Bitter Melon Studies » Isolation of Toxic and Non-Toxic Lectins from the Bitter Pear Melon Momordica charantia Linn

Isolation of Toxic and Non-Toxic Lectins from the Bitter Pear Melon Momordica charantia Linn

Filed under All Studies, Pre-Clinical Research
on August 10, 2006 at 12:27 pm

Toxicon Vol.16, pp.653-660

by Jung-Yaw Lin, Mei-Jane Hou, On-Chang Chen
Bitter pear melon, Momordica charantia Linn has been widely used in the Orient as a foodstuff or tonic. We present…

Isolation of Toxic and Non-Toxic Lectins from the Bitter Pear Melon Momordica charantia Linn

Author: Jung-Yaw Lin, Mei-Jane Hou, On-Chang Chen

Type of Publication: Pre-Clinical

Date of Publication:

Publication: Toxicon Vol.16, pp.653-660

Organization: Institute of Biochemistry, College of Medicine, National Taiean University, Taipei, Taiwan

INTRODUCTION

Bitter pear melon, Momordica charantia Linn has been widely used in the Orient as a foodstuff or tonic. We present in this paper methods for purification of toxic and non-toxic lectins from seeds of the bitter pear melon, as well as their chemical and biological properties. We name the pirified toxic lectin, momordin and the non-toxic lectin, momordica agglutinin. These are proteins, extracted from the seeds of the plant, which can agglutinate blood cells.

MATERIALS AND METHODS

Bovine serum albumin and cytochrome C were obtained from Sigma Chemical Co., Saint Louis, Missouri, U.S.A. Chymotrypsinogen and ovalbumin were purchased from Worthington Biochemical Co., Freehold New Jersey, U.S.A. Thymidine-methyl-³H with a specific activity of 6.7 Ci/mmole, uridine-2^-14 C with a specific activity of 55.3 mCi/mmole and L-leucine-4-5^-3H with a specific activity of 5 Ci/mmole were purchased from New England Nuclear Co., Boston, Mass., U.S.A.

The lethality of the toxic lectin was determined by i.p. injection into mice (Swiss White, 18  1 g). After 48 hr observation, the results were recorded.

The hemagglutinating activity of lectin was determined by two fold serial dilution. Lectin solution (0.5 ml) was added to 0.5% human O type red blood cell suspension prepared according to the method described by KABAT and MAYER (1961). After standing at room temperature for 2 hr, the results were recorded.

Gel filtration was carried out with a Sphadex G-100 column (2 x 100 cm), using 0.05 m phosphate buffer (pH 6.5) according to the method of ANDREW (1964).

Electrophoresis experiments were carried out with analytical apparatus Model DPS 200 from Savant Inc., Hicksville, N. Y., U.S.A. Polyacrylamide disc gel electrophoresis was carried out at pH 4.5 on standard 7% gels which were stained with Amidoblack. Polyacrylamide electrophoresis (10% gels) in sodium dodecyl sulfate was performed according to the method of WEBER and OSBORN (1972). Gels were stained with Commassie brilliant blue.

Protein was hydrolyzed at 105⁰ C for 24 hr in sealed, evacuated tubes with 6 N HCI containing 0.01 M 2-mercatoethanol. The hydrolysate were dried with an Evapo-Mix under reduced pressure and analyzed on a Beckman model 120 C amino acid autoanalyzer according to the method of SPECKMAN et al. (1958).

Protein was quantitatively measured by Folin phenol method of Lowry et al. (1951).

Ehrlich ascites tumor cells were used for measurement of protein, RNA and DNA biosynthesis. Five ml of cell suspension (10⁷ cells/ml) was incubated with various concentration of momordin at 37⁰ C for various time periods, and at the end of incubation, 0.2 ml of 10% TCA was added to terminate the reaction (Lin et al., 1970). The radioactivity was determined by a filter paper disc method (Mass and Novelli 1961). Samples were counted with a Nuclear-Chicago Model I liquid scintillation counter, Des Plaines, III. U.S.A.

Lectins were extracted from seeds (400 g) of Momordica charantia Linn with one litre of 0.8 N cold acetic acid containing 0.001 M EDTA, by homogenizing with a Waring blender. After centrifuging the homogenate with a Sorvall RC-2B refrigerated centrifuge at 19,6000 x g 20 min, the proteins in the supernatant were precipitated with 100% saturation of ammonium sulfate by centrifugation. The precipitates were dialyzed against 0.005 M phosphate buffer pH 6.4 at 4⁰ C for 36 hr. After removing the precipitate, which developed during dialysis, by centrifugation, the supernatant was applied on a CM-cellulose column (3.5 x 38 cm), which was pre-equilibrated with 0.005 M phosphate buffer. The column was successively eluted with 0.05 M, and 0.2 m phosphate buffer (pH 6.4). The results are shown in Fig.1. The proteins eluted with 0.1 M phosphate buffer have toxic and hemagglutinating activities and were further purified with the same column. The results are shown in Fig.2. The first peak has a strong hemagglutinating activity while the fourth peak contains a toxic protein.

RESULTS

Purification of lectins

Table 1 shows a typical experiment in which both lectins were purified simultaneously. The minimum amount of momordica agglutinin cause hemagglutination was decreased from 5 to 0.05 g. The _{LD 50} of momordin was decreased from 40 to 5 mg/kg body weight.

