Identification of proteins adducted by reactive naphthalene metabolites in vitro.
Metabolic activation of inert chemicals to electrophilic intermediates has been correlated with the incidence and severity of cytotoxicity. The current studies have identified several proteins adducted by reactive metabolites of the lung toxicant, naphthalene. Proteins isolated from microsomal incubations of (14)C-naphthalene were separated by 2-DE, proteins were blotted to PVDF membranes and radioactive proteins were localized by storage phosphor analysis. Adducted proteins were isolated from complimentary gels and identified by peptide mass mapping. A total of 18 adducted proteins were identified including: protein disulfide isomerase precursor, ER-60 protease, alpha actin, mouse urinary proteins, and cytochrome b5 reductase. In supernatant fractions, protein disulfide isomerase, heat shock protein 70, and alpha-actin were key proteins to which reactive naphthalene metabolites were bound. All of the proteins adducted, with the exception of cytochrome b5 reductase were sulfhydryl rich. Although several of the proteins found to be adducted in these studies have also been shown to be adducted by other electrophiles, several others have not been reported as common targets of reactive metabolites. These studies provide a basis for both in situ and in vivo work designed to follow the fate and formation of reactive metabolite protein adducts.
Isbell
Margaret A
MA
Department of Molecular Biosciences, School of Veterinary Medicine, University of California, Davis, CA 95616, USA. lacemagsca@yahoo.com
Morin
Dexter
D
Boland
Bridget
B
Buckpitt
Alan
A
Salemi
Michelle
M
Presley
Jack
J
D000595:Amino Acid Sequence; D000818:Animals; D002250:Carbon Radioisotopes; D015180:Electrophoresis, Gel, Two-Dimensional; D051379:Mice; D008862:Microsomes, Liver / Q000378:metabolism; D006899:Mixed Function Oxygenases / Q000378:metabolism; D008969:Molecular Sequence Data; D009281:Naphthalenes / Q000633:toxicity*; D010449:Peptide Mapping; D020543:Proteome / Q000378:metabolism*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
D002250:Carbon Radioisotopes; D009281:Naphthalenes; D020543:Proteome; C031721:naphthalene; D006899:Mixed Function Oxygenases