Cloning, sequencing and further characterization of acylpeptide hydrolase from porcine intestinal mucosa

Acylpeptide hydrolase was purified to homogeneity from porcine intestinal m ucosa using a seven-step procedure including ammonium sulfate precipitation , gel filtration as well as anion exchange and affinity chromatography. The specific activity of the enzyme reached 105 000 nmol/mg protein per min an d the purification was as high as 5500-fold. This tetrameric enzyme is comp osed of four apparently identical subunits, the molecular mass of which was estimated to be 75 kDa, based on the results of amino acid analysis and ge l electrophoresis performed under denaturing conditions. It is likely that the NH2-terminal residue may be acetylated, while serine was found to be th e COOH-terminal residue. The hydrolytic activity of the enzyme toward N-ace tyl-L-alanine p-nitroanilide at the optimum pH value was increased twofold in the presence of the chloride anion. The K-m value calculated from the ki netics of the hydrolysis of acetylalanyl peptides was found to be 0.7 +/- 0 .1 mM, whereas the V-max value decreased from 200 to 50 nmol/min per mu g o f enzyme, depending on the peptidic chain lengths. The V-max value of the s ynthetic substrate (250 nmol/min per mu g of enzyme) was 25-500% higher tha n those of the acetylalanyl peptides, depending on the peptide chain length , although the enzyme affinity was slightly lower (1.8 mM as compared with 0.7 mM). In line with data on other animal species and on various tissues, the enzyme seemed likely to be a serine protease, since it was readily inhi bited by diisopropyl fluorophosphate and diethyl pyrocarbonate. A 2377-nucl eotide long cDNA coding for the enzyme was isolated from pig small intestin e. The deduced amino acid sequence consisted of 731 residues and showed a s ingle different amino acid with that of the porcine liver APH, except the N -terminal amino acid which is still probably lacking. (C) 1999 Elsevier Sci ence B.V. All rights reserved.