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Two arylamine N-acetyltransferases from chicken pineal gland as identified by cDNA cloning
M Ohtomi1, M Sasaki, T Deguchi
1Department of Molecular Neurobiology, Tokyo Metropolitan Institute for Neurosciences, Japan.
Abstract:
A cDNA library prepared from the poly(A)-rich RNA of the chicken pineal gland obtained at night was screened with the 32P-labeled cDNA of arylamine N-acetyltransferase from the chicken liver recently isolated in this laboratory. Two positive clones (p-NAT-3 and p-NAT-10) that cross-hybridized with the liver cDNA were isolated. The cDNAs did not cross-hybridize each other under a high stringency, indicating that they corresponded to different mRNAs. When the cDNAs were inserted into an expression vector pcDL1 under the control of the early promoter of simian virus 40 and introduced into Chinese hamster ovary cells, both cDNAs expressed arylamine N-acetyltransferase activity in the transfected cells. The nucleotide sequences of the cDNAs were determined, from which amino acid sequences were deduced. Both cDNAs coded for 290 amino acids. Similarities in amino acid sequences were about 60% between p-NAT-3, p-NAT-10 and liver N-acetyltransferases. Poly(A)-rich RNA blot hybridization analysis indicated that p-NAT-3 cDNA detected a 2.2-kb band with the poly(A)-rich RNAs from the brain, gut and, less intensively, spleen, liver and kidney, while p-NAT-10 cDNA hybridized only with the poly(A)-rich RNA from the kidney. Neither cDNA detected any hybridization band with the poly(A)-rich RNA from the pineal gland, suggesting that the contents were low. Genomic Southern blot hybridization analysis showed that p-NAT-3, p-NAT-10 and liver N-acetyltransferases were encoded in a separate single gene. The properties of the enzymes expressed in the transfected cells were compared with N-acetyltransferases from the pineal gland, brain and kidney. On a DEAE-cellulose column, the kidney and p-NAT-10 enzymes appeared in the effluent fraction, whereas the brain and p-NAT-3 enzymes were eluted from the column with a gradient elution at 0.08 M NaCl. The supernatant of the pineal gland obtained in the daytime showed two peaks appearing in the effluent fraction and the eluate fraction at 0.08 M NaCl. The substrate specificity of these enzymes were examined with p-phenetidine, 2-aminofluorene, tryptamine and phenylethylamine as substrates. All the enzymes preferred arylamines to arylalkylamines, indicating that both p-NAT-3 and p-NAT-10 cDNAs encoded arylamine N-acetyltransferases.
Insights
Researchers identified two distinct chicken arylamine N-acetyltransferase (NAT) genes, p-NAT-3 and p-NAT-10. These genes encode enzymes with differing tissue expression and biochemical properties, expanding our understanding of NAT function.
Area of Science:
- Molecular Biology
- Biochemistry
- Genetics
Background:
- Arylamine N-acetyltransferase (NAT) is a crucial enzyme involved in the metabolism of various compounds.
- Understanding the diversity of NAT enzymes and their tissue-specific expression is important for comprehending metabolic pathways.
Purpose of the Study:
- To isolate and characterize novel chicken arylamine N-acetyltransferase (NAT) cDNAs from the pineal gland.
- To investigate the expression patterns and enzymatic properties of the identified NAT variants.
Main Methods:
- Screening of a chicken pineal gland cDNA library using a chicken liver NAT cDNA probe.
- Isolation and sequencing of positive clones (p-NAT-3 and p-NAT-10).
- Expression of cDNAs in Chinese hamster ovary cells and characterization of enzyme activity, including substrate specificity and ion-exchange chromatography.
Main Results:
- Two distinct NAT cDNAs, p-NAT-3 and p-NAT-10, were isolated and found to encode proteins with approximately 60% amino acid similarity to liver NATs.
- p-NAT-3 cDNA detected mRNA in brain, gut, spleen, liver, and kidney, while p-NAT-10 cDNA hybridized only to kidney mRNA.
- Both expressed enzymes showed arylamine N-acetyltransferase activity, with distinct chromatographic properties and substrate preferences, indicating functional differences.
Conclusions:
- Chicken pineal gland expresses at least two distinct arylamine N-acetyltransferase (NAT) genes, p-NAT-3 and p-NAT-10.
- These genes encode enzymes with differential tissue distribution and biochemical characteristics, suggesting specialized roles in amine metabolism.
- The findings contribute to a deeper understanding of NAT enzyme diversity and function in vertebrates.