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 ∼ 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 60 C respectively. Kinetic parameters K and V estimated with p-nitrophenyl α-mannopyranoside were 0.85 mM and 12.1 U/mg respectively. Swainsonine inhibits the enzyme activity (IC value was 50 nM). Secondary structural analysis at far UV (190-300 nm) showed 11.6 % α-helix and 36.5 % β-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.