The N alpha-acetylenkephalin carboxypeptidase activity of N-acetyltyrosine deacetylase from monkey kidney. Purification, characterization and substrate specificity.

N alpha-Acetylenkephalin carboxypeptidase was co-purified with N-acetyltyrosine deacetylase from monkey kidney. Almost 90% of the activity from the homogenate was recovered in a high-speed supernatant without the use of detergents. The crucial steps in the purification were Cibacron Blue F3GA--Sepharose chromatography (involving negative and positive binding sequentially) and metal chelate affinity chromatography. The purified enzyme showed three bands on gel electrophoresis under non-denaturing conditions. All the three bands exhibited both N-acetyltyrosine deacetylase and N-acetylenkephalin carboxypeptidase activity, indicating their co-migration, Sodium dodecyl sulphate/polyacrylamide-gel electrophoresis in the presence and absence of 2-mercaptoethanol gave a single protein band of mol.wt. 34 000. The native enzyme was a dimer of mol.wt. 66 000 as observed on Bio-Gel P-300 gel filtration. The carboxypeptidase removed two amino acids from the C-terminal end of either N-acetyl[Met5]- or N-acetyl[Leu5]-enkephalin. Non-acetylated enkephalins were less active as substrates. Peptides with their carboxy end blocked were inactive as substrates. Models suggested for carboxypeptidase A [Hartsuck & Lipscomb (1971) Enzymes 3, 1-56] support the idea that the kidney N-acetylated aromatic amino acid deacetylase or acylase III [Endo (1978) Biochim. Biophys. Acta 523, 207-217] can act as a carboxypeptidase on peptides having hydrophobic amino acids at the C-terminal end.