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°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 K(m) and V(max) 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 K(m) and 1.6 and 1.4 mU/mg of protein for V(max), respectively), whereas the stability on standing at 4°C or exposure to freezing and thawing cycles was greater for pH 4.2 sialidase. Both enzyme activities could be separated by Sepharose 6B column filtration in a unique fraction that was eluted between catalase and bovine serum albumin. These results suggest that (a) the plasma membrane fraction from pig forebrain contains two sialidases (pH 4.2 and 6.6) that are presumably linked via a glycosylphosphatidylinositol anchor, sensitive to PIPLC action, and (b) pH 6.6 sialidase may be in the membrane under the inhibition of a factor that is liberated by PIPLC treatment and removed during ammonium sulfate fractionation and subsequent dialysis.

solubilization of the membrane-bound sialidase from pig brain by treatment with bacterial phosphatidylinositol phospholipase C / A. Chiarini, A. Fiorilli, C. Siniscalco, G. Tettamanti, B. Venerando. - 55:5(1990), pp. 1576-1584. [10.1111/j.1471-4159.1990.tb04941.x]

solubilization of the membrane-bound sialidase from pig brain by treatment with bacterial phosphatidylinositol phospholipase C

A. Fiorilli
Secondo
;
B. Venerando
Ultimo
1990

Abstract

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°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 K(m) and V(max) 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 K(m) and 1.6 and 1.4 mU/mg of protein for V(max), respectively), whereas the stability on standing at 4°C or exposure to freezing and thawing cycles was greater for pH 4.2 sialidase. Both enzyme activities could be separated by Sepharose 6B column filtration in a unique fraction that was eluted between catalase and bovine serum albumin. These results suggest that (a) the plasma membrane fraction from pig forebrain contains two sialidases (pH 4.2 and 6.6) that are presumably linked via a glycosylphosphatidylinositol anchor, sensitive to PIPLC action, and (b) pH 6.6 sialidase may be in the membrane under the inhibition of a factor that is liberated by PIPLC treatment and removed during ammonium sulfate fractionation and subsequent dialysis.
membrane-bound sialidase; phospholipase C
Settore BIO/10 - Biochimica
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Utilizza questo identificativo per citare o creare un link a questo documento: http://hdl.handle.net/2434/197465
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