Purification and characterization of a sea squirt beta-galactosidase.
A beta-galactosidase was extracted from the internal organs of a sea squirt, Styela plicata, and purified 959-fold, with an 18% yield, by successive gel chromatography, anion-exchange chromatography, chromatofocusing, and affinity chromatography on a Con A-Sepharose column. The purified enzyme was fairly homogeneous, as judged on disc PAGE, SDS-PAGE, and gel chromatography on a Sephadex G-200 column. The molecular weight of the enzyme was estimated to be 77,000 and 75,000 by gel chromatography and SDS-PAGE, respectively, and its isoelectric point was determined to be 4.9 by the isoelectric focusing method. The enzyme was substantially stable in the pH range of 3.5 to 7.5, the optimum pH being 4.0. The enzyme was significantly inhibited by 9 mM HgCl2 and 9 mM DFP, while the inhibition by 0.9% PCMB was only 60% at 0 degrees C for 30 min. The purified beta-galactosidase apparently liberated galactose from a sea squirt antigen (H-antigen), two allergenically active glycopeptides (Gp-1 and Gp-2) derived from another sea squirt antigen (Gi-rep), asialo-ovomucoid glycopeptide, asialo-fetuin glycopeptide, GA1, CDH, and an ABEE-derivative (Gal beta 1----3ThrNAc-ABEE) of Gal beta 1----3GalNAc-ol isolated from bovine submaxillary gland mucin.
Shigeta
S
S
Department of Fermentation Technology, Faculty of Engineering, Hiroshima University.
Ono
K
K
Oka
S
S
D000818:Animals; D002240:Carbohydrate Sequence; D006020:Glycopeptides / Q000737:chemistry; D007526:Isoelectric Point; D008969:Molecular Sequence Data; D008970:Molecular Weight; D013379:Substrate Specificity; D014561:Urochordata / Q000201:enzymology*; D001616:beta-Galactosidase / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism
D006020:Glycopeptides; D001616:beta-Galactosidase