The rat kidney acylase I, characterization and molecular cloning - Differences with other acylases I

The soluble acylase I from rat kidney was purified to homogeneity using a f ive-step procedure. As the resulting protein was found to have a relative m olecular mass of 125 kDa based on size-exclusion chromatography and 44 kDa based on SDS/PAGE, the native protein was taken to consist of three subunit s. The amino-acid sequence of a peptide resulting from limited proteolysis of the polypeptide chain with proteinase K, which was determined by microse quencing (RHEFHALRAGFALDEGLA), was found to be very similar to the correspo nding sequence of porcine kidney acylase I. However, as N-furyl-acryloyl-l- methionine, a synthetic substrate for porcine acylases, was not hydrolyzed by the rat enzyme, it was suggested that the polypeptide chain might differ in other respects from those of the other acylases I. A full length cDNA c oding for the rat kidney acylase I was therefore isolated and found to cont ain a 1224-bp open reading frame encoding a protein consisting of 408 amino -acid residues, which corresponded to a calculated molecular mass of 45 847 Da per subunit. The deduced amino-acid sequence showed 93.6% and 87.2% ide ntity with that of the human liver and porcine kidney, respectively.