Purification, characterization and kinetics of beta-N-acetylhexosaminidases A and B from the slug Arion rufus L.
Two beta-N-acetylhexosaminidases have been purified to homogeneity and characterized, from the digestive gland of the slug A. rufus L., showing very high specific activities. Hexosaminidase A (Hex A) was purified 1300-fold with a yield of 12%, and hexosaminidase B (Hex B) was purified 1400-fold with a yield of 20%. Purified Hex A or Hex B run as a single protein band in polyacrylamide gel disc electrophoresis, showing different mobilities. The purified preparations do not show any of the other glycosidase activities present in the crude extract. beta-N-acetylglucosaminidase (GlcNAc-ase) and beta-N-acetylgalactosaminidase (GalNAc-ase) activities are always associated in a single peak for each enzyme form, with constant activity ratio, in all the purification steps, since they are catalyzed by the same enzyme (Hex A or Hex B). The optimal pH for both forms are 4.5 for GlcNAc-ase and 4.0 for GalNAc-ase activity. Hex B shows thermal and pH-stability higher than Hex A. The isoelectric points are 4.5 and 5.5 for A and B forms, respectively. The molecular weight is 150 000 for Hex A and 320 000 for Hex B. The amino acid composition of purified Hex A and B presents some differences concerning particularly Cys, Thr, Ser, Glu and Ile. The ratios Vmax/Km show that GlcNAc-ase is the main activity of both enzyme forms. beta-N-acetylglucosides and beta-N-acetylgalactosides completely compete for a common active site in mixed-substrates experiments. The Ki values are always coincident for GlcNAc-ase and GalNAc-ase activities, using competitive inhibitors (the corresponding lactones). These results strongly suggest that both activities are catalyzed by the same active site in both Hex A and B. Inhibition of the enzyme activities was found with the corresponding lactones, N-acetyl hexosamines, mannose, mannosides, HgCl2 and lead acetate; activation, with ribose, and with some chlorides and sulphates of divalent cations.
D000118:Acetylglucosaminidase / Q000378:metabolism; D000596:Amino Acids / Q000032:analysis; D000818:Animals; D004589:Electrophoresis, Disc; D054818:Hexosaminidase A; D054819:Hexosaminidase B; D006596:Hexosaminidases / Q000302:isolation & purification* / Q000378:metabolism; D007526:Isoelectric Point; D007700:Kinetics; D008970:Molecular Weight; D008974:Mollusca; D048828:beta-N-Acetyl-Galactosaminidase; D001619:beta-N-Acetylhexosaminidases
D000596:Amino Acids; D006596:Hexosaminidases; D000118:Acetylglucosaminidase; D054818:Hexosaminidase A; D054819:Hexosaminidase B; D001619:beta-N-Acetylhexosaminidases; D048828:beta-N-Acetyl-Galactosaminidase