Purification and characterization of sea squirt alpha-N-acetylgalactosaminidase.
Sea squirt alpha-N-acetylgalactosaminidase was purified to homogeneity. Its molecular weight was estimated to be approximately 160,000 by gel filtration and 40,000 by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) under reducing condition. The chromatographic and electrophoretic behaviors indicated that the enzyme was composed of four subunits. The optimum pH of the enzyme reaction was about 4.0 at 37 degrees C, while the enzyme was stable in the range of pH 5.0 to 6.0 during 4 h preincubation at 37 degrees C. Although the enzyme (0.1 unit) was stable at 0 degrees C for 30 min in the presence of 7.5 mM metal ions (Al3+, Ba2+, Ca2+, K+, Mn2+, Pb2+, Sr2+, and Zn2+), almost 40% of the enzyme activity was lost in the presence of Cu2+, Hg2+, monoiodoacetic acid, and EDTA. The enzyme hydrolyzed aryl N-acetyl-alpha-D-galactosaminide as well as GalNAcalpha1(-->4GalNAcalpha1-->)n 4GalNAc-p-aminobenzoic acid ethyl ester (ABEE) (n = 1-4), but GalNAcalpha1-->4GalNAc-ABEE only scarcely. Furthermore, an allergenic pentasaccharitol ABEE derivative, GalNAcalpha1-->2Fucalpha1-->3(GalNAcbeta1-->4) GlcNAcbeta1-->2(3-acetoamido-3-deoxy)L-threose-ABEE, the minimum structural unit for the sea squirt allergenicity was hydrolyzed to 95 mol% for 72 h incubation with the enzyme. The enzyme could be utilized as a powerful tool for the structural analyses of the carbohydrate epitopes of the sea squirt allergen molecules.
Shigeta
S
S
Department of Molecular Biotechnology, Graduate School of Advanced Sciences of Matter, Hiroshima University, 1-4-1 Kagamiyama, Higashi-Hiroshima 739-8527, Japan.
Suzuki
O
O
Aki
Y
Y
Kawamoto
S
S
Ono
K
K