Momordica charantia seed proteins - Purification, biochemical characterization of a class II α-mannosidase isoenzyme and its interaction with the lectin and protein body membrane.

Momordica charantia seeds contain a galactose specific lectin and mixture of glycosidases. These bind to lectin-affigel at pH 5.0 and are all eluted at pH 8.0. From the mixture, α-mannosidase was separated by gel filtration (purified enzyme Mr âˆ¼ 238 kDa). In native PAGE (silver staining) it showed three bands that stained with methylumbelliferyl substrate (possible isoforms). Ion exchange chromatography separated two isoforms in 0.5 M eluates and one isoform in 1.0 M eluate. In SDS-PAGE it dissociated to Mr ∼70 and 45 kDa subunits, showing antigenic similarity to jack bean enzyme. MALDI analysis confirmed the 70 kDa band to be α-mannosidase with sequence identity to the genomic sequence of Momordica charantia enzyme (score 83, 29 % sequence coverage). The pH, temperature optima were 5.0 and 60o C respectively. Kinetic parameters KM and Vmax estimated with p-nitrophenyl α-mannopyranoside were 0.85 mM and 12.1 U/mg respectively. Swainsonine inhibits the enzyme activity (IC50 value was 50 nM). Secondary structural analysis at far UV (190-300 nm) showed 11.6 % α-helix and 36.5 % ß-sheets. 2.197 mg of the enzyme was found to interact with 3.75 mg of protein body membrane at pH 5.0 and not at pH 8.0 suggesting a pH dependent interaction.

Purification and biochemical/biophysical characterization of two hexosaminidases from the fresh water mussel, Lamellidens corrianus.

Two thermostable isoforms of a hexosaminidase were purified to homogeneity from the soluble extract of fresh water mussel Lamellidens corrianus, employing a variety of chromatographic techniques. Hexosaminidase A (HexA) is a heterodimer with subunit masses of ~80 and 55 kDa. Hexosaminidase B (HexB) is a homodimer with a subunit mass of 55-60 kDa. Circular dichroism spectroscopic studies indicated that both HexA and HexB contain ß-sheet as the major secondary structural component with considerably lower content of α-helix. The temperature and pH optima of both the isoforms were found to be 60 °C and 4.0, respectively. The IC50 values for HexA with N-acetyl-d-galactosamine, N-acetyl-d-glucosamine, d-galactosamine, d-glucosamine, methyl α-d-mannopyranoside and d-mannose are 3.7, 72.8, 307, 216, 244 and 128 mM, respectively, whereas the corresponding IC50 values for HexB were estimated as 5.1, 61, 68, 190, 92 and 133 mM, respectively. Kinetic parameters KM and Vmax for HexA and B with p-nitrophenyl N-acetyl-ß-d-glucosaminide are 4 mM, 0.23 µmol·min-1·mL-1 and 2.86 mM, 0.29 µmol·min-1·mL-1, respectively, and with p-nitrophenyl N-acetyl-ß-d-galactosaminide are 4.5 mM, 0.054 µmol·min-1·mL-1 and 1.4 mM, 0.14 µmol·min-1·mL-1, respectively. GalNAc inhibited both isoforms in a non-competitive manner, whereas a mixed mode of inhibition was observed with GlcNAc with both forms.