Reversible Binding of the Tissue Plasminogen Activator With Fibronectin

Abstract

Fresh pig heart tissues were homogenized and deli-pidized with cold acetone. The dry acetone powder was extracted with 0.45 M potassium acetate pH 4.5, the extract was fractionated with ammonium sulfate to 60% saturation, successively followed by four chromatography steps: chromatography on CM-Sepharose CL-6B; gel filtration on Sephadex G-100; Fibrin-Sepharose affinity chromatography and chromatography on DEAE-Sepharose CL-6B. The fibrin plate method was used for the tissue plasminogen activator (t-PA) activity determination. The porcine t-PA purified was proved to be homogeneous either by SDS gel electrophoresis or by high performance liguid chromatography with a molecular weight of 67,000. Using the human melanoma t-PA antigen and its rabbit antiserum, the double immunodiffusion and immu-noelectrophoretic tests demonstrated that the porcine t-PA possessed a cross-reaction with the human t-PA. During the purification procedure, it was found that the porcine t-PA was contaminated and reversibly bound with the cell fibronectin. The binding ability depends on salt concentration. They could be separated from each other on Sepharose G-100 gel filtration at high salt concentration, this reversible binding was further confirmed by mixing these two purified components monitored on high performance liquid chromatography. The fibronectin-bound t-PA retained its ability to activate plasminogen, indicating that these two proteins may exist in a form of complex in the in vivo tissue. Some monoclonal antibodies against the porcine t-PA were obtained, being used to screen what domain of the enzyme is responsible for binding the cell fibronectin.