Cathepsin D from the liver of the Antarctic icefish Chionodraco hamatus exhibits unusual activity and stability at high temperatures

Cathepsin D was purified to homogeneity from the liver of Antarctic icefish by anion-exchange chromatography followed by affinity chromatography on co ncanavalin-A Sepharose. The purified enzyme showed a molecular mass of 40 k Da and displayed optimal activity at pH 3.0 with a synthetic chromogenic su bstrate. The N-terminal sequence of this proteinase was determined by autom ated Edman degradation and was used to design a primer for use in reverse-t ranscriptase polymerase chain reaction. The open reading frame of the clone d cDNA encoded an aspartic proteinase, which contained the experimentally d etermined N-terminal sequence. The predicted sequence (396 residues) had a high similarity with those of cathepsin D from various vertebrate sources, but was considerably different from that of nothepsin, a distinct aspartic proteinase described previously from Antarctic fish [1]. Determination of k inetic parameters for substrate hydrolysis showed that, at temperatures bet ween 8 and 50 degrees C, the icefish cathepsin D had a higher specificity c onstant (k(cat)/K-m) than human cathepsin D. The stability of both enzymes was measured at 50 degrees C and half-lives of 55 and 3 min were derived fo r icefish and human cathepsin D, respectively. (C) 1999 Elsevier Science B. V. All rights reserved.