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Purification and Characterization of Blood Group A-degrading Isoforms of α-N-Acetylgalactosaminidase from Ruminococcus torques Strain IX-70
To cleave blood group A immunodeterminants from erythrocytes (Hoskins, L. C., Larson, G., and Naff, G. B. (1995) Transfusion 35, 813-821), we purified and characterized α- N -acetylgalactosaminidase (EC 3.2.1.49) activity from culture supernatants of the human fecal bacterium Ruminococcus torques s...
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Published in: | The Journal of biological chemistry 1997-03, Vol.272 (12), p.7932 |
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Main Authors: | , , |
Format: | Article |
Language: | English |
Online Access: | Get full text |
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Summary: | To cleave blood group A immunodeterminants from erythrocytes (Hoskins, L. C., Larson, G., and Naff, G. B. (1995) Transfusion 35, 813-821), we purified and characterized α- N -acetylgalactosaminidase (EC 3.2.1.49) activity from culture supernatants of the human fecal bacterium Ruminococcus torques strain IX-70. Three isoforms separated during hydrophobic interaction chromatography. Hydroxyapatite chromatography further
resolved the most hydrophilic, isoform I, into isoforms IA and IB. The most hydrophobic, isoform III, differed from IA and
IB by a more acidic pH optimum, greater heat resistance, greater sensitivity to alkylating agents, and anomalous retardation
during gel filtration chromatography. Isoform IB differed from IA and III in N-terminal amino acid sequence and in sensitivity
to EDTA inhibition. Each cleaved nonreducing α(1â3)- N -acetylgalactosamine residues from human blood group A and AB mucin glycoproteins, Forssman hapten, and blood group A lacto
series glycolipids. The apparent molecular mass of denatured isoform subunits of IA, IB, and III-PII (158, 173, and 201 kDa,
respectively) bore no integer relationship to the apparent molecular mass of the native isoforms (265, 417, and 530 kDa),
but the latter bore a ratio of 1.96:3.09:3.93 to the weight-average apparent molecular mass of native IA (135 kDa), suggesting
that the isoforms are multimers of a 135-kDa sequence. Isoforms IA and III-PII had an identical N-terminal amino acid sequence
which showed homologies to the N-terminal sequence of sialidases produced by Bacteroides fragilis SBT3182, another commensal enteric bacterium. |
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ISSN: | 0021-9258 1083-351X |
DOI: | 10.1074/jbc.272.12.7932 |