Cloning and expression of cDNAs for polymorphic and monomorphic arylamine N-acetyltransferases from human liver

• Published in: The Journal of biological chemistry 1990-03, Vol.265 (8), p.4630-4634
• Main Authors: Ohsako, S, Deguchi, T
• Format: Article
• Language: English
• Subjects: Acetyltransferases - genetics; Amino Acid Sequence; Animals; Arylamine N-Acetyltransferase - genetics; Base Sequence; Cell Line; Cloning, Molecular; Cricetinae; DNA - genetics; DNA - isolation & purification; Gene Expression; genes; Humans; Immunoblotting; liver; Liver - enzymology; Molecular Sequence Data; Nucleic Acid Hybridization; Plasmids; Polymorphism, Restriction Fragment Length; Restriction Mapping; Transfection
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1016/s0021-9258(19)39609-7

To elucidate the molecular basis of human N-acetylation polymorphism, cDNA clones encoding human liver N-acetyltransferases (EC 2.3.1.5) were isolated from lambda gt10 cDNA libraries using the 32P-labeled cDNA of rabbit liver N-acetyltransferase recently cloned in this laboratory. Three types of cDNAs (D-14, O-7, and D-24) were isolated and their nucleotide sequences were determined, from which the amino acid sequences of human N-acetyltransferases were deduced. All the cDNAs coded for 290 amino acids. Between D-14 and O-7 cDNAs, there was only a single-base substitution in the coding region, which replaced glutamic acid in D-14 cDNA for glycine in O-7 cDNA. There were considerable differences between O-7/D-14 and D-24 cDNAs, with 80% homology in amino acid sequences. When the cDNAs were inserted into pEF321 expression vector and introduced into Chinese hamster ovary cells, N-acetyltransferase activity was expressed in three groups of the transfected cells. The activity in cells transfected with D-14 cDNA was only 9-17% of the activity in O-7 cells. Immunoblot analysis of the transfected cells indicated that the difference in the enzyme activity between O-7 and D-14 cells was possibly due to a difference in the amount of enzyme proteins. The substrate specificity of the expressed enzymes indicated that O-7 and D-14 cDNAs code for polymorphic N-acetyltransferase whereas D-24 cDNA codes for monomorphic enzyme. Southern blot analysis indicated that the polymorphic and monomorphic N-acetyltransferases were encoded in separate genes and that there was restriction fragment length polymorphism with KpnI digestion in the polymorphic N-acetyltransferase gene.