title: Destabilization of the Ca2+-ATPase of sarcoplasmic reticulum by thiol-specific, heat shock inducers results in thermal denaturation at 37 degrees C.
abstract: A number of protein reactive compounds, including the thiol reagents diamide and arsenite, are known inducers of heat shock protein (HSP) synthesis and thermotolerance. These compounds are thought to damage cellular protein, which has been proposed to serve as the signal for induction. The specific mechanism of protein damage and its relation to thermal denaturation are unknown. The Ca2+-ATPase of sarcoplasmic reticulum, a membrane protein that contains 24 cys residues, was used to determine the effect of diamide, arsenite, N-ethylmaleimide (NEM), and the cys-specific probes Br-DMC and IAEDANS, which label one or two specific cys residues, respectively, on protein conformation and stability. The Ca2+-ATPase was chosen because diamide has been shown to affect the thermal properties of a class of membrane proteins of CHO cells (Freeman et al., 1995). The labeling of one or two thiols has no effect on activity or conformation, while more extensive reaction (but with less than approximately five to eight groups titrated) results in destabilization of the Ca2+-ATPase such that it denatures thermally at 37 degrees C. Higher levels of titration result in greater destabilization such that the protein is no longer stable at room temperature, with the production of a state similar to the thermally denatured state as assayed 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 thermal denaturation, is directly proportional to inactivation of ATPase activity. Thus, inactivation of the Ca2+-ATPase by thiol reagents occurs because of denaturation not through oxidation of essential thiols. These results indicate that these thiol-specific heat shock inducers function by two mechanisms: (1) destabilization of proteins such that they thermally denature at 37 degrees C and (2) direct denaturation, apparently driven by thermal processes at room temperature, following more extensive reaction which results in extreme destabilization. We suggest that these are general mechanisms by which heat shock inducers damage proteins.
authors.affiliation: Guelph Waterloo Program for Graduate Studies in Physics and Department of Biology, University of Waterloo, Waterloo, Ontario N2L 3G1, Canada.
authors.forename: G A
authors.initials: GA
authors.lastname: Senisterra
authors.forename: S A
authors.initials: SA
authors.lastname: Huntley
authors.forename: M
authors.initials: M
authors.lastname: Escaravage
authors.forename: K R
authors.initials: KR
authors.lastname: Sekhar
authors.forename: M L
authors.initials: ML
authors.lastname: Freeman
authors.forename: M
authors.initials: M
authors.lastname: Borrelli
authors.forename: J R
authors.initials: JR
authors.lastname: Lepock
mesh_terms: D000818:Animals; D018053:Arsenites / Q000737:chemistry / Q000378:metabolism*; D000252:Calcium-Transporting ATPases / Q000737:chemistry / Q000378:metabolism*; D003958:Diamide / Q000737:chemistry / Q000378:metabolism*; D006358:Hot Temperature; D018482:Muscle, Skeletal / Q000201:enzymology* / Q000648:ultrastructure; D011489:Protein Denaturation; D011817:Rabbits; D012519:Sarcoplasmic Reticulum / Q000201:enzymology*; D013438:Sulfhydryl Compounds
chemical_list: D018053:Arsenites; D013438:Sulfhydryl Compounds; D003958:Diamide; D000252:Calcium-Transporting ATPases; C015001:arsenite