Action of Vibrio cholerae Neuraminidase (Sialidase) upon the Surface of Intact Cells and Their Isolated Sialolipid Components

Norman W Barton,Abraham Rosenberg

doi:10.1016/s0021-9258(19)43296-1

Norman W Barton, Abraham Rosenberg

Open Access

https://doi.org/10.1016/s0021-9258(19)43296-1

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Abstract

Abstract The effect of strong electrolyte upon the activity of Vibrio cholerae neuraminidase (VC neuraminidase) toward the surface of intact cells and toward their isolated, purified sialolipid components, was studied in detail. A linear dependence of the logarithm of initial velocity on the square root of ionic strength was observed for purified GM3 over a concentration range of 0 to 0.05 m NaCl in 0.01 m Tris-acetate, pH 6.5. The rate of decrease of the logarithm of the optimum initial velocity, obtained in 0.01 m Tris-acetate alone, with increasing square root of ionic strength was 3 times more rapid with GM1, GD1a, GD1b, and GT1 as substrate mixture than with the smaller sialolipid, GM3, as substrate. These data are consistent with previous results which indicate that ionic interactions upon the enzyme conformationally govern steric availability of the catalytic site. The effect of strong electrolyte upon the activity of neuraminidase toward the surface of intact cells was small in comparison with that observed with purified sialolipid substrates. Analysis of lipid extracts from untreated control and VC neuraminidase-treated samples by thin layer chromatography revealed no detectable difference between the sialolipid content of untreated control samples and samples treated with VC neuraminidase. These findings suggest that the sialolipid components in the intact cell surface are relatively unavailable to the extracellular action of VC-neuraminidase. This is supported by evidence obtained by detailed analysis of the sialyl components in four cell types (Green monkey kidney cells, Morris hepatoma, B-16 melanoma, and human neuroblastoma) before and after VC neuraminidase treatment. The total cellular sialic acid content of these cell types, untreated, varied from 2.3 to 7.1 µg of sialic acid per mg of protein; lipid-bound sialic acid content varied from 0.7 to 1.6 µg of sialic acid per mg of protein. Progress curves for sialic acid release from the surface of intact cells were hyperbolic and had reached a plateau within approximately 30 min. In the plateau region approximately 20% of the total cellular sialic acid had been released consistently, in each case. Analysis of the lipid-bound sialic acid content of the VC neuraminidase-treated intact cells revealed no major changes, all values being greater than 90% of the untreated, control values. The sialolipid composition of each cell type was examined by thin layer chromatography and found nevertheless to consist predominately of VC neuraminidase-labile components. Therefore, the sialolipids of the intact cell surface appear to be more deeply localized so as to be sterically inaccessible to VC neuraminidase, and release of sialic acid from intact cells by VC neuraminidase appears to be from surface sialoglycoproteins.

Highlights

EJ’ects of Strong Electrolyte on VC Neuraminidase Activity toward Purijed Sialolipid Subsfrates-Strong electrolyte (NaCl) concentration ranged from 0 to 0.05 x Assay mixtures consisted of specified quantities of salt and sialolipid substrate contained in a final volume of 1.00 ml of 0.01 M Tris-acetate, pH 6.5
Linear dependence of the logarithm of initial velocity on the square root of the ionic strength (p) of t.he assay medium was observed over a concentration range of 0 to 0.05 M NaCl in
No detectable difference in the sialolipid content of untreated control samples, as compared with VC neuraminidase-treated samples, was observable over the entire range of strong electrolyte concentrations employed in these studies. These findings suggest that the sialolipids in the intact cell surface are relatively unavailable to the extracellular action of VC neuraminidase

Summary

Methods

EJ’ects of Strong Electrolyte on VC Neuraminidase Activity toward Purijed Sialolipid Subsfrates-Strong electrolyte (NaCl) concentration ranged from 0 to 0.05 x Assay mixtures consisted of specified quantities of salt and sialolipid substrate contained in a final volume of 1.00 ml of 0.01 M Tris-acetate, pH 6.5.To each assay mixture, 5 units of VC neuraminidase were added.Assay mixtures were incubated at 37” for 7 min, which was determined experimentally to be within the duration of initial velocity for these substrates. EJ’ects of Strong Electrolyte on VC Neuraminidase Activity toward Purijed Sialolipid Subsfrates-Strong electrolyte (NaCl) concentration ranged from 0 to 0.05 x Assay mixtures consisted of specified quantities of salt and sialolipid substrate contained in a final volume of 1.00 ml of 0.01 M Tris-acetate, pH 6.5. 5 units of VC neuraminidase were added. Assay mixtures were incubated at 37” for 7 min, which was determined experimentally to be within the duration of initial velocity for these substrates. At the termination of the incubation period, the assay mixtures were cooled in an ice bath. Released, free sialic acid was estimated immediately by the Warren thiobarbituric acid procedure [8] modified by a proportional reduction in reagent volumes. All analyses were run in duplicate with corresponding reagent, enzyme, and substrate blanks

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Discussion

Conclusion