Action of endo-alpha-N-acetyl-D-galactosaminidase on synthetic glycosides including chromogenic substrates.
The synthetic glycosides, p-nitrophenyl- and o-nitrophenyl-2-acetamido-2-deoxy-3-O-beta-D-galactopyranosyl-alpha- D-galactopyranosides, were found to be effective chromogenic substrates for an endo-alpha-N-acetyl-D-galactosaminidase. We did not experience any problems when these substrates were used for the screening of column fractions during the purification of the endoenzyme from Diplococcus pneumoniae culture filtrates. However, it should be pointed out that a combination of exo-beta-galactosidase, capable of cleaving beta 1-->3 linkages, and an exo-alpha-N-acetyl galactosaminidase would also liberate nitrophenol from the above substrates. The enzyme had no action on several other synthetic glycosides tested indicating the strict specificity of this enzyme for the disaccharide Gal beta-->GalNAc linked via an alpha-linkage to the aglycone. The enzyme was inactive when the aglycone was methanol but shows activity against the glycosides of phenol, nitrophenols, serine, and threonine. The use of p-nitrophenyl-2-acetamido-2-deoxy-3-O-beta -D-galactopyranosyl-beta-D-galactopyranoside, which is a competitive inhibitor of the endoenzyme, as an affinity ligand for the purification of the enzyme is described.
Umemoto
J
J
Department of Biological Chemistry, Milton S. Hershey Medical Center, Pennsylvania State University, Hershey 17033, USA.
Matta
K L
KL
Barlow
J J
JJ
Bhavanandan
V P
VP
D000345:Affinity Labels; D002846:Chromatography, Affinity / Q000379:methods; D002863:Chromogenic Compounds; D006027:Glycosides / Q000138:chemical synthesis / Q000737:chemistry; D006596:Hexosaminidases / Q000302:isolation & purification / Q000378:metabolism*; D007700:Kinetics; D008024:Ligands; D013296:Streptococcus pneumoniae / Q000201:enzymology; D013379:Substrate Specificity; D048809:alpha-N-Acetylgalactosaminidase
D000345:Affinity Labels; D002863:Chromogenic Compounds; D006027:Glycosides; D008024:Ligands; D006596:Hexosaminidases; D048809:alpha-N-Acetylgalactosaminidase