Characterization of an activating factor required for hydrolysis of Gm2 ganglioside catalyzed by hexosaminidase A

Abstract

Separation of the hexosaminidase A (EC 3.2.1.52) and B isozymes of human liver by ion-exchange chromatography results in recovery of greater than 80% of the activity in crude extracts when synthetic substrates are used to monitor enzyme activity. Only 15% of hexosaminidase activity toward the N-acetylgalactosaminyl (N-acetylneuraminyl) galactosyl glucosylceramide (Gm2 ganglioside) substrate is recovered and all of this activity is associated with the hexosaminidase A fraction. The low level of Gm2 ganglioside hydrolase activity in the hexosaminidase A fraction could be enhanced by coincubation with column fractions which contain hexosaminidase B. The activating factor, which was partially purified by gel filtration, is a heat-stable protein with a MW of 36,000 and is without enzyme activity toward hexosaminidase substrates. Highly purified hexosaminidase A or crude hexosaminidase A recovered after gel filtration on Sephadex G-100 has no Gm2 ganglioside hydrolase activity. The Gm2 ganglioside hydrolase activity of these hexosaminidase A preparations can be completely restored by addition of the activating factor. The activating factor does not affect the rate of hydrolysis of synthetic substrate or asialo Gm2 ganglioside catalyzed by hexosaminidase A.