An exceptionally stable 1:2 complex [FeL(2)](3-) is formed from the ligand H(3)L and Fe(III). In contrast, the affinity of this ligand for other biometals is relatively small. These properties make H(3)L a highly promising candidate for medical applications (e.g. for the treatment of iron overload).

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The crystal structure of the molecular complex of eglin, a serine proteinase inhibitor from leeches, with subtilisin Carlsberg has been determined at 2.0 A resolution by the molecular replacement method. The complex has been relined by restrained-parameter least-squares. The present crystallographic R factor (=CI ]13,\-]1;1 I/ClF,I) is 0.183. Eglin is a member of the potato inhibitor 1 family, a group of serine proteinase inhibitors lacking disultide bonds. Eglin shows strong structural homology to CL2, a related inhibitor from barley seeds. The structure of subtilisin Carlsberg in this complex is very similar to the known structure of subtilisin novo, despite changes of 84 out of 274 amino acids. Potato inhibitor 1 Serine proteinase Molecular replacement method Hirudo medicinalisCrystallography

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Five protease inhibitors with different modes of action were found to reduce the growth of transformed mouse (Py3T3, SV3T3, and 3T12) and hamster (PyBHK) cells. Some of these inhibitors caused the transformed cells to cease growth at saturation densities characteristic for nontransformed cells.The protease inhibitors were strikingly selective with regard to the transformed cells; they had essentially no effect on the growth of the nontransformed cells. From this result, it is concluded that the inhibitors block a protease-like activity that is required for the unrestrained growth of transformed cells.The inhibitors exerted their effect directly on the cells; they did not affect growth by interacting with serum components of the medium.Normal cells in tissue culture cease to divide upon formation of a monolayer. This phenomenon, which has been termed contact inhibition (1) or density-dependent inhibition of growth (2), is observed to a much lesser degree in virally or spontaneously transformed cells.Proteolytic treatment temporarily releases normal fibroblasts from contact inhibition (3, 4), and induces the appearance of surface properties characteristic of transformed cells (5-9). This finding implies the existence of a proteolytic activity in transformed cells that might be responsible for some of the cell surface alterations associated with the release from contact inhibition of growth (5, 10). If this speculation were correct, inhibition of this proteolytic activity should lead to a decreased growth of transformed cells and, possibly, to an "induction" of contact inhibition.This report describes the strikingly selective inhibition of growth of transformed cells by five commercially available protease inhibitors, and suggests that these cells require a protease-like activity for escape from contact inhibition. Cell growth curves in the presence or absence of protease inhibitors were obtained as follows: the cells were plated in 3.5-cm petri dishes (Falcon) 48 hr before addition of the inhibitors. At the beginning of each experiment, and every 24 hr thereafter, the medium with or without inhibitors (1.5 ml per plate) was changed in each plate.Five cell counts were done on each of two petri dishes for each point. To do this, the cells were trypsinized in 1 ml of trypsin-EDTA (Gibco No. 530), and were counted in a Neubauer Hemocytometer.The following protease inhibitors were used: Tosyl-arginine methylester (TAME), Tosyl-phenylalanyl-chloromethylketone (TPCK) and Tosyl-lysyl-chloromethylketone (TLCK) all from Calbiochem; soybean trypsin inhibitor 1-S (Sigma), ovomucoid II 0 (Sigma), and Trasylol injectible (5000 units/ml) from Bayer. Trasylol was dialyzed against phosphate-buffered saline, pH 7.4 to remove a toxic bactericidal additive before use. RESULTS AND DISCUSSIONAs an example of the cell growth curves obtained in the presence or absence of protease inhibitors, the effect of TLCK on the proliferation of SV3T3 and 3T3 cells is shown in Fig. 1

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