Identification and characterization of cathepsin D in a highly purified sialidase from starfish A. pectinifera |
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Authors: | Kannappan Ramaswamy Satoh Youichi Iriyama Naoko Ando Masayuki Sawada Michiko Takagi Takahashi Nobuaki Furuhata Kimio Uda Yutaka |
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Institution: | Department of Health Chemistry, Faculty of Pharmaceutical Sciences, Niigata University of Pharmacy and Applied Life Sciences, Niigata 956-8603, Japan. |
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Abstract: | 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. |
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