Arylamine N-acetyltransferase from chicken liver. I. Monoclonal antibodies, immunoaffinity purification, and amino acid sequences.
Five monoclonal antibodies against arylamine acetyltransferase (EC 2.3.1.5) from the chicken liver were established by immunizing a mouse with a partially purified enzyme preparation. None of the antibodies cross-reacted with arylamine N-acetyltransferase from the livers of cow, rabbit, and rat, nor with arylalkylamine N-acetyltransferase from the chicken pineal gland, indicating a high specificity of the antibodies. By using the antibodies, two immunoaffinity purification procedures were elaborated: A partially purified enzyme preparation was incubated with the monoclonal antibody, and the resulting enzyme-IgG complex was separated by a protein A-Sepharose column. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed a single protein band with a molecular mass of 34 kDa in addition to the heavy and light chains of IgG. Secondly, an immunoaffinity column was prepared by immobilizing a monoclonal antibody to Sepharose 4B. After a partially purified enzyme preparation was absorbed on the column, N-acetyltransferase activity was eluted with 1 M NaCl and 1 M urea. The eluted sample contained a single 34-kDa protein. The purified enzyme preferred arylamines to arylalkylamines as substrates, indicating that it was arylamine N-acetyltransferase. The purified protein was subjected to digestion by lysylendopeptidase and separated by high performance liquid chromatography. Partial amino acid sequences of three peptides were determined by a gas-phase sequence analyzer.
D000101:Acetyl-CoA C-Acetyltransferase / Q000032:analysis / Q000302:isolation & purification*; D000123:Acetyltransferases / Q000302:isolation & purification*; D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal; D002645:Chickens; D002846:Chromatography, Affinity; D002851:Chromatography, High Pressure Liquid; D003429:Cross Reactions; D004591:Electrophoresis, Polyacrylamide Gel; D007525:Isoelectric Focusing; D008099:Liver / Q000201:enzymology*; D051379:Mice; D008969:Molecular Sequence Data; D010449:Peptide Mapping; D010870:Pineal Gland / Q000201:enzymology
D000911:Antibodies, Monoclonal; D000123:Acetyltransferases; D000101:Acetyl-CoA C-Acetyltransferase