Purification and characterization of hen oviduct N alpha-acetyltransferase

Hen oviduct N alpha-acetyltransferase was purified to homogeneity by ammonium sulfate fractionation and DEAE-cellulose, Sepharose 6B, hydroxylapatite, and CoA affinity chromatography. The molecular weights of the native N alpha-acetyltransferase and its protein subunit were estimated as 240,000 and 79,000, respectively. The purified enzyme exhibited a narrow pH optimum centered at 7.8. The enzyme was activated by dithiothreitol, cysteine, glutathione, and beta-mercaptoethanol, but inhibited by Fe2+, Mn2+, Zn2+, Ca2+, Mg2+, and all thiol-specific reagents tested. These findings suggest that a thiol group(s) is essential to the enzyme activity. Substrate specificity experiments of the purified enzyme revealed that (i) the minimal length of a peptide chain required for N alpha-acetylation is 10 residues, (ii) the amino acids, Ala, Ser, Met, and Gly, which are predominantly found in the N termini of N alpha-acetylated proteins, are not the sole determinant of N alpha-acetylation for 10 and more residue peptides, and (iii) N alpha-acetyltransferase recognizes a minute difference in the side chain structure at the N termini of ACTH1-18-NH2 and [Gly1]ACTH1-18-NH2, a productive and a nonproductive substrate, respectively.