Quenching of intrinsic tryptophan fluorescence in membranes of rat pituitary cells

The GH₄C₁ strain of hormone-producing rat pituitary cells has specific receptors for the tripeptide thyrotropin-releasing hormone (TRH). Membranes prepared from GH₄C₁ cells show intrinsic tryptophan fluorescence which was quenched by low concentrations (10–100 nM) of TRH and N^τ-methyl TRH but not by biologically inactive analogs of TRH. Membranes from GH₄C₁ cells were subjected to thermal denaturation. A conformational transition was noted above 40°C and an irreversible denaturation was observed at 52°C. TRH-induced quenching of intrinsic fluorescence was lost completely in membranes previously incubated for 10 min at 30°C while loss of ³H]-TRH binding was only about 20% at this temperature. Collisional quenching by iodide revealed that about 38% of the tryptophanyl residues in GH₄C₁ membranes were exposed to solvent. Quenching by TRH occurred with a shift in wavelength maximum from 336 to 342 nm suggesting that few of the tryptophanyl residues quenched by the tripeptide are totally exposed. Membranes prepared from cells preincubated with 20 nM TRH for 48 h, in which TRH receptors were decreased to 30% of control values, showed no quenching of tryptophan fluorescence in response to freshly added TRH. We conclude that the TRH-receptor interaction in GH₄C₁ cells is associated with a change in membrane conformation that can be measured by differential spectrofluorometry of intrinsic tryptophan fluorescence.