50) of 5.12 µM, 32.85 µM, 3.12 µM and 40.12 µM respectively after treatment for 24 hours. Flow cytometry with Annexin V and propidum iodide staining indicated apoptosis of MCF7 cells. Hoechst 33342 staining also indicated formation of apoptotic bodies in MCF7 cells after exposure to brown kidney bean lectin. Western blotting revealed that the lectin-induced apoptosis involved ER stress and unfolded protein response.

Show MeSH
Related in: MedlinePlus