Structure and function of microplasminogen: reconstitution of microplasminogen and microplasmin from isolated fragments.

Abstract

We describe limited chemical proteolysis of microplasminogen/microplasmin (mPlg/mPlm) and their reconstitution from isolated fragments. A V141-->M141 substitution in methionineless human mPlg/mPlm allowed the protein(s) to be cleaved in CNBr/formic acid. The resulting two fragments (141 and 118 residues, respectively), each internally disulfide bonded, were separated by preparative non-reducing gradient SDS-PAGE, and could then be mixed to reconstitute the characteristic mPlg/mPlm, including their activation by urokinase (uPA) and streptokinase (SK), and inhibition by macromolecular inhibitors. The isolated larger, N-terminal fragment, which contains the mPlg activation site in a normal disulfide configuration, was not cleaved by uPA in the absence of its smaller C-terminal companion, showing that the linear amino acid sequence is not by itself sufficient to confer substrate character, even when its conformation is constrained by the disulfide structure.