Purification and physical-chemical properties of acetyl-CoA:arylamine N-acetyltransferase from pigeon liver.
Acetyl-CoA:arylamine N-acetyltransferase (EC 2.3.1.5) from pigeon liver was purified by protamine sulfate precipitation, ion exchange chromatography on DEAE-A-25 Sephadex, gel filtration on Sephadex G-75, amethopterin-AH-Sepharose 4B affinity chromatography, and finally, gel filtration on Sephadex G-100. The enzyme preparation was homogeneous as judged by ultracentrifugation studies, SDS-polyacrylamide gel electrophoresis and gel filtration. The N-terminal amino acid was detected to be histidine and the complete amino acid composition is reported. The enzyme contains one disulfide bridge and two cysteine residues/mol monomer. The isoelectric point was estimated to be 4.8. The molecular weight was determined to be 32900 by high-speed sedimentation equilibrium analysis, 33000 by Sephadex G-100 gel filtration and 31600 by SDS-disc gel electrophoresis. The sedimentation coefficient from conventional sedimentation velocity runs was 3.1 S observed by ultraviolet optics. 'Active enzyme centrifugation' showed a sedimentation constant of 5.0 and 4.8 S for the purified enzyme and crude extract from pigeon liver, respectively, indicating that the enzyme forms a dimer under conditions of catalysis. It could be demonstrated that the inhibitor amethopterin was noncompetitive with respect to the acetyl donor and the acetyl acceptor. Acetyl-CoA:arylamine N-acetyltransferase was examined in different organs of pigeon. The enzyme was not inducible by 1,3-phenylenediamine and hexobarbital in vivo.
D000123:Acetyltransferases / Q000302:isolation & purification*; D000596:Amino Acids / Q000032:analysis; D000818:Animals; D001191:Arylamine N-Acetyltransferase / Q000037:antagonists & inhibitors / Q000302:isolation & purification*; D055598:Chemical Phenomena; D011232:Chemical Precipitation; D002627:Chemistry, Physical; D002845:Chromatography; D010856:Columbidae; D004355:Drug Stability; D008099:Liver / Q000201:enzymology*; D008727:Methotrexate / Q000494:pharmacology; D008970:Molecular Weight; D011479:Protamines; D014461:Ultracentrifugation
D000596:Amino Acids; D011479:Protamines; D000123:Acetyltransferases; D001191:Arylamine N-Acetyltransferase; D008727:Methotrexate