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Characterization of rat hepatic acetyltransferase
1Department of Chemical Carcinogenesis, Michigan Cancer Foundation, Detroit 48201.
Environmental Health Perspectives
|October 1, 1994
Summary
A single 32 kDa protein in rat liver cytosol performs N-acetylation, O-acetylation, and N,O-acetyltransfer. Monoclonal antibodies confirmed this single enzyme and revealed its instability, impacting acetylation activity ratios.
Area of Science:
- Biochemistry
- Enzymology
Background:
- Rat liver cytosol exhibits N-acetylation (NAT), O-acetylation (OAT), and N,O-acetyltransfer (AHAT) activities.
- These diverse enzymatic functions were previously attributed to multiple proteins.
Purpose of the Study:
- To identify the specific protein responsible for NAT, OAT, and AHAT in rat liver cytosol.
- To characterize the enzyme's properties and the impact of antibodies on its activity.
Main Methods:
- Gel filtration chromatography and SDS-PAGE/Western blotting were used.
- Monoclonal antibodies (mAbs) were employed to probe enzyme activity and specificity.
- Enzyme instability was assessed under varying salt concentrations and isoelectric focusing.
Main Results:
- A single 32 kDa protein was identified as responsible for all three acetylation activities (NAT, OAT, AHAT).
- Specific mAbs (1F2, 5F8) indicated the presence of a single enzyme by altering apparent molecular size and Western blot reactivity.
- The enzyme demonstrated instability in low salt solutions and through deamidation, affecting activity ratios.
Conclusions:
- A single 32 kDa protein catalyzes arylamine N-acetylation, arylhydroxylamine O-acetylation, and arylhydroxamic acid N,O-acetyltransfer.
- Enzyme conformation, influenced by factors like antibody binding and deamidation, significantly affects the balance of NAT, OAT, and AHAT activities.
- Understanding these conformational effects provides insight into species-specific differences in aromatic amine metabolism.