Abstract
We isolated, by immunological screening of a Uni-ZAP XR cDNA library constructed from rat liver mRNAs, a cDNA clone with 2212 base pairs encoding aminopeptidase B (EC 3.4.11.6). The open reading frame encodes a 649-amino acid protein with a theoretical molecular mass of 72,545 Da and bears the consensus sequence of the zinc metalloexopeptidases, indicating that the enzyme belongs to this family, which includes aminopeptidase A, aminopeptidase N, and leukotriene-A4 hydrolase. Escherichia coli SOLR cells infected with the pBluescript phagemid excised from the Uni-ZAP XR vector containing the aminopeptidase B cDNA had a high L-arginyl-beta-naphthylamidase activity. The recombinant protein was purified to homogeneity from the recombinant E. coli extracts. The enzyme had Cl--dependent aminopeptidase activity specifically restricted to the Arg and Lys derivatives and contained 1 mol of zinc per mol of the enzyme.
Highlights
Aminopeptidases (Ap1) play critical roles in processes such as protein maturation, protein digestion in its terminal stage, regulation of hormone levels, selective or homeostatic protein turnover, and plasmid stabilization [1]
The molecular mass of the insert cDNA and the nucleotide sequence of the 5Ј region of the insert cDNA of both clones were the same; we concluded that the clones were identical
The deduced amino acid sequence contains the HEXXH consensus sequence of zinc metallopeptidases with an additional glutamic acid 18 amino acids away, which constitutes the active site of metallopeptidases such as Ap-N, Ap-A, and leukotriene-A4 hydrolase [23]
Summary
Aminopeptidase; NA, -naphthylamide; PAGE, polyacrylamide gel electrophoresis. enzyme by interaction with a catalytic zinc ion in the active site. Aminopeptidase; NA, -naphthylamide; PAGE, polyacrylamide gel electrophoresis. Enzyme by interaction with a catalytic zinc ion in the active site. We cloned Ap-B cDNA and found that the enzyme belongs to the family of zinc metallopeptidases and contains the zinc-binding domain in its sequence. By an atomic absorption experiment we demonstrated that the recombinant enzyme contains a zinc ion, which we presume to be catalytic
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