Section 7
Chapter 6,459

Specificity of human liver hexosaminidase a ec and hexosaminidase b against glyco sphingo lipids g m2 and g a2 purification of the enzymes by affinity chromatography employing specific elution

Sandhoff, K.; Conzelmann, E.; Nehrkorn, H.

Hoppe Seyler's Zeitschrift fuer Physiologische Chemie 358(7): 779-788


Accession: 006458678

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The hexosaminidases A and B were extracted from human autopsy liver and purified to apparent homogeneity by a simple affinity chromatographic procedure. As ligand for the affinity gel a competitive inhibitor of the isoenzymes was synthesized, a thio derivative of N-acetyl-galactosamine:6-aminohexyl 2-acetamido-2-deoxy-1-thio-.beta.-D-galactopyranoside. The isoenzymes A and B adsorbed to the affinity gel were specifically eluted with 2-acetamido-2-deoxy-D-gluconolactone, a potent inhibitor of the enzymes, and subsequently separated by ion-exchange chromatography. The specificity of the hexosaminidases A and B, purified to apparent homogeneity, towards labeled glycolipids GM2 and GA2 was investigated. The enzymatic degradation of glycosphingolipid GA2 was greatly enhanced by detergents such as sodium taurodeoxycholate above their critical micellar concentration as well as by 2 activator protein preparations. Hexosaminidase B was more active than hexosaminidase A under these conditions. The cleavage of ganglioside GM2, the main storage compound in Tay-Sachs disease, by hexosaminidase A was greatly facilitated by the addition of sodium taurodeoxycholate or one of the activator preparations, whereas hexosaminidase B exhibited only a little activity in the presence of sodium taurodeoxycholate and none at all in the presence of the activator proteins. This specificity indicated a causal relationship between the absence of hexosaminidase A and the storage of ganglioside GM2 in Tay-Sachs disease. The different specificities of the 2 enzymes were even more pronounced in the absence of detergents or activator proteins against a water-soluble ceramide-free derivative of ganglioside GM2, GalNAc.beta.1 .fwdarw. 4Gal-[3 .rarw. 2 .alpha.NeuAc].beta.1 .fwdarw. 4 sorbitol, which was hydrolyzed by hexosaminidase A but not by hexosaminidase B.

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