The arylation of microsomal membrane proteins by acetaminophen is associated with the release of a 44 kDa acetaminophen-binding mouse liver protein complex into the cytosol.
When analyzed by Western blotting with affinity purified antibodies against acetaminophen, proteins of molecular weight 44 and 58 kDa appear to be the major macromolecular targets in livers of mice administered hepatotoxic concentrations of acetaminophen. In this study, we have examined the characteristics and biochemical properties of the 44 kDa acetaminophen-binding protein in mouse liver. Data are presented which indicate that the 44-kDa protein is the earliest detectable protein targeted by acetaminophen; 30 min after acetaminophen administration in vivo, the binding to the 44 kDa protein is primarily localized in the microsomal fraction. After 1 hr, the 44 kDa acetaminophen-binding protein can be detected in both the microsomes and the cytosol. Extractions of microsomes with Triton X-114 or 1 M NaCl suggests that the acetaminophen-bound 44-kDa protein behaves as a peripheral membrane protein associated with the endoplasmic reticulum by ionic interactions. The cytosolic and microsomal 44-kDa proteins possess similar biochemical properties; both exist natively as components of a protein complex of greater than 200 kDa and both consist of two major isovariants with isoelectric points of 7.0 and 7.1 on two-dimensional gels. When N-acetyl-p-benzoquinone imine, the reactive metabolite of acetaminophen, is incubated with cytosolic or microsomal fractions from control liver, targeting of a 44-kDa protein is only observed in the microsomes. However, when acetaminophen is activated in an NADPH-regenerating microsomal system in vitro, some of the microsomal 44-kDa protein complex can be solubilized and released into the cytosol. Thus, acetaminophen administration can alter the subcellular distribution of at least one protein target in the cell.
Birge
R B
RB
Department of Molecular, University of Connecticut, Storrs 06269.
Bulera
S J
SJ
Bartolone
J B
JB
Ginsberg
G L
GL
Cohen
S D
SD
Khairallah
E A
EA
D000082:Acetaminophen / Q000378:metabolism / Q000633:toxicity*; D000284:Administration, Oral; D000818:Animals; D003600:Cytosol / Q000187:drug effects / Q000378:metabolism; D007425:Intracellular Membranes / Q000187:drug effects / Q000378:metabolism; D008297:Male; D008565:Membrane Proteins / Q000187:drug effects* / Q000378:metabolism; D051379:Mice; D008810:Mice, Inbred C57BL; D008813:Mice, Inbred ICR; D008862:Microsomes, Liver / Q000187:drug effects* / Q000378:metabolism
D008565:Membrane Proteins; D000082:Acetaminophen