Homogenicity

As shown in Fig.3(a and b), momordica agglutinin and momordin each migrate as a single band during disc gel eletrophoresis at pH 4.5 and sodium dodecyl sulfate polyacrylamide gel electrophoresis with or without mercaptoethanol.

Molecular weight

The mol. wt of momordin and momordica agglutinin were determined separated by gel filtration and SDS polyacrylamide gel electrophoresis (Fig.4 and 5). The mol. wt of momordin was found to be 23,000 and 22,000 by sodium dodecyl sulfate polyacrylamide gel electrophoresis and gel filtration respectively while that of momordica agglutini was determined to be 30,000 by both methods.

Amino acid composition

The amino acid composition of momordin and momordica agglutinin are presented in Table2. According to the results of gel filtration and sodium dodecyl sulfate polyacrylamide gel electrophoresis, the molecular weights of momordin and momordica agglutinin were calculated from the results of Table 2 to be 23,678 and 31,762 respectively.

Hemagglutination

The two lectins showed a remarkable difference in their ability to agglutinate human O tyoe red blood cells. At concentration above 0.5 g per ml, momordin produced agglutination but momordica agglutinin caused agglutination at 0.05 g per ml. The agglutination caused by momordica agglutinin was inhibited by D-galactose or those sugars containing D-galactose such as lactose or raffinose. The concentration required for complete inhibition of hemagglutination caused by agglutinin were 10 ^–₃ M D galactose and raffinose and 10^-₄ M lactose.

Biosynthesis of macromolecules

Momordin has an inhibitory effect on the protein biosynthesis of Ehrlich ascites tumor cells whereas it has little effect on DNA and RNA biosynthesis (Fig.6). At a concentration of 500 g/ml, momordin inhibited about 65% of protein biosynthesis, 10% of RNA biosynthesis and had no effect on DNA biosynthesis.

Ehrlich ascites tumor cells are resistant to momordin during the frist 10 min, and then become gradually sensitive to momordin which inhibits about 65% of proteinbiosynthesis of the tumor cells after 40 min incubation (Fig.7).

DISCUSSION

Two lectins, isolated from Momordica charantia Linn, were each found to have a single peptide chain (sodium dodecyl sulfate polyacrylamide gel electrophoresis). These lectins are quite different from those isolated from Abrus precatoriuos and Ricinus communis which were demonstrated to consist of two subunits, namely effectomer and haptomer (Olsnes et al., 1974). The single polypeptide chain of momordin probably has both the binding site and the active site which are respectively responsible for binding to receptors of tumor cells and inhibiting the protein biosynthesis.

The toxic has a moderate inhibitory effect on protein biosynthesis and slight or no effect on RNA and DNA biosynthesis. There is a 10 min lag period for the toxic lectin ricin which are require 5 –10 min to penetrate into reticulocytes and inhibit their protein biosynthesis. Momordica agglutinin causes the the hemagglutination of human O type red blood cells as well as Ehrlich ascites tumor cells; it requires 0.25 g per ml to cause the agglutinination of tumor cells and 0.5 g per ml for red blood cells. The hemagglutination caused by momordica agglutinin by galactose or its derivatives; results which are identical with those reported by Tomita et al.(1972).

Acknowledgements- We thank Miss Mei-Ling Chen, Mr. Lee-Hong Chang and Mr. Hon-Lee Haung for their skillful technical assistance.

REFERENCES

Andrew, P. (1964) Examination of the molecular weights of proteins by Sephadex gel filtration. Biochem. J. 91, 222.

Kabat and Mayer (1961) Experimental Immunochemistry, 2^nd edn., p.114, Springfiled: Charles C. Thomas.

Lin, J. Y., Kao, W. Y. Tserng, K. Y., Cheng, C. C. and Tung, T. C. (1970) Effects of crystalline abrin in the biosynthesis of protein, RNA and DNA in experimental tumors. Cancer Res. 30, 2431.

Lowry, O. H., Rosenbrough, N. J., Farr, A. L. and Randall, R. J. (1951) Protein measurement with the Folin Phenol Reagent. J. biol. Chem 227, 292.

Mans, R. J. and Novelli, G. D. (1961) Measurement of the incorporation of radioactive amino acids into proteins by filter paper disk method. Archs Biochem. Biophys. 94, 48.

Olsnes, S., refsnes, K. and Phil, A. (1974) Mechanism of action of the toxic lectins abrin and ricin. Nature 249, 627.

Speckman, D. H., Stein, W. H. and Moor, S. (1958) Automatic recording apparatus for use in the chromatography of amino acids. Analyt. Chem. 30, 1190.

Tomita, M., Kurokawa, T., Onozaki, K., Ichiki, N., Osawa, T. and Ukita, T. (1972) Purification of galactose-binding phytoagglutinins and phytotoxin by affinity column chromatography using Sepharose. Experienta 28 (1) 84.

Weber, K., Pringle, J. R. and Osborn, M. (1972) Measurement of molecular weight by electrophoresis in SDS-acrylamide gel. In: Methods in Enzymology XXVI pp.3-27 (Hirs, C. H. W. and Tunasgeff, S. N., Eds.).

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Isolation of Toxic and Non-Toxic Lectins from the Bitter Pear Melon Momordica charantia Linn

RESULTS

Purification of lectins

Homogenicity

Molecular weight

Amino acid composition

Hemagglutination

DISCUSSION

REFERENCES

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