title: Mitochondrial membrane protein thiol reactivity with N-ethylmaleimide or mersalyl is modified by Ca2+: correlation with mitochondrial permeability transition.
abstract: The content of mitochondrial membrane protein thiol groups accessible to react with the monofunctional thiol reagents mersalyl or N-ethylmaleimide (NEM) was determined using Ellman's reagent. Deenergized mitochondria incubated in the presence of Ca2+ (0-500 microM) undergo a very significant decrease in the content of membrane protein thiols accessible to NEM, and an increase in the content of thiols accessible to mersalyl. This process is time-dependent and inhibited by Mg2+, ruthenium red and ADP, but not by cyclosporin A. This suggests that Ca2+ binding to the inner mitochondrial membrane promotes extensive alterations in the conformation of membrane proteins that result in location changes of thiol groups. The relationship between these alterations and mitochondrial membrane permeability transition was studied through the effect of NEM and mersalyl on mitochondrial swelling induced by Ca2+ plus t-butyl hydroperoxide (t-bOOH) or Ca2+ plus the thiol cross-linkers 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS) or phenylarsine oxide (PhAsO). We observed that the hydrophobic thiol reagent NEM inhibits the effects of t-bOOH, DIDS and PhAsO, while the hydrophilic thiol reagent mersalyl inhibits only the effect of DIDS. Permeability transition in all the situations studied is accompanied by a significant decrease in the total membrane protein thiol content. In addition, mitochondrial membrane permeabilization induced by PhAsO is inhibited by EGTA, but not by ruthenium red. This result suggests that PhAsO leads to permeability transition through a mechanism independent of intramitochondrial Ca2(+)-induced alterations of thiol group reactivity, but dependent on Ca2+ binding to an extramitochondrial site. This site is sensitive to extramitochondrial Ca2+ concentrations in range of 1-50 microM.
authors.affiliation: Departamento de Bioquímica, Universidade Estadual de Campinas, SP, Brazil.
authors.forename: A J
authors.initials: AJ
authors.lastname: Kowaltowski
authors.forename: A E
authors.initials: AE
authors.lastname: Vercesi
authors.forename: R F
authors.initials: RF
authors.lastname: Castilho
mesh_terms: D000818:Animals; D001152:Arsenicals / Q000494:pharmacology; D001665:Binding Sites; D002118:Calcium / Q000378:metabolism / Q000494:pharmacology*; D005033:Ethylmaleimide / Q000494:pharmacology; D066298:In Vitro Techniques; D007425:Intracellular Membranes / Q000187:drug effects / Q000378:metabolism; D008565:Membrane Proteins / Q000378:metabolism*; D008634:Mersalyl / Q000494:pharmacology; D008930:Mitochondria, Liver / Q000187:drug effects / Q000378:metabolism*; D008933:Mitochondrial Swelling / Q000187:drug effects; D010539:Permeability; D010545:Peroxides / Q000494:pharmacology; D051381:Rats; D017208:Rats, Wistar; D013438:Sulfhydryl Compounds / Q000378:metabolism*; D013439:Sulfhydryl Reagents / Q000494:pharmacology; D020122:tert-Butylhydroperoxide
chemical_list: D001152:Arsenicals; D008565:Membrane Proteins; D010545:Peroxides; D013438:Sulfhydryl Compounds; D013439:Sulfhydryl Reagents; C029341:oxophenylarsine; D008634:Mersalyl; D020122:tert-Butylhydroperoxide; D005033:Ethylmaleimide; D002118:Calcium