A protein activator for the enzymic hydrolysis of GM2 ganglioside.

A protein activator (GMz-activator) specific for stimulating the hydrolysis of G M ~ ganglioside (GalNAcb1 4GalC3 + 2aNeuAc]/?l-+ 4Glcb1-+ 1’Cer) by &hexosaminidase A has been purified over 1OS-fold with a high yield from human liver. The purification procedure includes the adjustment of the pH of liver extract to pH 4.3, followed by ammonium sulfate precipitation, Sephadex G-200 filtration, and column chromatographies on DEAE-Sephadex A-50, Matrex Gel Blue A, and octyl-Sepharose 4B. The physical properties of the GMzactivator are: (a) moderately heat-stable up to 50 “C in crude states, but very unstable in the purified form even at 37 “C; (b) molecular weight, about 23,500; and (c) isoelectric point about 4.8. Chemical analysis identifies this activator as a protein. GMe-activator is very specific for stimulating the hydrolysis of GMz ganglioside, but only slightly effective in stimulating the hydrolysis of asialo-Gmz or globotetraosylceramide catalyzed by p-hexosaminidase A. Unlike bile salts such as sodium taurodeoxycholate, this activator does not stimulate the hydrolysis of the above three glycosphingolipids catalyzed by 8-hexosaminidase B. It does not stimulate the hydrolysis of synthetic substrates such as 4-methylumbelliferyl-/3-GalNAc and p-nitrophenyl-P-GalNAc. The oligosaccharide derived from GMz ganglioside is not hydrolyzed by 8-hexosaminidase A or B in the presence of either GMz-activator or sodium taurodeoxycholate. The rate of the hydrolysis of GMz ganglioside is affected by both the amount of GMz-aCtiVatOr and &hexosaminidase A. The molar ratio of enzyme to GMz-activator for obtaining the maximal hydrolysis of GMz ganglioside is close to 1:1, while the molar ratio of Gnrz ganglioside to GMz-activator is about 300:l. These results may suggest that GM2-activator interacts with &hexosaminidase A rather than the lipid substrate.