title: Isolation, purification, and biochemical characterization of two forms of lysosomal β-N-acetylhexosaminidase from the invertebrate Unio.
abstract: Lysosomal hexosaminidases are glycosyl hydrolases that remove the terminal hexosamine residues of glycoconjugates. Though mammalian hexosaminidases are well characterized, the biochemical nature of these enzymes among invertebrates remains elusive. In this study, we purified two thermostable N-acetyl β-D-hexosaminidases (hex A and B) to homogeneity from soluble extracts of whole Unio animal tissue by a combination of chromatographic procedures. Purified hex A and hex B migrated as a single protein species on native PAGE and exhibited enzyme activity. However on SDS-PAGE, hex A dissociated into two subunits of molecular masses about 75 kDa and 30 kDa respectively, while hex B showed a molecular mass of 40 kDa. Hex A and B were recognized by the affinity purified mannose 6-phosphate receptor 46 on ligand blot analysis. This specific interaction was similar to what is known for the vertebrate receptors and lysosomal enzymes. The enzymes showed different K(M) values with respect to the substrates p-nitrophenyl N-acetyl-β-D-glucosaminide and p-nitrophenyl N-acetyl-β-D-galactosaminide. The enzymes were thermally stable up to 80 °C and showed pH optima between 5.0 and 6.0. This is the first report on the purification of two forms of hexosaminidases from Unio.
authors.affiliation: Protein Biochemistry and Glycobiology Laboratory, Department of Biochemistry, University of Hyderabad, India.
authors.forename: Ashapogu
authors.initials: A
authors.lastname: Venugopal
authors.forename: Nadimpalli
authors.initials: N
authors.lastname: Sivakumar
mesh_terms: D000818:Animals; D054818:Hexosaminidase A / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism*; D054819:Hexosaminidase B / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008247:Lysosomes / Q000201:enzymology*; D008360:Mannosephosphates / Q000378:metabolism; D012995:Solubility; D013696:Temperature; D049881:Unio / Q000166:cytology* / Q000201:enzymology*
chemical_list: D008360:Mannosephosphates; C027693:mannose-6-phosphate; D054818:Hexosaminidase A; D054819:Hexosaminidase B