Isolation of plasminogen activator inhibitor-2 (PAI-2) from human placenta. Evidence for vitronectin/PAI-2 complexes in human placenta extract.
Plasminogen activator inhibitor-2 (PAI-2), found in human placenta and pregnancy plasma, was prepared in a highly purified and functionally active form from human placenta. The purification was achieved by a combination of Rivanol and ammonium sulfate precipitation, followed by chromatography on DEAE Affigel Blue, hydroxylapatite and phenylalanine-Sepharose. PAI-2, which is precipitated by low Rivanol concentrations, can be selectively redissolved from the pellet by increasing the Rivanol concentration in the presence of a reducing agent, i.e. dithiothreitol. The purified protein shows a molecular mass of 45 kDa in SDS PAGE, cross-reacts with monoclonal antibodies against PAI-2 (Mab'PAI-2), and inhibits the amidolytic activity of urokinase-type plasminogen activator (u-PA) towards the chromogenic substrate Glu-Gly-Arg-pNA (S-2444). The specific activity of the purified inhibitor was 52,300 units/mg, attaining 71,000 units/mg in peak fractions. In the immunopurification of placental extract on anti-PAI-2 Sepharose, the eluate showed the expected reaction with Mab' PAI-2, and it also cross-reacted with anti-vitronectin serum. In order to complement these results, anti-vitronectin Sepharose was used for immunopurification of placenta extract. In Western Blot experiments the eluates of anti PAI-2 Sepharose and anti-vitronectin Sepharose both showed a heterogeneous pattern of high molecular weight bands recognized by either polyclonal antiserum against vitronectin or Mab'PAI-2. In either case, reduction of the eluates releases mainly a 45-kDa band, which is recognized by Mab'PAI-2, or 80-kDa and 76-kDa bands recognized by anti-serum against vitronectin. These data suggest that the predominant form of PAI-2 in placenta extract is heterogeneous and of high molecular mass, containing complexes in which vitronectin is covalently bound to PAI-2 by disulfide bridges.
Radtke
K P
KP
Forschungslaboratorien der Behringwerke AG, Marburg/Lahn.
Wenz
K H
KH
Heimburger
N
N
D015153:Blotting, Western; D002845:Chromatography / Q000379:methods; D004591:Electrophoresis, Polyacrylamide Gel; D004975:Ethacridine / Q000031:analogs & derivatives; D005260:Female; D006023:Glycoproteins / Q000302:isolation & purification*; D006801:Humans; D007120:Immunochemistry; D008970:Molecular Weight; D010920:Placenta / Q000737:chemistry*; D015849:Plasminogen Inactivators / Q000032:analysis / Q000302:isolation & purification*; D011247:Pregnancy; D019096:Vitronectin
D006023:Glycoproteins; D015849:Plasminogen Inactivators; D019096:Vitronectin; D004975:Ethacridine