Open AccessActivation of Human Plasminogen by an Insoluble Derivative of Urokinase
Author(s) -
Wiman Björn,
WallÉN Per
Publication year - 1973
Publication title -
european journal of biochemistry
Language(s) - English
Resource type - Journals
eISSN - 1432-1033
pISSN - 0014-2956
DOI - 10.1111/j.1432-1033.1973.tb02880.x
Subject(s) - plasmin , chemistry , amino acid , cysteine , methionine , urokinase , cleavage (geology) , biochemistry , peptide , stereochemistry , autocatalysis , size exclusion chromatography , enzyme , catalysis , biology , paleontology , fracture (geology) , genetics
Activation of plasminogen was performed by perfusing a plasminogen solution through a small column loaded with insolubilized urokinase. The effluent was immediately collected in an excess of plasmin inhibitor ( p ‐nitrophenyl‐ p ‐guanidino‐benzoate). This provides a practically instantaneous inhibition of the plasmin formed and autocatalytic degradation is thus reduced to a minimum. Different degrees of activation are obtained by changing the perfusion rate. Dodecylsulfate‐polyacrylamide electrophoresis, amino acid and NH 2 ‐terminal amino acid analyses show that the activation of human plasminogen involves cleavage of at least two peptide bonds, the first event being the release of a peptide of 64 amino acid residues from the NH 2 ‐terminal end. An intermediate inactive product with a molecular weight of about 86000 and methionine as NH 2 ‐terminal amino acid is formed. Cleavage at the second site results in formation of a molecule with two chains connected by disulfide bridges. One chain which has a molecular weight of about 63000 is very sensitive to proteolytic degradation in its NH 2 ‐terminal part. However, there is evidence that methionine is the true NH 2 ‐terminal amino acid of this fragment. The other chain has a molecular weight of about 25000 and has valine as NH 2 ‐terminal amino acid.