Revised Manuscript Received January 18, 7995® abstract: Recently we reported the isolation and cloning of a novel plasma procarboxypeptidase B that binds plasminogen [Eaton, D. L., Malloy, BE, Tsai, S. P., Henzel, W., & Drayna, D.(1991) J. Biol. Chem. 266, 21833—21838]. This plasma procarboxypeptidase is structurally similar to tissue procarboxypeptidases, and initial substrate studies showed that this plasma protein behaves like a basic carboxypeptidase and is now known as human plasma procarboxypeptidase B (pro-pCPB). However, unlike the tissue procarboxypeptidases, pro-pCPB is extremely unstable to trypsin activation. Trypsin cleaves pro-pCPB at two sites: Arg-92 and Arg-330. Cleavage at Arg-92 releases the activation peptide and generates an active enzyme. However, cleavage at Arg-330 inactivates pCPB. This renders the characterization of pCPB difficult. We have found that 6-amino-n-hexanoic acid (eACA), a compeptitive inhibitor of basic carboxypeptidases, selectively limits trypsin cleavage of pro-pCPB. In the presence of eACA, trypsin cleavage at Arg-330 is significantly limited while the cleavage at Arg-92 is unaffected. Using this approach, active pCPB can now be obtained. Kinetic characterizationshows that pCPB behaves like other known basic carboxypeptidases. pCPB is more specific for substrates with C-terminalarginine than those with C-terminal lysine for all the natural and synthetic peptides tested. It also hydrolyzes the synthetic ester substrate more efficiently than the synthetic peptide substrate, especially at high pH. The active site Zn2+ can be replaced with other metals with change in substrate specificity. Binding studies using either Lys-plasminogen or Glu-plasminogen with pro-pCPB or pCPB show that pro-pCPB has a 10-fold higher affinity to both forms of plasminogen than pCPB. This suggests that the glycosylated activation peptidemediates the high-affinity binding of pro-pCPB to plasminogen. Ligand blot binding studies show that the binding site for pro-pCPB to plasminogen is inhibited by 0.2-antiplasmin, suggesting a similar site of interaction.

Recently, we reported the purification and cloning of a novel procarboxypeptidase B from human plasma that specifically binds plasminogen (Eaton et al., 1991). This human plasma procarboxypeptidase B (pro-pCPB)* 1*** is synthesized in the liver and released as a glycosylated zymogen into the circulation. pro-pCPB is structurally similar to tissue procarboxypeptidases in that it shares~ 40% sequence identity and contains the amino-terminal activation peptide of~ 95 residues followed by an~ 300 amino acid catalytic domain. The catalytic domains of pCPB and tCPB share~ 50% sequence identity (Eaton et al., 1991). Unlike tissue carboxypeptidase B (tCPB), whose function is to serve as a digestive enzyme, the physiological function of pCPB is unknown. However, its binding to plasminogen suggests a role in fibrinolysis.