Aspartic proteinase from Antarctic fish. A biochemical and molecular approach.

Cathepsin D was purified to homogeneity from the liver of Antarctic icefish by anion-exchange chromatography followed by affinity chromatography on concanavalin-A Sepharose. The N-terminal sequence of this proteinase was used to design a primer to be employed in reverse-transcriptase polymerase chain reaction together with liver RNA from two Antarctic Notothenioidei, the red-blooded Trematus bernacchii and the haemoglobinless icefish Chionodraco hamatus. The reaction mixture analysed by agarose gel electrophoresis, showed two bands of 1400 bp and 1200 bp. The open reading frame of the cloned cDNA, containing the 1400 bp fragment, encoded an aspartic proteinase showing sequence similarity with cathepsin D of other vertebrates. The nucleotide sequence of the 1200 bp fragment showed 58% identity with aspartic proteinase from other sources, but also features not found in homologous enzymes from other organisms. The temperature dependence of kinetic parameters was determined for the purified icefish cathepsin D. At temperatures between 8 and 50° C, the icefish proteinase had a higher specificity constant (kcat/Km) than human cathepsin D. The stability of both enzymes was measured at 50° C and half-lives of 55 and 3 min were derived for icefish and human cathepsin D, respectively.