Characterization of human liver alpha-D-mannosidase purified by affinity chromatography.
Human liver acidic alpha-D-mannosidase was purified 1400-fold by a relatively short procedure incorporating chromatography on concanavalin A-Sepharose and affinity chromatography on Sepharose 4B-epsilon-aminohexanoylmannosylamine. In contrast with the acidic enzymic activity the neutral alpha-mannosidase did not bind to the concanavalin A-Sepharose so the two types of alpha-mannosidase could be separated at an early stage in the purification. The only significant glycosidase contaminant after affinity chromatography on the mannosylamine ligand was alpha-L-fucosidase, which was selectively removed by affinity chromatography on the corresponding fucosylamine ligand. The final preparation was free of other glycosidase activities. The pI of the purified enzyme was increased from 6.0 to 6.45 on treatment with neuraminidase. Although the pI and the mol.wt. (220 000) suggested that alpha-mannosidase A had been purified selectively, ion-exchange chromatography on DEAE-cellulose indicated that the preparation consisted predominantly of alpha-mannosidase B. This discrepancy is discussed in relation to the basis of the multiple forms of human alpha-mannosidase. The purified enzyme completely removed the alpha-linked non-reducing terminal mannose from a trisaccharide isolated from the urine of a patient with mannosidosis. A comparison of the activity of the pure enzyme towards the natural substrate and synthetic substrates suggests that the same enzymic activity is responsible for hydrolysing all the substrates. These results validate the use of synthetic substrates for determining the mannosidosis genotype. They are also further evidence that mannosidosis is a lysosomal storage disease resulting from a deficiency of acidic alpha-mannosidase.
D004186:Disaccharidases / Q000302:isolation & purification*; D004492:Edetic Acid / Q000494:pharmacology; D006801:Humans; D007525:Isoelectric Focusing; D008099:Liver / Q000201:enzymology*; D008361:Mannosidases / Q000302:isolation & purification* / Q000378:metabolism; D008970:Molecular Weight; D014312:Trisaccharides / Q000378:metabolism; D015032:Zinc / Q000494:pharmacology
D014312:Trisaccharides; D004492:Edetic Acid; D004186:Disaccharidases; D008361:Mannosidases; D015032:Zinc