Studies on trypsin-modified bovine and human lens acylpeptide hydrolase

Acylpeptide hydrolase removes the N-acetylated amino acids from the peptide substrates but not from intact proteins. Cleavage between amino acid resid ues 203-204 of the native acylpeptide hydrolase results in the formation of a 55 kDa truncated active enzyme in the bovine lens, in vivo. In this stud y we explored the hydrolytic properties of the truncated enzyme using lens beta- and gamma -crystallins as substrates. SDS-PAGE analysis indicated tha t the beta B2-crystallin was cleaved by truncated acylpeptide hydrolase int o several protein fragments (10-26 kDa). No cleavage of the gamma -crystall ins was observed under similar conditions. Both the acylpeptide hydrolase a ctivity and the protease activity of the 55 kDa enzyme were completely inhi bited by diisopropylfluorophosphate, p-chloromercuribenzoate and ebelactone , and moderately inhibited by N-tosyl phenylalanine chloromethyl ketone. SD S-PAGE analysis followed by fluorography of (H-3) diisopropylfluorophosphat e labeled human lens acylpeptide hydrolase preparation showed the presence of the 55 kDa truncated form of the enzyme, as observed in the bovine lens. The peptide (d)-AIKGDQFL-NH2-the amino acid sequence 200-207 of the native bovine acylpeptide hydrolase with an in vivo cleavage site of native prote in-was hydrolysed by the lens protease(s) suggesting that the in vivo gener ation of the 55 kDa acylpeptide hydrolase may be mediated through a proteol ytic processing. The protease(s) responsible for the cleavage of this pepti de was inhibited by diisopropylfluorophosphate and p-chloromercuribenzoate. (C) 2001 Academic Press.