Purification and properties of the hexosaminidase A-activating protein from human liver

Abstract

Human liver extracts contain an activating protein which is required for hexosaminidase A[EC 3.2.1.30]-catalyzed hydrolysis of the N-acetylgalactosaminyl linkage of GM2 ganglioside [N-acetylgalactosaminyl-(N-acetylneuraminyl)galactosylglucosylceramide]. A partially purified preparation of human liver hexosaminidase A that is substantially free of GM2 ganglioside hydrolase activity is used to assay the activating protein. The procedures of heat and alcohol denaturation, ion-exchange chromatography and gel filtration were used to purify the activating protein over 100-fold from crude human liver extracts. When the purified activating protein is analyzed by polyacrylamide-gel disc electrophoresis, 2 closely migrating protein bands are seen. When purified activating protein is used to reconstitute the GM2 ganglioside hydrolase activity, the rate of reaction is proportional to the amount of hexosaminidase A used. The activation is specific for GM2 ganglioside and hexosaminidase A. The activating protein did not stimulate hydrolysis of asialo-GM2 ganglioside by either hexosaminidase A or B. Hexosaminidase B did not catalyze hydrolysis of GM2 ganglioside with or without the activator. Kinetic experiments suggest the presence of an enzyme-activator complex. The Kd of this complex is decreased when higher concentrations of substrate are used, suggesting the formation of a ternary complex between enzyme, activator and substrate. Determination of the MW of the activating protein by gel-filtration and sedimentation-velocity methods gave values of 36,000 and 39,000, respectively.