Solubilization of the membrane-bound sialidase from pig brain by treatment with bacterial phosphatidylinositol phospholipase C.
The total pellet from pig forebrain (from which the cytosolic sialidase was completely washed out) was treated with phosphatidylinositol phospholipase C (PIPLC) and centrifuged at high speed. The supernatant contained sialidase and 5'-nucleotidase activities. The greatest liberation of sialidase was obtained after incubation for 20 min with PIPLC at 37 degrees C using pH 6.0 and a ratio between PIPLC (as units) and protein of 1.6. Under these conditions, the release of sialidase, 5'-nucleotidase, and protein was 22, 50, and 18.5%, respectively. On treatment with PIPLC, a purified preparation of pig brain neuronal (synaptosomal) membranes released 28% of its sialidase whereas a purified preparation of pig brain lysosomes did not liberate any sialidase activity. The pH optimum of sialidase present in the supernatant obtained after PIPLC treatment of the total pellet was 4.2, the same as that of the enzyme embedded in the membrane. When this supernatant was subjected to ammonium sulfate fractionation, 88% of its sialidase, having a pH optimum of 4.2, was recovered in the fraction precipitated between 20 and 45% of salt saturation and subsequently dialyzed. Ammonium sulfate treatment caused the appearance of a second sialidase activity, having a pH optimum of 6.6 and behaving on fractionation similarly to the pH 4.2 sialidase. The Km and Vmax values of pH 4.2 and pH 6.6 sialidase were similar (1.48 x 10(-4) and 0.98 x 10(-4) M for Km and 1.6 and 1.4 mU/mg of protein for Vmax, respectively), whereas the stability on standing at 4 degrees C or exposure to freezing and thawing cycles was greater for pH 4.2 sialidase.(ABSTRACT TRUNCATED AT 250 WORDS)
Department of Medical Chemistry and Biochemistry, Medical School, University of Milan, Italy.
A
A
Chiarini
A
A
Fiorilli
C
C
Siniscalco
G
G
Tettamanti
B
B
Venerando
D000818:Animals; D001419:Bacteria / Q000201:enzymology*; D001921:Brain / Q000378:metabolism*; D003600:Cytosol / Q000201:enzymology; D008066:Lipolysis; D009439:Neuraminidase / Q000378:metabolism*; D010447:Peptide Hydrolases / Q000378:metabolism; D010716:Phosphatidylinositols / Q000378:metabolism*; D012995:Solubility; D013552:Swine; D010738:Type C Phospholipases / Q000378:metabolism*
D010716:Phosphatidylinositols; D010738:Type C Phospholipases; D009439:Neuraminidase; D010447:Peptide Hydrolases