PURIFICATION AND CHARACTERIZATION OF A RAT HEPATIC ACETYLTRANSFERASE THAT CAN METABOLIZE AROMATIC AMINE DERIVATIVES

Rat liver cytosol is capable of N-acetylation of arylamines, O-acetyla tion of arylhydroxylamines and N,O-acyltransfer of arylhydroxamic acid s. The objective of this study was to characterize the enzyme(s) respo nsible for these reactions. A partially purified acetyltransferase pre paration from rat liver cytosol was used to produce rive mouse monoclo nal IgG1s that bound to acetyltransferase on Western blots and affecte d one or more of the acetylation reactions. Two immunoaffinity columns were prepared by covalently cross-linking monoclonal antibodies to pr otein A-Sepharose. The first column permitted recovery of a single, im munoreactive 32 kDa protein that was capable of catalyzing all three r eactions, while the second removed all three acetylation activities fr om a partially purified enzyme preparation and yielded a single, immun oreactive 32 kDa protein on elution. The harsh conditions necessary fo r elution from the latter column precluded recovery of an active enzym e. Although Western blots from SDS-PAGE at all stages of purification showed a single 32 kDa protein, purification was associated with the p roduction of multiple, immunochemically reactive peptides with higher pIs. Direct enzymatic assays of these immunochemically reactive compon ents after isoelectric focusing on polyacrylamide gels demonstrated th at a single 32 kDa, pl 4.5 protein is capable of all three cytosolic a cetylation activities. A second 32 kDa protein, pI 4.8, was able to ca rry out N-acetylation but not N,O-acetyltransfer. Immunoreactive compo nents with pIs > 4.8 that were formed during purification were catalyt ically inactive. However, isoelectric focusing in solution of cytosoli c preparations that had been subjected only to gel filtration gave a s ingle 32 kDa immunoreactive peptide that was capable of all three acet ylation reactions. Buffer concentration differentially affected the en zymatic activities of the enzyme, i.e. as a pH 7.4 buffer was decrease d from 50 mM sodium pyrophosphate to 2 mM, the ability to N-acetylate arylamines was lowered while the abilities for O-acetylation and N,O-a cetyltransfer were unaffected. It has been shown that a single 32 kDa protein carries out all of the acetylation reactions in rat liver cyto sol. Although it cannot be ruled out that other similarly sized and cl osely related enzymes that share antigenic sites are also capable of t hese acetylation reactions, these studies suggest that instabilities o f the major peptide responsible for these activities, as reflected in changes in isoelectric point, may be responsible for changes in the en zymatic potentials of this peptide.