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Journal of Biochemistry Advance Access originally published online on October 30, 2007
Journal of Biochemistry 2008 143(1):117-122; doi:10.1093/jb/mvm199
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© 2007 The Japanese Biochemical Society.

Identification and Characterization of Cathepsin D in a Highly Purified Sialidase from Starfish A. pectinifera

Ramaswamy Kannappan1, Youichi Satoh1, Naoko Iriyama1, Masayuki Ando1, Michiko Takagi Sawada2, Nobuaki Takahashi3, Kimio Furuhata4 and Yutaka Uda1,*

1Department of Health Chemistry, Faculty of Pharmaceutical Sciences, Niigata University of Pharmacy and Applied Life Sciences, Niigata 956-8603; 2Evaluation Department, National Institute of Advanced Industrial Science and Technology, Tsukuba, Ibaraki 305-8568; 3Marine Biomedical Institute, Sapporo Medical University, School of Medicine, Risirifuji, Hokkaodo 097-0101; and 4Scohool of Pharmaceutical Sciences, Kitasato University, Minato-ku, Tokyo 108-8641, Japan

*To whom correspondence should be addressed. Tel: +81-250-25-5270, Fax: +81-250-25-5021, E-mail: uda{at}nupals.ac.jp

Received June 16, 2007; Accepted October 10, 2007


  Abstract

A sialidase [EC 3.2.1.1 [EC] 8] 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.

Key Words: Asterina pectinifera, cathepsin D, enzymatic properties, sialidase, ovary of starfish

Abbreviations: 4MU-NeuAc, 4-methylumbelliferyl-{alpha}-D-N-acetylneuraminic acid; HPGFC, high-performance gel-filtration chromatography; IPG, immobilized pH gradient; Neu I, neuraminidase I; PPCA, protective protein/cathepsin A; PVDF, polyvinylidine difluoride; β-Gal, beta-galactosidase; TBS, tris-buffered saline


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