Substrate specificity of rat brain neurolysin disclosed by molecular display system and putative substrates in rat tissues |
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Authors: | Tetsuya Kadonosono Michiko Kato Mitsuyoshi Ueda |
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Institution: | (1) Division of Applied Life Sciences, Graduate School of Agriculture, Kyoto University, Sakyo, Kyoto 606-8502, Japan |
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Abstract: | To search for the substrates, other than neurotensin, of rat brain neurolysin, a novel method of determining peptidase activity
was developed using a yeast molecular display system. This is a useful and convenient method of handling homogenously pure
proteins to evaluate the properties of neurolysin. The neurolysin gene was ligated to the C-terminal half of the α-agglutinin
gene with a FLAG tag sequence and a yeast cell-surface molecular displaying plasmid was constructed. Display of neurolysin
with correct folding and appropriate activity was verified by immunofluorescence staining and activity measurement of a bradykinin-related
peptide. The cleavage sites of peptides were determined by high-performance liquid chromatography (HPLC) and matrix assisted
laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). The results showed the amino acid preferences
of hydrophobic, aromatic, and basic residues, which were the same as those of soluble neurolysin. Moreover, this method clearly
showed the presence of two recognition motifs in neurolysin. By using these motifs, novel substrate candidates of neurolysin
in rat tissues were screened, and several bioactive peptides that regulate feeding were found. We also discussed the ubiquitous
distribution of neurolysin in rat tissues and the functions of substrate candidate peptides. |
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Keywords: | Molecular display Neurolysin Cell surface display Rat brain Peptidase |
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