Halothane metabolism: immunochemical evidence for molecular mimicry of trifluoroacetylated liver protein adducts by constitutive polypeptides.
A monoform antibody [anti-TFA antibody] against TFA-protein adducts (TFA-adducts) was obtained by affinity purification of a polyclonal antiserum, raised in rabbits against TFA-rabbit serum albumin, on a N-epsilon-TFA-L-lysine matrix coupled to Affi-Gel 102. The anti-TFA antibody did recognize TFA-adducts of distinct molecular mass on Western blots of hepatocyte homogenates or microsomal membranes obtained from rats pretreated with halothane. The anti-TFA antibody also recognized cross-reactive polypeptides with apparent molecular masses of 52 kDa and 64 kDa on Western blots of hepatocyte homogenates obtained from rats not treated with halothane or metabolites thereof. The 52-kDa and 64-kDa cross-reactive polypeptides were localized in the 3,000 x g particulate fraction of liver homogenates. Recognition, on Western blots, of TFA-adducts and both the 52-kDa and 64-kDa cross-reactive polypeptides by anti-TFA antibody was sensitive to competition by N-epsilon-TFA-L-lysine (IC50 less than 100 microM) and N-epsilon-acetyl-L-lysine (IC50 approximately 10 mM). Treatment with piperidine (1 M) did abolish the recognition of TFA-adducts but not that of the 52-kDa and the 64-kDa cross-reactive polypeptides by anti-TFA antibody on Western blots. In antibody-exchange experiments, anti-TFA antibody was affinity-adsorbed on Western blots to the 52-kDa or the 64-kDa cross-reactive polypeptides of the rat heart, followed by spontaneous transfer to target TFA-adducts present on Western blots of rat liver microsomal membranes. The majority of these target TFA-adducts were recognized by anti-TFA antibody transferring from the source 52-kDa or 64-kDa cross-reactive polypeptides. When examined up to 10 days after exposure of rats to a single dose of halothane, no influence on the constitutive level of expression, in the liver, of either cross-reactive polypeptide was observed. In contrast, TFA-adducts were persistent for greater than 90 hr but less than 10 days. In addition to the liver, the 52-kDa and the 64-kDa cross-reactive polypeptides were prominently expressed in the heart and the kidney and, to a much lesser degree, in the spleen, the thymus, the lung, and skeletal muscle of the rat. Considerable variation in the level of expression of the 52-kDa and the 64-kDa cross-reactive polypeptides was recognized in livers of the six human individuals tested so far.(ABSTRACT TRUNCATED AT 250 WORDS)
Christen
U
U
Department of Pharmacology, Biocenter of the University, Basel, Switzerland.
Bürgin
M
M
Gut
J
J
D000818:Animals; D015153:Blotting, Western; D003429:Cross Reactions; D000939:Epitopes / Q000032:analysis; D006221:Halothane / Q000378:metabolism*; D006801:Humans; D007150:Immunohistochemistry; D008099:Liver / Q000378:metabolism*; D008297:Male; D009928:Organ Specificity; D010455:Peptides / Q000032:analysis* / Q000276:immunology; D011506:Proteins / Q000378:metabolism*; D011817:Rabbits; D051381:Rats; D011919:Rats, Inbred Strains; D012709:Serum Albumin / Q000378:metabolism; D014269:Trifluoroacetic Acid / Q000276:immunology / Q000378:metabolism*
D000939:Epitopes; D010455:Peptides; D011506:Proteins; D012709:Serum Albumin; D014269:Trifluoroacetic Acid; D006221:Halothane