The determination and localization of sialic acid in guinea-pig granulocytes

Publisher Website
Google Scholar
Abstract
When intact guinea-pig granulocytes (polymorphonuclear leukocytes) disrupted by sonication or with detergent were treated with neuraminidase from Vibrio cholerae, 3.1-3.2 nmol of sialic acid/107 cells was released. By using a chromatographic procedure for the specific determination of total cell sialic acid, this releasable portion constituted 70% of the total sialate. All of the neuraminidase-releasable sialic acid of the cells could be removed by enzymic treatment of intact cells with neuraminidase. The neuraminidase-releasable sialic acid may be all on the cell surface. To make sure that the result was not due to entry of neuraminidase into the cells, the enzyme was bound covalently to Sepharose 6B, and intact polymorphonuclear leukocytes were treated with the bound enzyme. All neuraminidase-releasable sialic acid could still be removed, though more slowly. The cells remained intact and only 1.5-2% of the bound enzyme was released from the Sepharose during incubation. Freed enzyme could have been responsible, at the very most, for release of 18% of the sialic acid. Fractionation studies showed that the nucleus and cytoplasm contain low amounts of sialic acid and that the neuraminidase-releasable sialic acid distributes in a manner similar to the distribution of 5''-nucleotidase, an unambiguous marker for the plasma membrane in these cells. Neuraminidase-releasable sialate constitutes a clear marker for the membrane of polymorphonuclear leukocytes. Most neuraminidase-insensitive sialate was present in the granule fraction. Removal of sialic acid from intact polymorphonuclear leukocytes did not affect their ecto-AMPase, -ATPase and -p-nitrophenyl phosphatase activities.