TY - JOUR
T1 - Presence of two endo-β-N-acetylglucosaminidases in human kidney
AU - DeGasperi, R.
AU - Li, Y. T.
AU - Li, S. C.
PY - 1989
Y1 - 1989
N2 - We have isolated for the first time two kinds of endo-β-N-acetylglucosaminidases (E-β-GNases) simultaneously from human kidney. E-β-GNase 1 was purified by water extraction, ammonium sulfate fractionation, and chromatography on Sephadex-G-200, DEAE-Sephadex, concanavalin A-Sepharose and Hypatite C columns. After the DEAE-Sephadex step, 107 units of E-β-GNase 1 with a specific activity of 0.53 units/mg was obtained and after hydroxyapatite column, the enzyme recovery was 26 units with a specific activity of 10.4 units/mg. This enzyme hydrolyzed the high mannose-type asparaginylglycopeptide efficiently and had little activity toward the complex-type glycopeptide. This enzyme had an pH optimum at about 4.5 and was not inhibited by acetate ion. The Asn residue in a glycopeptide appeared not be an important recognition site for E-β-GNase 1 to express its activity because the acetylation or the dansylation of Asn residues as well as the elimination of Asn residue from the glycopeptide did not change the susceptibility of the oligosaccharide to E-β-GNase 1. E-β-GNase 2 was purified by water extraction, ammonium sulfate fractions, and chromatography on Sephadex G-200, DEAE-Sephadex, concanavalin A-Sepharose, and Mono S columsn. This enzyme was purified about 110-fold with 6.6% recovery. E-β-GNase 2 was found to be a novel type of E-β-GNase that hydrolyzed both the high mannose-type and the complex-type oligosaccharide with chitobiosyl group at the reducing end and without the Asn. E-β-GNase 2 activity was found to be dependent on a L-aspartamido-β-D-N-acetylglucosamine amidohydrolase (Asn-GNase) for the hydrolysis of asparaginylglycopeptide. Asn-GNase cleaved off the Asn reisdue from the glycopeptide, and the resulting oligosaccharide was hydrolyzed by E-β-GNase 2. Because the acetylation or the dansylation of Asn residue in a glycopeptide rendered the glycopeptide resistant to Asn-GNase, the use of the modified asparaginylglycopeptide could not reveal the existence of E-β-GNase 2 activity. The pH optimum of E-β-GNase was found to be about 3.5. Like β-hexosaminidases, this enzyme was inhibited by acetate ion, suggesting the recognition of GlcNAc moiety by this enzyme.
AB - We have isolated for the first time two kinds of endo-β-N-acetylglucosaminidases (E-β-GNases) simultaneously from human kidney. E-β-GNase 1 was purified by water extraction, ammonium sulfate fractionation, and chromatography on Sephadex-G-200, DEAE-Sephadex, concanavalin A-Sepharose and Hypatite C columns. After the DEAE-Sephadex step, 107 units of E-β-GNase 1 with a specific activity of 0.53 units/mg was obtained and after hydroxyapatite column, the enzyme recovery was 26 units with a specific activity of 10.4 units/mg. This enzyme hydrolyzed the high mannose-type asparaginylglycopeptide efficiently and had little activity toward the complex-type glycopeptide. This enzyme had an pH optimum at about 4.5 and was not inhibited by acetate ion. The Asn residue in a glycopeptide appeared not be an important recognition site for E-β-GNase 1 to express its activity because the acetylation or the dansylation of Asn residues as well as the elimination of Asn residue from the glycopeptide did not change the susceptibility of the oligosaccharide to E-β-GNase 1. E-β-GNase 2 was purified by water extraction, ammonium sulfate fractions, and chromatography on Sephadex G-200, DEAE-Sephadex, concanavalin A-Sepharose, and Mono S columsn. This enzyme was purified about 110-fold with 6.6% recovery. E-β-GNase 2 was found to be a novel type of E-β-GNase that hydrolyzed both the high mannose-type and the complex-type oligosaccharide with chitobiosyl group at the reducing end and without the Asn. E-β-GNase 2 activity was found to be dependent on a L-aspartamido-β-D-N-acetylglucosamine amidohydrolase (Asn-GNase) for the hydrolysis of asparaginylglycopeptide. Asn-GNase cleaved off the Asn reisdue from the glycopeptide, and the resulting oligosaccharide was hydrolyzed by E-β-GNase 2. Because the acetylation or the dansylation of Asn residue in a glycopeptide rendered the glycopeptide resistant to Asn-GNase, the use of the modified asparaginylglycopeptide could not reveal the existence of E-β-GNase 2 activity. The pH optimum of E-β-GNase was found to be about 3.5. Like β-hexosaminidases, this enzyme was inhibited by acetate ion, suggesting the recognition of GlcNAc moiety by this enzyme.
UR - http://www.scopus.com/inward/record.url?scp=0024334141&partnerID=8YFLogxK
M3 - Article
C2 - 2498329
AN - SCOPUS:0024334141
SN - 0021-9258
VL - 264
SP - 9329
EP - 9334
JO - Journal of Biological Chemistry
JF - Journal of Biological Chemistry
IS - 16
ER -