Action of Vibrio cholerae neuraminidase (sialidase) upon the surface of intact cells and their isolated sialolipid components.

نویسندگان

  • N W Barton
  • A Rosenberg
چکیده

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 GM8 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, GDla, GDlb, and GT1 as substrate mixture than with the smaller sialolipid, GM,, 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 eflect 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 VCneuraminidase. 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 pg of sialic acid per mg of protein; lipid-bound sialic acid content varied from 0.7 to 1.6 pg 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

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عنوان ژورنال:
  • The Journal of biological chemistry

دوره 248 21  شماره 

صفحات  -

تاریخ انتشار 1973