Kinetics of rat liver GM2 ganglioside-beta-D-N-acetylhexosaminidase.
The kinetics of beta-D-N-acetylhexosaminidase against GM2 ganglioside were examined. We used a crude preparation of rat liver as the enzyme source because purification of beta-D-N-acetylhexosaminidase results in a decrease in specific activity against GM2 ganglioside. Kinetic plots were not linear but showed a break. At substrate concentrations less than 50 microM the Vmax was 6 pmol GM2 hydrolyzed per hour per micromole 4-MU-GlcNAc hydrolyzed per hour (pmol GM2/mumol 4-MU-GlcNAc) and the Km was 5 microM. At substrate concentrations greater than 50 microM, the Vmax was 7 pmol GM2/mumol 4-MU-GlcNAc and the Km was 14 microM. The critical micelle concentration of GM2 ganglioside was 20-25 microM as determined by spectral shifts of the dye pinacyanol chloride in association with GM2, and 10-15 microM from electrical conductivity measurements which also showed the end of the monomer-micelle transition to occur at 40-50 microM GM2. The increasing excess of micellar substrate at greater than 50 microM GM2 explains the discontinuity in the kinetic plots. Sodium taurocholate had a critical micelle concentration of 9-11 mM using pinacyanol chloride and 2.5-3 mM using electrical conductivity. When included in the assay mixture at a concentration of 10 mM, sodium taurocholate produced a linear kinetic plot. This is probably due to the formation of mixed micelles of detergent and GM2 ganglioside. The Vmax was 200 pmol GM2/MUmol 4-MU-GlcNAc and the Km was 93 microM. The data suggest that ganglioside hydrolysis occurs more readily when the substrate is incorporated into a membrane-like environment.
D000818:Animals; D005678:G(M2) Ganglioside; D005732:Gangliosides; D006596:Hexosaminidases / Q000378:metabolism*; D007700:Kinetics; D008099:Liver / Q000201:enzymology*; D008297:Male; D008823:Micelles; D051381:Rats; D013379:Substrate Specificity; D013656:Taurocholic Acid; D001619:beta-N-Acetylhexosaminidases
D005732:Gangliosides; D008823:Micelles; D005678:G(M2) Ganglioside; D013656:Taurocholic Acid; D006596:Hexosaminidases; D001619:beta-N-Acetylhexosaminidases