alpha-chymotrypsin and dexamethasone-21-methanesulfonate

Two independently derived rat hepatoma cell lines, HTC and Fu5-5, differ in their sensitivities to both glucocorticoids and antiglucocorticoids, despite virtually identical number and affinity of glucocorticoid receptors. The present study further examined both receptors for differences that could account for the nonidentical responses of the two cell lines. HTC and Fu5-5 cell receptors that were covalently labeled with [3H] dexamethasone 21-mesylate ([3H]DM) had the same mol wt of about 97,000 on sodium dodecyl sulfate-polyacrylamide gel electrophoresis and the same isoelectric point of about 6.4 by nonequilibrium pH gradient electrophoresis. Limited proteolysis of receptor-[3H]DM complexes with three different proteases generated identical protease-specific digestion patterns regardless of the cellular origin of the receptors. Receptor-[3H]dexamethasone complexes prepared from either Fu5-5 or HTC cells bound calf thymus DNA with the same affinity in vitro. In intact cells, the intracellular distribution of receptor-dexamethasone or receptor-DM complexes at equilibrium was almost identical in the two cell lines. Thus, we detected no differences in the size, sequence, or net charge of Fu5-5 or HTC cell receptors; additionally, there were no significant differences in steroid uptake, receptor binding, or activation, translocation, and nuclear binding of receptor-steroid complexes. However, the DM labeling efficiency, calculated as the percentage of total receptors covalently labeled by DM, was higher in HTC cells (65.9 +/- 12.9%; n = 5) than in Fu5-5 cells (39.3 +/- 7.7%; n = 5). The labeling efficiency of DM correlated inversely with its ability to induce tyrosine aminotransferase activity, suggesting that DM forms noncovalent, as well as covalent, complexes in vivo which mediate the glucocorticoid and antiglucocorticoid activities of DM, respectively. Further research is required to identify the factor(s) that influences DM labeling efficiency, thereby affecting the amount of DM agonist activity and, possibly, the sensitivity of the cells to glucocorticoids.

Topics: Affinity Labels; Animals; Cell Nucleus; Chymotrypsin; Cytoplasm; Cytosol; Dexamethasone; DNA; Electrophoresis; Electrophoresis, Polyacrylamide Gel; Glucocorticoids; Hydrogen-Ion Concentration; Isoelectric Focusing; Liver Neoplasms, Experimental; Peptide Fragments; Rats; Receptors, Glucocorticoid; Serine Endopeptidases; Trypsin; Tumor Cells, Cultured

Purified rat liver glucocorticoid receptor was covalently charged with [3H]glucocorticoid by photoaffinity labeling (UV irradiation of [3H]triamcinolone acetonide-glucocorticoid receptor) or affinity labeling (incubation with [3H]dexamethasone mesylate). After labeling, separate samples of the denatured receptor were cleaved with trypsin (directly or after prior succinylation), chymotrypsin, and cyanogen bromide. Labeled residues in the peptides obtained were identified by radiosequence analysis. The peaks of radioactivity corresponded to Met-622 and Cys-754 after photoaffinity labeling with [3H]triamcinolone acetonide and Cys-656 after affinity labeling with [3H]dexamethasone mesylate. The labeled residues are all positioned within hydrophobic segments of the steroid-binding domain. The patterns of hydropathy and secondary structure for the glucocorticoid receptor are highly similar to those for the progestin receptor and similar but less so to those for the estrogen receptor and to those for c-erb A.

Topics: Affinity Labels; Amino Acid Sequence; Animals; Chymotrypsin; Cyanogen Bromide; Dexamethasone; Liver; Peptide Fragments; Protein Denaturation; Rats; Receptors, Glucocorticoid; Receptors, Steroid; Triamcinolone Acetonide; Trypsin

Glucocorticoid receptors in wild type and mutant S49 mouse lymphoma cells were affinity labeled with [3H]dexamethasone 21-mesylate and analyzed directly by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The molecular weight of receptors in cytosol from wild type cells and nuclear transfer decreased (nt-) mutants was 97,000 (97 kDa). The molecular weight of receptors in cytosol from nuclear transfer increased (nti) mutants was 48 kDa. The 97 kDa receptor in cytosol from wild type cells was digested by chymotrypsin to a 40 kDa steroid-binding receptor fragment but the 48 kDa receptor in cytosol from nti mutants was resistant to digestion by chymotrypsin. In addition to the 48 kDa receptor, cytosol from nti mutants contained 40 and 18 kDa receptor fragments. Cytosol from the nt- mutants also contained 18 kDa receptor fragments. The 40 and 18 kDa receptor fragments were present in multiple subclones of a nti mutant cell line. Formation of these receptor fragments was not prevented by protease inhibitors and was not increased by extended incubation of cytosol samples. Both 48 and 40 kDa forms of the receptor, but not the 18 kDa form, could be activated and bound by DNA-cellulose.

Topics: Affinity Labels; Animals; Cell Line; Chymotrypsin; Dexamethasone; DNA; DNA-Binding Proteins; Lymphoma; Mice; Molecular Weight; Mutation; Peptide Fragments; Receptors, Glucocorticoid

[3H]Dexamethasone 21-mesylate affinity-labeled glucocorticoid receptors were subjected to controlled proteolysis by trypsin, chymotrypsin, and Staphylococcus aureus V8 protease and then analyzed on denaturing constant percentage or gradient polyacrylamide gels. The molecular weights (Mr congruent to 98 000) and cleavage patterns for rat liver and HTC cell receptors indicated extensive homology between the glucocorticoid receptors from normal rat liver and a transformed rat liver cell line. The major DNA-binding species generated by chymotrypsin treatment was found to be a 42K fragment that was accompanied by several unresolved, slightly lower molecular weight fragments. The meroreceptors obtained after trypsinization were comprised of two species of Mr 30 000 and 28 000. Each of the three proteases, despite their differing specificities, generated fragments with molecular weights close to 42 500, 30 500, and 27 000. Nevertheless, each of the three proteases gave rise to a distinctive "ladder" of labeled fragments. No differences could be detected in the digestion patterns of unactivated and activated HTC cell complexes for all three proteases. Also, native and denatured receptor-steroid complexes yielded surprisingly similar digestion patterns with each enzyme. Digestion of denatured complexes readily generated large amounts of a fragment of Mr congruent to 15 000 that was much smaller than the protease-resistant meroreceptors formed from native complexes. The presence of these approximately 15K fragments suggested that the [3H]dexamethasone 21-mesylate labeling of the steroid-binding cavity is restricted to a relatively small segment of the receptor.

Topics: Animals; Chymotrypsin; Dexamethasone; Endopeptidases; Liver; Liver Neoplasms, Experimental; Macromolecular Substances; Molecular Weight; Peptide Hydrolases; Protein Conformation; Rats; Receptors, Glucocorticoid; Receptors, Steroid; Serine Endopeptidases