Studies on Trypsin-modified Bovine and Human Lens Acylpeptide Hydrolase

Abstract

Acylpeptide hydrolase removes the N -acetylated amino acids from the peptide substrates but not from intact proteins. Cleavage between amino acid residues 203–204 of the native acylpeptide hydrolase results in the formation of a 55kDa truncated active enzyme in the bovine lens, in vivo. In this study we explored the hydrolytic properties of the truncated enzyme using lens β- and γ-crystallins as substrates. SDS–PAGE analysis indicated that the βB2-crystallin was cleaved by truncated acylpeptide hydrolase into several protein fragments (10–26kDa). No cleavage of the γ-crystallins was observed under similar conditions. Both the acylpeptide hydrolase activity and the protease activity of the 55kDa enzyme were completely inhibited by diisopropylfluorophosphate, p -chloromercuribenzoate and ebelactone, and moderately inhibited by N -tosyl phenylalanine chloromethyl ketone. SDS–PAGE analysis followed by fluorography of (3H) diisopropylfluorophosphate labeled human lens acylpeptide hydrolase preparation showed the presence of the 55kDa 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 protein—was hydrolysed by the lens protease(s) suggesting that the in vivo generation of the 55kDa acylpeptide hydrolase may be mediated through a proteolytic processing. The protease(s) responsible for the cleavage of this peptide was inhibited by diisopropylfluorophosphate and p -chloromercuribenzoate.