Arylamine N-acetyltransferase from chicken liver. I. Monoclonal antibodies, immunoaffinity purification, and amino acid sequences

J Biol Chem. 1988 Jun 5;263(16):7528-33.

Abstract

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Acetyl-CoA C-Acetyltransferase / analysis
  • Acetyl-CoA C-Acetyltransferase / isolation & purification*
  • Acetyltransferases / isolation & purification*
  • Amino Acid Sequence
  • Animals
  • Antibodies, Monoclonal*
  • Chickens
  • Chromatography, Affinity
  • Chromatography, High Pressure Liquid
  • Cross Reactions
  • Electrophoresis, Polyacrylamide Gel
  • Isoelectric Focusing
  • Liver / enzymology*
  • Mice
  • Molecular Sequence Data
  • Peptide Mapping
  • Pineal Gland / enzymology

Substances

  • Antibodies, Monoclonal
  • Acetyltransferases
  • Acetyl-CoA C-Acetyltransferase