Open AccessThe rat kidney acylase I, characterization and molecular cloning
Author(s) -
Giardina Thierry,
Perrier Josette,
Puigserver Antoine
Publication year - 2000
Publication title -
european journal of biochemistry
Language(s) - English
Resource type - Journals
eISSN - 1432-1033
pISSN - 0014-2956
DOI - 10.1046/j.1432-1327.2000.01712.x
Subject(s) - molecular mass , complementary dna , biochemistry , peptide sequence , amino acid , proteolysis , methionine , biology , enzyme , open reading frame , microbiology and biotechnology , chemistry , gene
The soluble acylase I from rat kidney was purified to homogeneity using a five‐step procedure. As the resulting protein was found to have a relative molecular 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 subunits. The amino‐acid sequence of a peptide resulting from limited proteolysis of the polypeptide chain with proteinase K, which was determined by microsequencing (RHEFHALRAGFALDEGLA), was found to be very similar to the corresponding 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 coding for the rat kidney acylase I was therefore isolated and found to contain 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% identity with that of the human liver and porcine kidney, respectively.