Purification and characterization of alpha-N-acetylgalactosaminidase from skipjack liver.
By means of a simple procedure involving two gel filtrations and an ion-exchange chromatography, alpha-N-acetylgalactosaminidase was purified to an electrophoretically homogeneous form from skipjack liver, in which the enzyme is the dominant glycosidase. The final alpha-N-acetylgalactosaminidase preparation contained practically no other glycosidase activities except alpha-galactosidase activity, which amounted to 0.8% of the alpha-N-acetylgalactosaminidase activity and may be an intrinsic activity of the enzyme. The molecular weight of the enzyme was estimated to be 80,000 at pH 4.2 and 40,000 at pH 7.2 by molecular sieve chromatography, and to be 35,000 by SDS-polyacrylamide gel electrophoresis. The enzyme was most active at pH 4 and was inactive above pH 7. These results suggest that skipjack alpha-N-acetylgalactosaminidase exists as an active dimer at acidic pH and as inactive monomer at neutral or alkaline pH. The enzyme efficiently liberated the N-acetylgalactosamine unit from ovine submaxillary glycoprotein which had been desialylated by neuraminidase. The Km value and maximum velocity were 4.28 mM and 409 mumol/min X mg for p-nitrophenyl alpha-N-acetylgalactosaminide, and 0.0543 mM and 1.19 mumol/min X mg for ovine submaxillary asialoglycoprotein.
D000096:Acetone; D000818:Animals; D001212:Asialoglycoproteins / Q000378:metabolism; D002850:Chromatography, Gel; D005399:Fishes; D006596:Hexosaminidases / Q000302:isolation & purification* / Q000378:metabolism; D006358:Hot Temperature; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008099:Liver / Q000201:enzymology*; D008970:Molecular Weight; D013379:Substrate Specificity; D048809:alpha-N-Acetylgalactosaminidase
D001212:Asialoglycoproteins; D000096:Acetone; D006596:Hexosaminidases; D048809:alpha-N-Acetylgalactosaminidase