Purification and partial characterization of porcine liver alpha-N-acetylgalactosaminidase.

Abstract

a-N-Acetyl-D-galactosaminidase (EC 3.2.1.49)was purified from porcine liver by acid and ammonium sulfate precipitation, followed by successive chromatography on DEAE-cellulose, concanavalin A-Sepharose, DEAEcellulose with ampholyte elution, Sephadex G-150, and hydroxylapatite.The p-nitrophenyl-a-N-acetylgalactosaminide and Forssman hapten hydrolyzing activities were purified 3,300-fold and 19,600-fold, respectively.Native gel electrophoresis with 7% polyacrylamide at pH 8.3 gave two protein bands with p-nitropheny1-a-Nacetylgalactosaminide hydrolyzing activity.The native enzyme and its subunits were estimated to have molecular weights of 102,000 and 52,000, respectively.The kinetic behavior of this enzyme preparation was tested towards different substrates with a-N-acetylgalactosamine and a-galactose residues at the nonreducing end, including p-nitrophenyl-a-N-acetylgalactosaminide, Forssman hapten, N-acetyl-sphingosyl-Forssman pentasaccharide, porcine submaxillary mucin, N-acetylgalactosaminyl-(al-6)-1,2:3,4-di-0-isopropylidenegalactose and 4-methylumbelliferyl-a-galactoside.Apparent K,,, values for these substrates were 2.5 X 2.6 X 2.5 X 2.3 X 4.8 X and 1 X lo-' M, and the V,, values were 14.8,4.2,0.81, 0.42, 1.1, and