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The transcription factor NF-B activates a number of genes whose protein products are proinf lammatory. In quiescent cells, NF-B exists in a latent form and is activated via a signal-dependent proteolytic mechanism in which the inhibitory protein IB is degraded by the ubiquitinproteasome pathway.

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The natural product lactacystin exerts its cellular antiproliferative effects through a mechanism involving acylation and inhibition of the proteasome, a cytosolic proteinase complex that is an essential component of the ubiquitin-proteasome pathway for intracellular protein degradation. In vitro, lactacystin does not react with the proteasome; rather, it undergoes a spontaneous conversion (lactonization) to the active proteasome inhibitor, clasto-lactacystin beta-lactone. We show here that when the beta-lactone is added to mammalian cells in culture, it rapidly enters the cells, where it can react with the sulfhydryl of glutathione to form a thioester adduct that is both structurally and functionally analogous to lactacystin. We call this adduct lactathione, and like lactacystin, it does not react with the proteasome, but can undergo lactonization to yield back the active beta-lactone. We have studied the kinetics of this reaction under appropriate in vitro conditions as well as the kinetics of lactathione accumulation and proteasome inhibition in cells treated with lactacystin or beta-lactone. The results indicate that only the beta-lactone (not lactacystin) can enter cells and suggest that the formation of lactathione serves to concentrate the inhibitor inside cells, providing a reservoir for prolonged release of the active beta-lactone.

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Cell surface proteins of cultured cells are disulfide bonded to a greater degree than are total cellular proteins. In particular, the "large external transformation-sensitive" (LETS) protein, a major surface protein, is present almost exclusively in disulfide-bonded complexes including homodimers and also higher aggregates held together bydisulfide bonds or noncovalent interactions. Other cell surface proteins also appear to be involved in disulfide bonding, both intramolecular and intermolecular. In virally transformed cells, LETS protein and its disulfide complexes are absent and certain other disulfidebonded proteins are also not observed. Much recent research suggests that the surface of mammalian cells undergoes a change in organization on oncogenic transformation. Electron microscopic studies show altered surface morphology (1, 2). Studies with lectins indicate that the surface proteins of many transformed cells have greater freedom of lateral mobility than do those of their normal counterparts (3). It has been suggested frequently that these changes in surface properties are related to changes in cytoskeleton (4-8). Alterations in the organization of surface proteins also could be due to interactions between surface molecules (9). Whichever is the case, it is necessary to learn more about the molecular nature of the cell surface and the changes in its structure that occur on transformation. Numerous investigations have been made of cell surface proteins in normal and transformed cells (reviewed in ref. 10). The most prominent alteration in cell surface proteins is loss of a large external transformation-sensitive (LETS) glycoprotein (10-14). Few investigations have dealt with interactions between cell surface proteins except in erythrocytes (15-18). We report here that the surface of normal mammalian cells has extensive disulfide bonding, particularly involving the LETS protein. MATERIALS AND METHODSCells and Culture Conditions. The normal hamster cell line, NIL8, and a derivative transformed by hamster sarcoma-virus (NIL8-HSV) were cultured as described (11). Cultures were used as confluent monolayers. For labeling, cells were allowed to incorporate [35S]methionine (22 Ci/mmol) or [3H]glucosamine (10 Ci/mol; New England Nuclear) at 50 and 25 taCi/ml.The medium was modified by decreasing the methionine or glucose concentrations to 10% of the usual levels to improve incorporation.Lactoperoxidase-Catalyzed lodination. This was carried out on monolayers as previously described (11); this procedure labels only surface proteins in these cells.Oxidation of Sulfhydryl Groups. Oxidation to disulfide bonds was carried out with o-phenanthroline and copper sulfate (17,18) (18,20), samples were run in 3-mm-diameter cylindrical gels. The first-dimension cells were placed at the top of a slab gel and sealed in place with 0.5% (vol/vol) agarose in electrophoresis buffer. For reduction prior to the second dimension, the agarose contained 10% 2-mercaptoethanol. Markers for the second dimension were applied polymer...

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