The removal of cell surface material by enzymes used to dissociate mammary-gland cells |
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Authors: | Ceriani R L Peterson J A Abraham S |
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Institution: | (1) Bruce Lyon Memorial Research Laboratory, Children's Hospital Medical Center, 94609 Oakland, California |
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Abstract: | Summary Treatment of mouse mammary epithelial cells (MMEC) with various enzymes used for dispersing and transferring cells results
in extensive digestion of materials on the cell surfaces. MMEC biosynthetially labeled with 3H]fucose, 14C]fucose and 3H]amino acids or with125I by the lactoperoxidase method were exposed to either collagenase plus hyaluronidase, followed by pronase, or to trypsin
in concentrations and conditions currently used for cell dispersion. Whereas the latter enzyme preparation solubilized 76%
of the trichloroacetic acid precipitable radioactive fucose and 96% of the protein-bound125I, collagenase plus hyaluronidase treatment released lesser amounts of each label. Subsequent treatment of the cells with
pronase removed additional surface-labeled materials, but the total amounts released were still less than when the trypsin
preparation alone was employed. Released cell surface materials were analyzed by gel chromatography. Some of the peaks obtained
also were examined by polyacrylamide gel electrophoresis. The labeled materials that remained attached to the MMEC after enzymatic
treatment were investigated by these two methods as well. We could show that collagenase plus hyaluronidase solubilized three
main glycoprotein components from the cell surface. In addition, we could show that the extensive cell surface damage caused
by these two enzyme preparations was due to the high proteolytic activity present in these preparations as judged by their
ability to hydrolyze rabbit gamma globulin labeled with125I. Even though their membranes were extensively damaged by the enzyme treatments, the dispersed cells could be cultured successfully
in vitro and could incorporated fucose into their surfaces in a manner similar to that by intact tissue. Through the use of
gel-filtration (cochromatography of 14C]fucose and 3H]fucose cell surface materials), we could demonstrate the identity of cell surface glycoproteins synthesized by cultured
cells and by intact tissue.
This work was supported by Grant Nos. CA 11736 and CA 19455 from the National Cancer Institute, and Biomedical Research Support
Grant No. RR05467 from the National Institutes of Health, DHEW. |
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Keywords: | glycoproteins collagenase hyaluronidase pronase trypsin gel filtration polyacrylamide electrophoresis mammary gland |
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