Structure and function of microplasminogen: reconstitution of microplasminogen and microplasmin from isolated fragments.
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.
de los Santos
T
T
Department of Pharmacological Sciences, State University of New York at Stony Brook 11794-8651, USA.
Wang
J
J
Reich
E
E
D000595:Amino Acid Sequence; D004355:Drug Stability; D005341:Fibrinolysin / Q000737:chemistry* / Q000502:physiology; D006801:Humans; D008969:Molecular Sequence Data; D009154:Mutation; D010446:Peptide Fragments / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000502:physiology; D010958:Plasminogen / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000235:genetics / Q000502:physiology; D013329:Structure-Activity Relationship
D010446:Peptide Fragments; C111879:microplasminogen protein, human; C054561:microplasmin; D010958:Plasminogen; D005341:Fibrinolysin