Abstract
We have observed that the approximately 90-kDa non-steroid-binding component of nonactivated glucocorticoid receptors purified from WEHI-7 mouse thymoma cells (which has been identified as the approximately 90-kDa heat shock protein) consistently migrates as a doublet during polyacrylamide gel electrophoresis under denaturing and reducing conditions. It has recently been reported that murine Meth A cells contain a tumor-specific transplantation antigen (TSTA) which is related or identical to the approximately 90-kDa heat shock protein (Ullrich, S.J., Robinson, E.A., Law, L.W., Willingham, M., and Appella, E. (1986) Proc. Natl. Acad. Sci. U.S.A. 83, 3121-3125). The observation that TSTA and the approximately 90-kDa heat shock protein isolated from these cells exists as two isoforms of similar molecular mass and charge has suggested to us that the doublet we observed is also due to the existence of two isoforms. However, unlike TSTA, which appears to contain the two isoforms in similar relative abundance, nonactivated glucocorticoid-receptor complexes seem to contain predominantly the lower molecular mass isoform. We have therefore conducted this study to determine whether TSTA and the approximately 90-kDa component of glucocorticoid receptors are indeed related, to establish whether the receptor preferentially binds one isoform of the approximately 90-kDa heat shock protein, and to investigate the stoichiometry of the nonactivated receptor complex. By comparing Meth A TSTA and the approximately 90-kDa component of the receptor in their reactions with the AC88 monoclonal antibody (specific for the approximately 90-kDa heat shock protein) and a polyclonal antibody directed against Meth A TSTA, we found that these two proteins are indistinguishable and probably identical. We then used the BuGR1 (directed against the steroid-binding subunit of glucocorticoid receptors) and AC88 monoclonal antibodies to purify, respectively, receptor-associated and free approximately 90-kDa heat shock protein from WEHI-7 cells grown for 48 h with [35S]methionine to metabolically label proteins to steady state. Following analysis of the proteins by polyacrylamide gel electrophoresis under denaturing and reducing conditions, the relative amounts of the two isoforms in each sample were determined from the 35S counts and the known methionine content of each isoform. We found that approximately three-quarters of both the receptor-associated and the free approximately 90-kDa heat shock protein is present as the lower molecular weight isoform, indicating no preferential binding of either isoform in the receptor. The long-term metabolic labeling approach has also enabled us to direc
Highlights
(which hasbeen identified as the -90-kDa heat shock The long-term metabolic labeling approach has protein) consistently migrateass a doublet during poly- enabled us to directly determintehat thepurified nonacrylamide gel electrophoresis under denaturing and activated glucocorticoid receptor contains a single stereducing conditions
The observationthat transplantation antigen (TSTA) and the-90- stoichiometric ratio of steroid bindingto -90-kDa prokDa heat shock protein isolated from these cells exitsetisn is observed supports the view that the -90-kDa as two isoforms of similar molecular mass and charge heat shock protein is a true component of nonactivated has suggested to us that thdeoublet we observeids glucocorticoid-receptor complexes
We tions have been raised about whether it isa true component used the BuGRl and AC88 mono- cytosolic proteinandsteroidreceptorsarethoughtto be clonal antibodies to purify, respectively, receptor-as- “sticky”(seeKing, 1986 for review)
Summary
~-[~'S]Methionin(egreater than 800 Ci/mmol) and Iz5I-sheep (F(ab'), fragment) anti-mouse immunoglobulin (10-50 pCi/pg) were purchased from Amersham Corp., and '251-proteinA (2-10 pCi/gg) from ICN. In which we calculate the relative amounts of proteins based on their methionine content and their incorporation of the [35S]methioninelabel, it is necessary to attaina steady-statelabeling of the proteins We have done this by increasing the labeling time to 48 h and by increasing the total methionine concentration of the medium to its normal value(100 p ~ ) A. s a result of these two modifications,the cellsundergo at least three cell divisionsin medium containing tracer amounts of [35S]methionineat a constant specific activity (assuming that theoriginal cells provide a negligible amount of methionine compared with the total methionine in the medium). Analysis of Purified Complexes by SDS-PAGE-Antibody-receptor or antibody-Hsp 90 complexes were eluted from protein A columns as previously described (Mendel et aL, 1986)by heating the columns for 5 min in a boiling water bath in 125 pl of SDS-PAGE sample buffer.
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