1. Proteolytic cleavage of the puromycin-sensitive aminopeptidase generates a substrate binding domain.
- Author
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Ma Z, Daquin A, Yao J, Rodgers D, Thompson MW, and Hersh LB
- Subjects
- Aminopeptidases isolation & purification, Aminopeptidases metabolism, Binding Sites, Binding, Competitive, Enzyme Activation, Enzyme Stability, Escherichia coli chemistry, Escherichia coli enzymology, Escherichia coli genetics, Humans, Hydrolysis, Molecular Probe Techniques, Opioid Peptides chemistry, Peptide Fragments isolation & purification, Peptide Fragments metabolism, Protein Binding, Protein Conformation, Protein Folding, Protein Structure, Tertiary, Recombinant Fusion Proteins chemistry, Recombinant Fusion Proteins isolation & purification, Recombinant Fusion Proteins metabolism, Serine Endopeptidases chemistry, Substrate Specificity, Aminopeptidases chemistry, Peptide Fragments chemistry
- Abstract
The puromycin-sensitive aminopeptidase was found to be resistant to proteolysis by trypsin, chymotrypsin, and protease V8 but was cleaved into an N-terminal 60-kDa fragment and a C-terminal 33-kDa fragment by proteinase K. The two proteinase K fragments remain associated and retained enzymatic activity. Attempts to express the 60-kDa N-terminal fragment in Escherichia coli produced inclusion bodies. A hexa-histidine fusion protein of the 60-kDa N-terminal fragment was solubilized from inclusion bodies with urea and refolded by removal of the urea through dialysis. The refolded protein was devoid of aminopeptidase activity as assayed with arginine-beta-naphthylamide. However, the refolded protein bound the substrate dynorphin A(1-9) with a stoichiometry of 0.5 mol/mol and a K(0.5) value of 50 microM. Dynorphin A(1-9) binding was competitively inhibited by the substrate dynorphin B(1-9), but not by des-Tyr(1)-leucine-enkephalin, a poor substrate for the enzyme.
- Published
- 2003
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