Limited proteolysis of covalently labeled glucocorticoid receptors as a probe of receptor structure.

[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.

Isolation and structural properties of murine SAA--the acute phase serum precursor of amyloid AA.

The murine serum protein SAA, has been found to have a structure similar to human SAA, the precursor of human secondary amyloid fibril protein AA. SAA is detected by its cross-reaction in radioimmunoassay with antibodies raised to denatured amyloid fibrils of protein AA isolated from tissues of mice with amyloidosis. Murine SAA exists in the native state as a 160,000 molecular weight species, and can be isolated as a 12,500 molecular weight moiety, SAAL, by gel filtration in 10% formic acid. The quaternary structure of SAA is such that its AA determinants are relatively inaccessible for immunoreaction. Unfolding of these determinants can occur spontaneously; however, it is promoted by dissociation of SAA to SAAL.

Conformational flexibility of the serum amyloid precursor SAA.

SAA is a normal acute-phase serum protein and is thought to be the precursor of amyloid protein AA which is deposited as insoluble beta-pleated sheet fibrils in secondary amyloidosis. Native SAA has a molecular weight of 160,000 and has not been isolated; it has been most frequently purified as a species (designated SAAL) of 12,500 mol. wt. by gel filtration in dissociating solutions. The conformational properties of SAA proteins in patients with and without amyloidosis have been compared in an effort to determine the factors involved in the induction of the beta-pleated sheet conformation in the amyloid SAA protein prior to fibril deposition. Amyloid and nonamyloid SAA proteins are similar in that they readily undergo conformational changes which result in the formation of heterogenous mol. wt. SAA species and in an increased exposure of antigenic determinants which cross-react with AA fibril proteins. Amyloid and nonamyloid SAA are different, however, in that amyloid SAA is more resistant to dissociation to SAAL. Amyloid SAAL, while similar to nonamyloid SAAL in immunoreactivity, shows a greater tendency toward aggregation. The relative resistance of both amyloid SAA and SAAL to complete dissociation may play an important role in amyloid fibril formation from these precursors.