Kinetics of rat liver GM2 ganglioside-β-D-N-acetylhexosaminidase

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Abstract
The kinetics of β-D-N-acetylhexosaminidase against GM2 ganglioside were examined. We used a crude preparation of rat liver as the enzyme source because purification of β-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 μM the Vmax was 6 pmol GM2 hydrolyzed per hour per micromole 4-MU-GlcNAc hydrolyzed per hour (pmol GM2/μmol 4-MU-GlcNAc) and the Km was 5 μM. At substrate concentrations greater than 50 μM, the Vmax was 7 pmol GM2/μmol 4-MU-GlcNAc and the Km was 14 μM. The critical micelle concentration of GM2 ganglioside was 20–25 μM as determined by spectral shifts of the dye pinacyanol chloride in association with GM2, and 10–15 μM from electrical conductivity measurements which also showed the end of the monomer–micelle transition to occur at 40–50 μM GM2. The increasing excess of micellar substrate at greater than 50 μM 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/μmol 4-MU-GlcNAc and the Km was 93 μM. The data suggest that ganglioside hydrolysis occurs more readily when the substrate is incorporated into a membrane-like environment.