Proteolytic cleavage of the puromycin-sensitive aminopeptidase generates a substrate binding domain

Zhangliang Ma, Alex Daquin, Jia Yao, David Rodgers, Michael W. Thompson, Louis B. Hersh

Research output: Contribution to journalArticlepeer-review

7 Scopus citations


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 0.5mol/mol and a K0.5 value of 50μM. 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.

Original languageEnglish
Pages (from-to)80-86
Number of pages7
JournalArchives of Biochemistry and Biophysics
Issue number1
StatePublished - Jul 1 2003

Bibliographical note

Funding Information:
This work was supported in part by NIDA/NIH Grant DA02243.


  • Domain structure
  • Peptidase
  • Protein folding
  • Substrate binding

ASJC Scopus subject areas

  • Biophysics
  • Biochemistry
  • Molecular Biology


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