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Kinetics of rat liver GM 2 ganglioside-β- D -N-acetylhexosaminidase

1980; Canadian Science Publishing; Volume: 58; Issue: 1 Linguagem: Inglês

10.1139/o80-011

0008-4018

Anton Novak, J. A. Lowden,

Carbohydrate Chemistry and Synthesis

The kinetics of β-D-N-acetylhexosaminidase against GM 2 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 GM 2 ganglioside. Kinetic plots were not linear but showed a break. At substrate concentrations less than 50 μM the V max was 6 pmol GM 2 hydrolyzed per hour per micromole 4-MU-GlcNAc hydrolyzed per hour (pmol GM 2 /μmol 4-MU-GlcNAc) and the K m was 5 μM. At substrate concentrations greater than 50 μM, the V max was 7 pmol GM 2 /μmol 4-MU-GlcNAc and the K m was 14 μM. The critical micelle concentration of GM 2 ganglioside was 20–25 μM as determined by spectral shifts of the dye pinacyanol chloride in association with GM 2 , and 10–15 μM from electrical conductivity measurements which also showed the end of the monomer–micelle transition to occur at 40–50 μM GM 2 . The increasing excess of micellar substrate at greater than 50 μM GM 2 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 GM 2 ganglioside. The V max was 200 pmol GM 2 /μmol 4-MU-GlcNAc and the K m was 93 μM. The data suggest that ganglioside hydrolysis occurs more readily when the substrate is incorporated into a membrane-like environment.