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

Purification and biochemical characterization of beta-hexosaminidase from freshwater cnidarian Hydra vulgaris Ind-Pune

“…Further, the immunoblotting experiments using soluble extract suggested that the isoform of Hex in H. vulgaris Ind-Pune is HexB. Unlike several other species studied including humans, where the presence of two to three isoforms of Hex (HexA, HexB, and HexS) are evident [8,34], our results hint at HexB being the major isoform present in this strain of Hydra, though further extensive studies are essential to con rm the presence of other isoforms. Further, a two-step sequential chromatographic method involving phenyl-Sepharose hydrophobic interaction chromatography followed by S-200 gel ltration chromatography resulted in the puri cation of HexB to homogeneity.…”

“…Our laboratory has been elucidating the lysosomal enzymes and their sorting receptors in several species of invertebrates with a long-term goal of delineating evolutionarily conserved mechanisms of lysosomal biogenesis [5,29,34,[42][43][44]. Earlier reports on Hex from our group have shown that the freshwater mussel Unio, Lamellidens marginalis as well as Lamellidens corrianus possesses both HexA and HexB, which are the major forms of Hex isoenzymes characterized in mammals including humans.…”

“…Interestingly, Hex from another marine cnidarian Zoanthid Palythoa caribaeorum showed an apparent molecular mass of 25 kDa [46]. The HexB puri ed from Unio (L. corrianus) had a molecular mass in the range of 55-60 kDa, whose native molecular mass was found to be ~ 130 kDa [34]. In a 10 % native PAE Hydra HexB migrated as a single band with a molecular mass of ~ 130 kDa.…”

“…The pH optima for Hex from other cnidarians such as A. aurita and P. caribaeorum were observed to be 4.0 and 5.0, respectively [45,46]. While HexB from Unio showed maximum activity at pH 4.0, Hex from the soil bacterium Stackebrandtia nassauensis exhibited peak activity at pH 6.0 [34,49]. Hex puri ed from Sf-9 cell cultures of the lepidopteran insect Spodoptera frugiperda showed maximum activity at pH 5.5 [14].…”

“…The authenticity of the puri ed enzyme was con rmed also by in-gel activity staining. After resolving the protein on 7.5% native PAGE, the gel was brie y washed with 50 mM sodium acetate pH 5.0 and incubated with 0.1 mM 4-methylumbelliferyl-β-D-glucosaminide for 10 min at 37°C, and the gel was illuminated with UV light to visualize the in-gel enzyme activity [34].…”