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  • Title: In vitro production of corticosteroid binder IB in the presence of proteolytic inhibitors.
    Author: Mayer M, Schmidt TJ, Barnett CA, Miller A, Litwack G.
    Journal: J Steroid Biochem; 1983 Feb; 18(2):111-20. PubMed ID: 6843114.
    Abstract:
    The effect of proteolytic inhibitors on the temperature-dependent formation of corticosteroid binder IB in rat kidney cytosol was examined. Antipain increased the apparent binding of [3H]-triamcinolone acetonide in the cytosol. Leupeptin, chymostatin, soya bean trypsin inhibitor and lima bean trypsin inhibitor did not affect total binding, while L-1-tosylamide-2-phenylethyl chloromethyl ketone, N alpha-p-tosyl-L-lysine chloromethyl ketone and phenylmethylsulfonyl fluoride markedly reduced the charcoal resistant steroid binding. However, none of the inhibitors added during tissue homogenization, steroid binding or activation affected the extent of heat-dependent conversion of the [3H]-triamcinolone acetonide-receptor complexes to the IB form, which was characterized by its exclusion from DEAE-Sephadex ion exchanger. In contrast, sodium molybdate (10 mM) effectively inhibits IB formation without inhibiting protease activity of rat kidney cytosol. These observations indicate that the temperature-dependent formation of corticosteroid binder IB in vitro does not involve proteolytic transformation of unbound or steroid-bound cytosolic proteins. Addition of antipain (3 mM) to the cytosol markedly increased the radioactivity in the buffer prewash of DEAE-cellulose columns (apparent IB) only when the inhibitor was added prior to charcoal adsorption. However, a similar peak in the prewash also was obtained with receptor-free cytosol. Antipain had no effect on the rate of dissociation of performed [3H]-triamcinolone-acetonide-receptor complexes nor did it increase the amount of receptor adsorbed to hydroxylapatite. Chromatography on Sephadex G-25 and P-2 columns showed that the increased activity in the charcoal-resistant fraction in the presence of antipain is due to unbound steroid. Thus, antipain interferes with the ability of charcoal to remove unbound steroid from the cytosol.
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