The release of radioactive nucleic acids and mucoproteins by trypsin and ethylenediaminetetra-acetate treatment of baby-hamster cells in tissue culture
- 1 October 1970
- journal article
- Published by Portland Press Ltd. in Biochemical Journal
- Vol. 119 (4), 707-714
- https://doi.org/10.1042/bj1190707
Abstract
Monolayers of baby-hamster kidney cells were grown on glass in tissue culture and harvested with trypsin or EDTA in order to investigate the cell surface macromolecules removed by these cell-disaggregating agents. The release of nucleic acids from the cells during the harvesting procedure was monitored by labelling the cellular RNA with [5-3H]uridine and the cellular DNA with [2-14C]thymidine. Treatment of the cells with EDTA was found to cause an increase in the permeability of the plasma membrane with 7.6% of the cellular RNA, but less than 1% of the cellular DNA, being released. Moreover, 61% of the cells harvested with EDTA were permeable to Trypan Blue. With crude trypsin, lysis of the cell occurred with the release of similar amounts of RNA and DNA amounting to about 11% of the total cellular nucleic acid. In contrast, crystalline trypsin released only 1% of the cellular nucleic acids. Since virtually all the cells (99%) after harvesting in crystalline trypsin were impermeable to Trypan Blue, this method was suitable for obtaining cell surface macromolecules without contamination by intracellular damage. [1-14C]Glucosamine was incorporated by the cells only into bound hexosamines and sialic acids. [By monitoring the release of radioactivity in high-molecular-weight material in such experiments a measure of the release of macromolecules containing amino sugars was obtained.] Of the total macromolecules containing amino sugars in the cells 33%, 24% and 13% were released when the cells were harvested with crude trypsin, crystalline trypsin or EDTA respectively. Crystalline trypsin also released 39% of the total sialic acid of the cell, whereas less than 1% of the cellular sialic acid was present in the EDTA-treated fraction. It is concluded that the macromolecules containing amino sugars released with crude trypsin and EDTA are likely to be heavily contaminated with intracellular material. However, the macromolecules released by crystalline trypsin appear to come from the cell surface.Keywords
This publication has 21 references indexed in Scilit:
- Nonviability of sodium tetraphenylboron-dissociated cellsExperimental Cell Research, 1969
- Changes in the surface properties of embryonic chick neural retina cells after dissociationExperimental Cell Research, 1969
- Organizational difference of cell surface “hematoside” in normal and virally transformed cellsBiochemical and Biophysical Research Communications, 1968
- Protein synthesis and aggregation of embryonic cellsExperimental Cell Research, 1968
- Cell Proliferation: Enhancement by Extracts from Cell Surfaces of Polyoma-Virus-Transformed CellsScience, 1968
- Production of heparin related glycosaminoglycans by an established mammalian cell lineJournal of Cellular Physiology, 1968
- Regeneration of sialic acid on the surface of chinese hamster cells in culture. II. Incorporation of radioactivity from glucosamine-l-14CJournal of Cellular Physiology, 1967
- Blood-Group SubstancesScience, 1966
- Intracellular distribution of sialic acid and its relationship to membranesBiochimica et Biophysica Acta, 1962
- Isolation and Determination of Neuraminic (Sialic) AcidsPublished by Wiley ,1960