Purification and characterization of a lysosomal aspartic protease with cathepsin D activity from the mosquito |
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| Institution: | 1. School of Life Sciences, Chongqing University, Chongqing 400044, China;2. College of Life Science, China West Normal University, Nanchong 637002, China;3. College of Forestry and Life Sciences, Chongqing University of Arts and Sciences, Yongchuan 402160, China;1. Evolutionary Biology, University of Potsdam, Karl-Liebknecht Str. 24-25, 14476 Potsdam, Germany;2. Max Planck Institute of Molecular Plant Physiology, Am Mühlenberg 1, 14476 Potsdam, Germany;3. Leibniz Institute for Zoo and Wildlife Research, Alfred-Kowalke-Straße 17, 10315 Berlin, Germany;4. School of Environmental Sciences, University of Guelph, 50 Stone Road East, N1G 2W1 Guelph, ON, Canada;5. Institute for Biochemistry and Biology, University of Potsdam, Karl-Liebknecht-Str. 24-25, 14476 Potsdam, Germany;1. Institute for Cell Biology, University of Bonn, Ulrich-Haberland-Strasse 61a, 53121 Bonn, Germany;2. Institute for Genetics and Cologne Excellence Cluster on Cellular Stress Responses in Aging-Associated Diseases (CECAD), University of Cologne, Joseph-Stelzmann-Strasse 26, 50931 Cologne, Germany;1. Medical College, Shenzhen University, Shenzhen 518000, Guangdong, China;2. Department of Pathophysiology, Southern Medical University, Guangzhou 510515, China;3. Cardiology Division, Department of Medicine, The University of Hongkong, Hong Kong, China;4. Department of Anatomy, Hebei Medical University, Hebei 050017, China;5. Department of Endocrinology, The First Affiliated Hospital of Shenzhen University, Shenzhen 518060, China;1. CBMA – Molecular and Environmental Research Centre, Department of Biology, University of Minho, Braga, Portugal;2. Faculty of Administration, University of Ljubljana, Ljubljana, Slovenia;3. IBB – Institute for Biotechnology and Bioengineering, Centre of Biological Engineering, University of Minho, Braga, Portugal;4. Escola Superior de Biotecnologia, Universidade Católica Portuguesa, Porto, Portugal |
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| Abstract: | A lysosomal aspartic protease with cathepsin D activity, from the mosquito, Aedes aegypti, was purified and characterized. Its isolation involved ammonium sulfate (30–50%) and acid (pH 2.5) precipitations of protein extracts from whole previtellogenic mosquitoes followed by cation exchange chromatography. Purity of the enzyme was monitored by SDS-PAGE and silver staining of the gels. The native molecular weight of the purified enzyme as determined by polyacrylamide gel electrophoresis under nondenaturing conditions was 80,000. SDS-PAGE resolved the enzyme into a single polypeptide with Mr = 40,000 suggesting that it exists as a homodimer in its non-denatured state. The pI of the purified enzyme was 5.4 as determined by isoelectric focusing gel electrophoresis. The purified enzyme exhibits properties characteristic of cathepsin D. It utilizes hemoglobin as a substrate and its activity is completely inhibited by pepstatin-A and 6M urea but not by 10 mM KCN. Optimal activity of the purified mosquito aspartic protease was obtained at pH 3.0 and 45°C. With hemoglobin as a substrate the enzyme had an apparent Km of 4.2 μ M. Polyclonal antibodies to the purified enzyme were raised in rabbits. The specificity of the antibodies to the enzyme was verified by immunoblot analysis of crude mosquito extracts and the enzyme separated by both non-denaturing and SDS-PAGE. Density gradient centrifugation of organelles followed by enzymatic and immunoblot analyses demonstrated the lysosomal nature of the purified enzyme. The N-terminal amino acid sequence of the purified mosquito lysosomal protease (19 amino acids) has 74% identity with N-terminal amino acid sequence of porcine and human cathepsins D. |
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