Activated human plasma carboxypeptidase B is retained in the blood by binding to alpha(2)-macroglobulin and pregnancy zone protein

A 66-kDa glycosylated carboxypeptidase, plasma procarboxypeptidase B (pro-plasma CPB), has recently been identified in human blood (Eaten, D, L,, Malloy, B, E., Tsai, S. P,, Henzel, W,, and Drayna, D, (1991) J. Biol, Chem. 266, 21833-21838), The pro-enzyme binds to plasminogen and the active enzyme is specific for COOH-terminal Lys or Arg residues, These properties implicate a role in the fibrinolytic or coagulation system, However, we show that the molecular mass of the active plasma CPB is approximately 36 kDa, which is below the glomerular filtration limit. Since activated plasma CPB no longer binds plasminogen, the active enzyme may not be retained in the circulation. To investigate this, we performed plasma elimination studies in mice which showed that I-125-plasma CPB remains in the circulation despite its small size, Native polyacrylamide gel electrophoresis of blood samples removed from the mice revealed that plasma CPB migrated as a high molecular weight band, Similar bands were observed in vitro when I-125-plasma CPB was added to plasma from humans and other species, The plasma CPB-binding proteins were purified from human plasma and identified as alpha(2)-macroglobulin (alpha(2)M) and pregnancy zone protein, Only the active enzyme bound to the two alpha-macroglobulins, and the interaction was specific for alpha(2)M in its native conformation, but not its receptor recognized forms, The complex between human alpha(2)M and plasma CPB dissociated during SDS-polyacrylamide gel electrophoresis and transverse urea gel electrophoresis suggesting that the interaction was noncovalent and depended on the tertiary structure of the native alpha(2)M molecule. The catalytic activity of plasma CPB was not significantly affected by its binding to alpha(2)M, The specific binding of plasma CPB to alpha-macroglobulins suggest that these proteins may function as a ''shuttle'' in vivo to modulate the clearance of plasma CPB from the circulatory system.