Open AccessBinding and internalization of thrombin by normal and transformed chick cells.
Author(s) -
Bruce R. Zetter,
Lan Bo Chen,
John M. Buchanan
Publication year - 1977
Publication title -
proceedings of the national academy of sciences of the united states of america
Language(s) - English
Resource type - Journals
eISSN - 1091-6490
pISSN - 0027-8424
DOI - 10.1073/pnas.74.2.596
Subject(s) - rous sarcoma virus , thrombin , trypsin , cytoplasm , cell culture , microbiology and biotechnology , sodium dodecyl sulfate , biochemistry , intracellular , biology , cell , incubation , chemistry , enzyme , immunology , platelet , genetics , gene
Thrombin stimulates cell proliferation in cultures of normal chick embryo fibroblasts but not in cells transformed with Rous sarcoma virus. Analysis of medium conditioned by Rous-sarcoma-virus-transformed cultures demonstrates that these cells do not secrete molecules that can inhibit or inactivate thrombin. The interaction of thrombin with these cells was investigated with enzymatically active 125I-thrombin. The amount of cell-associated 125I-thrombin was found to be three times greater with normal cells than with transformed cells. In both types of cell, greater than 50% of the total cell-associated 125I-thrombin was found as a component that was not dissociated from the cells by trypsin treatment, an observation suggesting that a significant portion was not on the cell surface. The amount of the trypsin-insensitive fraction increases with time up to 12 hr, whereas the trypsin-sensitive fraction is saturated after 1-4 hr. Autoradiography of thin sections of 125I-thrombin-treated cells observed by electron microscopy reveals that after 10 hr incubation greater than 70% of the label is localized in the cytoplasm of both normal and transformed cells. Autoradiograms of sodium dodecyl sulfate/polyacrylamide slab gels demonstrate that 40% of the intracellular label is the size of native thrombin with the remainder in two large fragments of 22,000 and 19,500 daltons.