1980 Experentia 36, pp.525-527

by S.S., L. Li
Summary. 2 different lectin have been prurified from the seeds of Momordica charantia by gel-filtration and ion-exchange chromatography. These 2…

Purification and Partial Characterization of Two Lectins from Momordica charantia

Author: S.S., L. Li

Type of Publication: Pre-Clinical

Date of Publication: 1980

Publication: Experentia 36, pp.525-527, 1980

Organization: Department of Microbiology, Mount Sinai School of Medicine, New York, Environmental Mutagenesis, NIEHS, NIH Research Triangle Park

Summary. 2 different lectin have been prurified from the seeds of Momordica charantia by gel-filtration and ion-exchange chromatography. These 2 lectins appear to be composed of 2 subunits of 26,000 daltons. Protein fraction I, but not showed agglutinating activity toward human type-O red blood cells. The amino acid composition and amino-terminal sequences of these homologous proteins are quite different.

The fruit of Momordica charantia is widely used in the orient, although the seeds are not eaten. The D-galactose-binding agglutinin from Momordica charantia has been shown to agglutinate human-O red blood cells, but not Yoshida sarcoma cells. Recently, toxic momordin and non-toxic momordica agglutinin have also been separated by Cm-cellulose chromatography, and the momordin inhibits protein biosynthesis of Ehrrlich ascites tumor cells. In this report, 2 lectins have been purified from the seeds of Momordica charantia, and their molecular weights, amino acid composition and aminoterminal sequence of 27 residues have been determined.

Materials and methods. The seeds of Momordica charantia were obtained from the Chan Man Hop Seed Co., Hong Kong. The proteins were isolated as previously described. Haemagglutination assays were performed in microtiter plates with human type-O red blood cells. Polyacrylamide gel electrophoresis in sodium dodecyl sulfate was performed on 12.5% slab gel in Tris-glycine buffer, pH 8.3. The gels were stained for protein with Coomassie brilliant blue and for carbohydrate with periodic acid-Schift reagent.

The proteins were hydrolyzed in 6 HCI at 110^o C for 24, 48 and 72 h, and the hydrolysates were analyzed with automatic amino acid analyzer (Beckman 121). Cystein and/ or half-cysteine were determined as cyteic acid after performic acid oxidation. Although Edman degradation were performed with the Beckman protein sequencer using N, N’-dimethylallylamine buffer and single acid cleayage. Phenylthiohydantoin-amino acids were identified by GLC, TLC, and/ or amino acid analysis after back hydrolysis with 6 N HCI or 56% HI. Phenylthiohydantoin arginine was also identified by the phenanthrene quinone spot test.

Results. The crude protein extract was chromatographed of a column of DEAE-Sephadex (Fig.1a) followed by gel filtration on a Sephadex G-150 column (Fig.1b). The proteins under peak G1 were further separated into fractions I and II on CM-cellulose using a linear gradient of sodium phosphate buffer (Fig.1c). The proteins under peak G2 were shown to be low-molecular-weight storage proteins, and have been described previously.

The proteins from DEAE-A50 and fraction G1 on G-150 had hemagglutinating titers of 1280 and 320, relatively, toward human type-O red blood cells. The titled protein fraction I agglutinated human type-O red blood cells at the concentration of about 2-4 g/ml, while protein fraction II did not show agllutination even at 2-4 g/ml. This hemagglutination could be inhibited by 10 mm D-galactose.

The reduced samples of proteins were run on SDS-polyarcylamide gel electrophoresis, and the gels were stained protein and carbohydrate. Both protein fractions I and showed single protein band corresponding mol. wt. of 1000 daltons (Fig.2). Both proteins were also periodic Schiff positive, indicating the presence of carbohydrate (data are not presented). The mol. wt of proteins after peak G1 (Fig.1b) was estimated to be approximately 49,000 daltons by gel-filtration on a calibrated Sephadex G-150 column. These results indicate that both proteins consist of 2 subunits of approximately 26,000 daltons.

Amino acid composition of both proteins (Table 1) was calculated from the molar ration and the assumed mol. wt of 15,000 daltons . These 2 proteins contain quite different amino acid composition, although they have very simlar mol. wts. The amino acid-terminal sequences of 27 residue of these 2 proteins were deduced from 2 runs of automatic many degradations. The residue identified are summarized in Table 2 and both sequences are compared among the 27 residues compared.

The protein fractions I and II showed very similar nearly absorption spectra with a typical maximum around 80 mm and the ratio of A₂₈₀ to A₂₆₀ being 2.0. These spectra are quite different from those of momordica storage proteins reported previously.

Discussion. 2 D-galactose-binding lectins were purified from Momordica charantia, and only protein fraction I, not hemagglutinated human red blood cells. On the basis of hemagglutinating activities and the behavior on CM-cellulose chromatography, the protein fractions I and II appear to correspond to the previously reported momordica agglutinin and toxic momrdin, respectively. However, the mol. wts and amino acid composition of both momordin proteins, I and II, determined in this study differ from published data. The larger mol. wt. and higher glutanin content of the previously isolated momordica agglutinin might be due to the contaminated momordica storage protein. Momoridca storage protein was eluted from cellulose column at similar position as the case of momordica agglutinin and it has an apparent mol. wt of 55000 daltons and very high content (32-34 moles%) of glutanin acid. It may be noted that both protein fractions I and like the D-galactose binding sophora agglutinin and E, did not bind to sepharose 4B column.

The partial amino acid sequences of both proteins and with their amino acid composition indicated that 2 homologous proteins have very different primary structures. 5 threonine residues are present at position nos. and 24 of protein fraction II while theronine is in the first 27 residues of protein fraction I. It will be interest to define the exact correlation of the structural sequences with their dinstinct agglutinating and toxic properties.

Acknowledgements. The skillful assistance of Ms. M. Diane Forde is greatly acknowledged. Part of this work was supported by NIH grants CA18621 and A109810 when the author was the Mount Sinai, School of Medicine.

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