Purification, antibody production, and partial amino acid sequence of the 58-kDa acetaminophen-binding liver proteins.
Immunochemical analysis of electrophoretically resolved liver proteins from mice administered hepatotoxic doses of acetaminophen has identified two proteins of 44 and 58 kDa as major targets for acetaminophen arylation. In the present study the 58-kDa acetaminophen-binding protein (58-ABP) was purified from mouse liver cytosol by gel permeation chromatography, preparative isoelectric focusing, and polyacrylamide gel electrophoresis. The acetaminophen adducts were visualized on immunoblots using affinity-purified anti-acetaminophen antibodies after each step of the purification. Gel permeation chromatography, under nondenaturing conditions, indicated that the protein is a monomer. Two-dimensional gel electrophoresis demonstrated that the 58-ABP consists of a cluster of four immunochemically reactive isoforms with isoelectric points ranging from 6.2 to 6.6. V-8 protease digestion of the isoforms suggested that they contained similar peptide fragments. The purified 58-ABP was utilized to produce polyclonal antibodies and to determine the amino acid composition and partial sequence of the protein. These antibodies revealed a protein cluster of similar molecular weight and isoelectric points in the cytosol of a human liver specimen. Amino acid analysis of the purified protein indicated that it contains eight cysteine residues (about 1.4% by weight). This low cysteine content raises the possibility that at hepatotoxic doses acetaminophen may also bind to non-thiol sites on the protein. The amino acid sequence of two cyanogen bromide/tryptic peptide fragments revealed that the major immunochemically detectable acetaminophen target in the cytosol is homologous to a selenium-binding protein which has been recently sequenced.
Bartolone
J B
JB
Department of Molecular and Cell Biology, University of Connecticut, Storrs 06269-3125.
Birge
R B
RB
Bulera
S J
SJ
Bruno
M K
MK
Nishanian
E V
EV
Cohen
S D
SD
Khairallah
E A
EA
D000082:Acetaminophen / Q000378:metabolism*; D000595:Amino Acid Sequence; D000818:Animals; D002352:Carrier Proteins / Q000276:immunology / Q000302:isolation & purification* / Q000378:metabolism; D002851:Chromatography, High Pressure Liquid; D007120:Immunochemistry; D008099:Liver / Q000378:metabolism*; D008297:Male; D051379:Mice; D008969:Molecular Sequence Data
D002352:Carrier Proteins; D000082:Acetaminophen