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Proteolytic cleavage of the puromycin-sensitive aminopeptidase generates a substrate binding domain
- Source :
-
Archives of Biochemistry & Biophysics . Jul2003, Vol. 415 Issue 1, p80. 7p. - Publication Year :
- 2003
-
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–β-naphthylamide. However, the refolded protein bound the substrate dynorphin A(1-9) with a stoichiometry of 0.5 mol/mol and a <f>K0.5</f> value of 50 μM. Dynorphin A(1-9) binding was competitively inhibited by the substrate dynorphin B(1-9), but not by des-Tyr1-leucine-enkephalin, a poor substrate for the enzyme. [Copyright &y& Elsevier]
- Subjects :
- *PEPTIDASE
*FERROMAGNETIC materials
*MAGNETIC domain
Subjects
Details
- Language :
- English
- ISSN :
- 00039861
- Volume :
- 415
- Issue :
- 1
- Database :
- Academic Search Index
- Journal :
- Archives of Biochemistry & Biophysics
- Publication Type :
- Academic Journal
- Accession number :
- 9948535
- Full Text :
- https://doi.org/10.1016/S0003-9861(03)00200-5