Monomer‐Micelle Transition of the Ganglioside GM1 and the Hydrolysis by Clostridium perfringens Neuraminidase

Publisher Website
Google Scholar
Abstract
The action of C. perfringens neuraminidase (EC 3.2.1.18) on the [rat brain] ganglioside GM1 tritiated in the ceramide moiety was studied. The rates of hydrolysis of the GM1 ganglioside were determined from radioactivity in the neutral glycolipid product, which was separated from the substrate on DEAE-Sephadex columns. To study the physical state of the substrate in the conditions used in the neuraminidase treatment, the critical micelle concentrations (CMC) of the GM1 ganglioside were determined using formation of the triiodide anion in aqueous iodine solution as an indicator. The CMC were also obtained by determining the non-sedimenting radioactivity at different concentrations of the labeled ganglioside per total volume used in ultracentrifugation experiments. The concentrations of the monomeric ganglioside were concluded from the ultracentrifugation results. The increase in the reaction rate of the GM1 hydrolysis as the function of the substrate concentration was leveled off at 25-28 .mu.M ganglioside. The abrupt change at this concentration is interpreted as reflecting the monomer-micelle transition of the ganglioside in the conditions used (50 mM sodium acetate buffer, pH 4.6). The CMC was 29 .mu.M on the basis of the triiodide test, and ultracentrifugation revealed the CMC 28 .mu.M. The reaction velocity of the hydrolysis was decreased immediately above the CMC, and became constant at higher concentrations of the ganglioside. A close correlation to these changes in the reaction rate may exist in the concentrations of the monomeric GM1 ganglioside. Saturation of the buffer used in the neuraminidase assays with butanol effected a striking change in the plot of reaction rate vs ganglioside concentration. The reaction rate increased up to 100-110 .mu.M GM1 ganglioside. The shift of the inflexion point in the rate plot from 25-28 .mu.M to 100-110 .mu.M ganglioside concentration may be due to a respective change in the CMC effected by butanol. N-Acetylneuraminyllactosyl ceramide, lactosyl ceramide and asialo-GM1 ganglioside had an inhibitory effect on the reaction. In contrast, N-acetylneuraminyllactose, lactose and some other free saccharides were not inhibitory. Factors other than the saccharide structure must be taken into account when substrate specificity of a glycosidase is studied using competition experiments. The inhibition effected by the glycolipids may be due to an increase in the micellar state of the GM1 ganglioside.