Interactions of pig brain cytosolic sialidase with gangliosides. The formation of catalytically inactive enzyme-ganglioside complexes requires homogeneous ganglioside micelles and is a reversible phenomenon.
Cytosolic sialidase A, obtained from pig brain and purified, interacts with ganglioside GT1b giving two catalytically inactive enzyme-ganglioside complexes. Treatment of these complexes with Triton X-100 under given conditions (1% detergent; 1 h at 37 degrees C; 0.1 M acetic acid-sodium acetate buffer, pH 4.8) leads to the liberation of part of the enzyme (about 47%) in a free and fully active form. Reversible inactivation of cytosolic sialidase requires the presence of homogeneous micelles of GT1b or of mixed micelles (for instance Triton X-100 and GT1b) with a high GT1b content. Triton X-100/ganglioside mixed micelles with a molar ratio above 50, as well as small unilamellar vesicles of egg yolk lecithin and GT1b (7-15 mol%), did not inactivate the enzyme at all; on the contrary these forms of ganglioside dispersion behaved as excellent substrates for the enzyme. It is to be concluded that under in vitro conditions the ability of ganglioside to interact with cytosolic sialidase, giving rise to catalytically inactive complexes or to Michaelis-Menten enzyme-substrate complexes, depends on the supramolecular organization of the ganglioside molecules. Arrangements of tightly packed molecules with strong side-side interactions facilitate the formation of complexes with the enzyme; arrangement with separated and loosely interacting molecules facilitates binding at the catalytically active site of the enzyme.
Department of Medical Chemistry and Biochemistry, Medical School, University of Milan, Italy.
B
B
Venerando
A
A
Fiorilli
L
L
Caimi
G
G
Tettamanti
D000818:Animals; D001921:Brain / Q000201:enzymology*; D003102:Colloids; D003600:Cytosol / Q000201:enzymology; D003902:Detergents / Q000494:pharmacology; D005677:G(M1) Ganglioside / Q000378:metabolism; D005732:Gangliosides / Q000378:metabolism* / Q000494:pharmacology; D006923:Hymecromone / Q000031:analogs & derivatives / Q000378:metabolism; D007700:Kinetics; D008823:Micelles; D009439:Neuraminidase / Q000037:antagonists & inhibitors / Q000378:metabolism*; D017830:Octoxynol; D011092:Polyethylene Glycols / Q000494:pharmacology; D013552:Swine
D003102:Colloids; D003902:Detergents; D005732:Gangliosides; D008823:Micelles; C021298:ganglioside, GD1a; D005677:G(M1) Ganglioside; D006923:Hymecromone; D011092:Polyethylene Glycols; C016481:trisialoganglioside GT1; C021224:2'-(4-methylumbelliferyl)-alpha-D-N-acetylneuraminic acid; D017830:Octoxynol; D009439:Neuraminidase