Identification and characterization of cathepsin D in a highly purified sialidase from starfish A. pectinifera.

A sialidase [EC 3.2.1.18] from the ovary of starfish Asterina pectinifera was isolated and highly purified by preparative PAGE. The SDS-PAGE separation of the purified enzyme revealed two natures of protein bands, upper (50 kDa) and a lower (47 kDa). To identify the protein, N-terminal amino acid sequence of the upper band was done. The sequence matched with the N-terminal amino acid sequence of human lysosomal mature cathepsin D and cathepsin D activity was also found in all the preparation steps. Protease inhibitor pepstatin A inhibited the proteolysis activity of cathepsin D against a synthetic substrate. The two enzymes sialidase and cathepsin D were separated from each other by using high-performance gel-filtration chromatography. The Western blot analysis and isoelectric focusing showed the co-purified cathepsin D is a 50 kDa protein with a PI value of 4.2.