Modulation of neuraminidase activity by the physical state of phospholipid bilayers containing gangliosides Gd1a and Gt1b.
The thermotropic behavior of large unilamellar dipalmitoylphosphatidylcholine vesicles containing the disialoganglioside Gd1a and the trisialoganglioside Gt1b on their outer surface has been studied as a function of the ganglioside molar fraction and Ca2+ concentration by using high-sensitivity differential scanning calorimetry and steady-state fluorescence spectroscopy. These studies indicate that both gangliosides have an ordering effect on the hydrocarbon region of the bilayer and that this effect is enhanced by the presence of Ca2+ ions. The calorimetric experiments also indicate that ganglioside Gt1b has an intrinsic tendency to phase separate into compositional-rich ganglioside domains even in the absence of Ca2+. Ganglioside Gd1a, on the other hand, only phase separates at Ca2+ concentrations equal to or higher than 10 mM. These studies have allowed us to identify and evaluate the factors affecting the rates of hydrolysis of gangliosides by the soluble neuraminidase from Clostridium perfringens. The data presented in this paper indicate that the rates of hydrolysis of membrane-bound gangliosides are correlated to the physical state of the membrane and the state of aggregation of the ganglioside molecules within the lipid bilayer. For membrane-bound gangliosides, maximal activation energies were found at temperatures slightly below the lipid phase transition temperature. The rates of hydrolysis of the soluble substrate sialyllactose or that of the micellar ganglioside is independent of Ca2+ concentration, whereas the rates of hydrolysis of membrane-bound ganglioside are inhibited by Ca2+ especially under conditions in which the clustering effect of Ca2+ is maximal. These studies suggest that the soluble neuraminidases from Clostridium perfringens prefer ganglioside substrates that are dispersed within the membrane and not forming part of largely aggregated clusters.
D002118:Calcium / Q000502:physiology; D002151:Calorimetry; D003016:Clostridium perfringens / Q000201:enzymology; D005454:Fluorescence Polarization; D005732:Gangliosides / Q000502:physiology*; D008051:Lipid Bilayers; D008560:Membrane Fluidity; D009439:Neuraminidase / Q000378:metabolism*; D013696:Temperature
D005732:Gangliosides; D008051:Lipid Bilayers; D009439:Neuraminidase; D002118:Calcium