DESTABILIZATION OF THE CA2-ATPASE OF SARCOPLASMIC-RETICULUM BY THIOL-SPECIFIC, HEAT-SHOCK INDUCERS RESULTS IN THERMAL-DENATURATION AT 37 DEGREES-C()

A number of protein reactive compounds, including the thiol reagents d iamide and arsenite, are known inducers of heat shock protein (HSP) sy nthesis and thermotolerance, These compounds are thought to damage cel lular protein, which has been proposed to serve as the signal for indu ction. The specific mechanism of protein damage and its relation to th ermal denaturation are unknown. The Ca2+-ATPase of sarcoplasmic reticu lum, a membrane protein that contains 24 cys residues, was used to det ermine the effect of diamide, arsenite, N-ethylmaleimide (NEM), and th e cys-specific probes Br-DMC and IAEDANS, which label one or two speci fic cys residues, respectively, on protein conformation and stability. The Ca2+-ATPase was chosen because diamide has been shown to affect t he thermal properties of a class of membrane proteins of CHO cells (Fr eeman et al., 1995). The labeling of one or two thiols has no effect o n activity or conformation, while more extensive reaction (but with le ss than approximately five to eight groups titrated) results in destab ilization of the Ca2+-ATPase such that it denatures thermally at 37 de grees C. Higher levels of titration result in greater destabilization such that the protein is no longer stable at room temperature, with th e production of a state similar to the thermally denatured state as as sayed by activity, differential scanning calorimetry, ANS binding, and light scattering. The fractional denaturation induced by these thiol reagents, determined by the decrease in the heat absorbed during therm al denaturation, is directly proportional to inactivation of ATPase ac tivity, Thus, inactivation of the Ca2+-ATPase by thiol reagents occurs because of denaturation not through oxidation of essential thiols. Th ese results indicate that these thiol-specific heat shock inducers fun ction by two mechanisms: (1) destabilization of proteins such that the y thermally denature at 37 degrees C and (2) direct denaturation, appa rently driven by thermal processes al room temperature, following more extensive reaction which results in extreme destabilization. We sugge st that these are general mechanisms by which heat shock inducers dama ge proteins.