Abstract
Human a-thrombin was modified by four different procedures to identify specific active site regions required for receptor binding and stimulation of cell division. Conjugation of a-thrombin with diisopropylphosphofluoridate (DIP-F) or methylsulfonyl fluoride (MS-F) yielded catalytically inactivated preparations. Nitration or limited proteolysis of a-thrombin led to nitro-a-thrombin or y-thrombin preparations, respectively. Both possessed very little clotting activity but retained significant esterase activity; they were modified at regions necessary for the binding recognition of fibrinogen. Measurements of specific binding of these modified thrombins to cultured mouse, hamster, chicken, and human fibroblasts revealed no significant binding of nitro-a-thrombin or y-thrombin. Thus, binding of athrombin to each of the cell types examined involved regions of a-thrombin distinct from the catalytic apparatus that are related, if not identical, to regions required for fibrinogen recognition. Binding experiments with catalytic site-conjugated DIPor MS-athrombins revealed significant differences in the athrombin receptor among the four cell types; these thrombin forms bound as effectively as a-thrombin to mouse and hamster cells, but did not bind significantly to chick or human cells. Thus, thrombin binding to chick and human cells required the thrombin catalytic apparatus or adjacent active site regions, whereas binding to mouse or hamster cells did not. The four cell types examined all responded to the mitogenic action of a-thrombin. However, the derivative thrombin forms did not stimulate division of any of the cells significantly, with the exception of nitro-athrombin which possessed some residual activity for mouse cells. Since enzymatically inactive DIPand MSa-thrombins bound to mouse and hamster cells as effectively as active a-thrombin, the mitogenic activity of a-thrombin on these cells requires the intact catalytic apparatus of the enzyme to interact with and presumably cleave a specific protein component.
Highlights
Quired for receptor binding and stimulationof cell di- 9),and CHL cells [10]
80 pg of Binding Assays-For all cell types, the medium on nondividing a-thrombin and 1.0 mCi of Nalz5I(100 Ci/ cultures was changed to bindingmedium were added to0.1 M NaCl buffered with containing 0.1% bovine serum albumin buffered with 0.015 M Hepes
Modified thrombin forms enabled evaluatioonf cells requires theactive catalytic site. This conclusion is based the requirements for the catalytic apparatus and adjacent on thefinding that neither I2'1-DIP- nor "'I-MS-a-thrombins active site regions of a-thrombin in its binding to cellular bound to CE or HF cells, a result in close agreement with receptors and stimulationof cell division
Summary
Fraction I11 paste (obtained through the courteosfyDr. Fred Feldman as gifts from Armour Pharmaceutical Co., Kankakee, Ill.) and characterized as previously described [15,16,17]. 80 pg of Binding Assays-For all cell types, the medium on nondividing a-thrombin (or other thrombin forms) and 1.0 mCi of Nalz5I(100 Ci/ cultures was changed to bindingmedium (serum-free DV medium mM, carrier-free; Amersham) were added to0.1 M NaCl buffered with containing 0.1% bovine serum albumin buffered with 0.015 M Hepes. The mixture was added to a medium was changed to binding medium containing the indichloroglycouril-treated tube and incubated for 10 to 20 min a t 4°C. cated concentration of '2sII-labeleda-thrombin or "'I-labeled throm-. Relative specific binding was measured as the amount of radioactivity bound esterase activities with the turnover substrate TosArgMwe ere deter- toculturesafterincubation in bindingmediumcontaining excess mined by the spectrophotometric methoodf Hummel[25]except that unlabeled a-thrombin (110 nM) in addition to the'2511-labeletdhromthe buffer solution consisted of 0.2 M Tris and mM CaCl, at pH8.1. CEcells were prepared from the body walls of 9-day-oldchick embryos as previously de-
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