HUMAN AND PORCINE AMINOACYLASE-I OVERPRODUCED IN A BACULOVIRUS EXPRESSION VECTOR SYSTEM - EVIDENCE FOR STRUCTURAL AND FUNCTIONAL IDENTITY WITH ENZYMES ISOLATED FROM KIDNEY

Aminoacylase I (EC 3.5.1.14) is one of the most abundant enzymes in th e cortical region of mammalian kidney. Both the porcine and the human enzyme were overexpressed using baculovirus expression vector systems and purified by hydrophobic interaction chromatography and anion-excha nge chromatography. The resulting preparations were analyzed for struc tural and functional identity with the corresponding enzymes isolated from kidney. The dansyl method as well as mass spectroscopy confirmed N-terminal blocking, For the porcine enzyme, atomic absorption spectro scopy yielded the correct metal content (one zinc per subunit). Kineti c analyses showed identical IQ values for the expression products and the enzymes isolated from kidney. By contrast, the porcine enzyme when overexpressed in Escherichia coli had a much lower specific activity. Comparative substrate specificity studies with natural and recombinan t human aminoacylase and 16 different N-acetyl-L-amino acids showed th at, among the derivatives of proteinogenic amino acids, N-acetyl-L-met hionine was the best substrate, followed by acetylated glutamate, leuc ine, alanine, and valine. These amino acids are also the most abundant residues at the N-termini of acetylated proteins. This suggests that kidney aminoacylase may be involved in the salvage of amino acids by h ydrolyzing acetyl amino acids released from proteins. (C) 1998 Academi c Press.