Purification and characterization of a rat hepatic acetyltransferase that can metabolize aromatic amine derivatives

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Abstract
Rat liver cytosol is capable of N-acetylation of arylamines, O-acetylation of arylhydroxylamines and N, O-acyltransfer of arylhydroxamic acids. The objective of this study was to characterize the enzyme(s) responsible for these reactions. A partially purified acetyltransferase preparation from rat liver cytosol was used to produce five mouse monoclonal IgG1s that bound to acetyltransferase on Western blots and affected one or more of the acetylation reactions. Two immunoaffinity columns were prepared by covalently cross-linking monoclonal antibodies to protein A - Sepharose. The first column permitted recovery of a single, immunoreactive 32 kDa protein that was capable of catalyzing all three reactions, while the second removed all three acetylation activities from a partially purified enzyme preparation and yielded a single, immunoreactive 32 kDa protein on elution. The harsh conditions necessary for elution from the latter column precluded recovery of an active enzyme. Although Western blots from SDS-PAGE at all stages of purification showed a single 32 kDa protein, purification was associated with the production of multiple, immunochemically reactive peptides with higher pIs. Direct enzymatic assays of these immunochemically reactive components after isoelectric focusing on polyacrylamide gels demonstrated that a single 32 kDa, pI 4.5 protein is capable of all three cytosolic acetylation activities. A second 32 kDa protein, pI 4.8, was able to carry out N-acetylation but not N, O-acetyltransfer. Immunoreactive components with pIs >4.8 that were formed during purification were catalytically inactive. However, isoelectric focusing in solution of cytosolic preparations that had been subjected only to gel filtration gave a single 32 kD immunoreactive peptide that was capable of all three acetylation reactions. Buffer concentration differentially affected the enzymatic activities of the enzyme, i.e. as a pH 7.4 buffer was decreased 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-acetyltransfer were unaffected. It has been shown that a single 32 kDa protein carries out all of the acetylation reactions in rat liver cytosol. Although it cannot be ruled out that other similarly sized and closely related enzymes that share antigenic sites are also capable of these acetylation reactions, these studies suggest that instabilities of the major peptide responsible for these activities, as reflected in changes in isoelectric point, may be responsible for changes in the enzymatic potentials of this peptide.

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