Differential effects of arylating and oxidizing analogs of N-acetyl-p-benzoquinoneimine on red blood cell membrane proteins.
Incubation of human red blood cell membranes (white ghosts) with N-acetyl-p-benzoquinone imine (NAPQI), a toxic metabolite of acetaminophen, or with either an arylating or an oxidizing analog of NAPQI, resulted in the inhibition of membrane ion transporting systems and the modification of cytoskeletal proteins. NAPQI and 2,6-dimethyl-NAPQI, which primarily arylates protein thiols, inhibited the calmodulin-activated Ca pump ATPase activity, the basal (calmodulin-independent) Ca pump ATPase activity and the Na,K pump ATPase activity. In contrast, 3,5-dimethyl-NAPQI, which primarily oxidizes protein thiols, caused selective inhibition of the calmodulin-activated Ca pump ATPase activity. Sodium dodecyl sulfate gel electrophoresis of red blood cell (RBC) membrane proteins revealed that NAPQI and 2,6-dimethyl-NAPQI, but not 3,5-dimethyl-NAPQI, decreased the intensity of band 3 corresponding to the anion transporter, whereas NAPQI as well as 2,6-dimethyl-NAPQI, and to a lesser extent 3,5-dimethyl-NAPQI, caused a decrease of cytoskeletal protein bands, including spectrin, actin, and bands 4.1 and 4.2. These modifications were associated with increased formation of high molecular weight protein aggregates that did not enter the gel. Treatment of 3,5-dimethyl-NAPQI-exposed ghosts with the reducing agent dithiothreitol (DTT), resulted in the recovery of the affected cytoskeletal protein bands. Conversely, the modifications caused by NAPQI and 2,6-dimethyl-NAPQI were only partially reversed by DTT treatment. Taken together our results suggest that NAPQI and its two analogs modified ion transporting systems and cytoskeletal proteins by reacting with protein thiols. Both oxidation and arylation of protein thiols can alter the functional properties of important RBC membrane proteins. Of the two reactions, arylation appeared to be the less specific and more damaging event.
Nicotera
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Department of Toxicology, Karolinska Institutet, Stockholm, Sweden.
Hinds
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Nelson
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Vincenzi
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D000251:Adenosine Triphosphatases / Q000097:blood*; D016227:Benzoquinones / Q000494:pharmacology*; D000252:Calcium-Transporting ATPases / Q000097:blood; D002147:Calmodulin / Q000494:pharmacology; D004910:Erythrocyte Membrane / Q000187:drug effects / Q000378:metabolism*; D006801:Humans; D007097:Imines / Q000494:pharmacology*; D007700:Kinetics; D008565:Membrane Proteins / Q000097:blood*; D013329:Structure-Activity Relationship
D016227:Benzoquinones; D002147:Calmodulin; D007097:Imines; D008565:Membrane Proteins; D000251:Adenosine Triphosphatases; D000252:Calcium-Transporting ATPases; C028473:N-acetyl-4-benzoquinoneimine