Characterization and fibrin-binding properties of different molecular forms of pro-urokinase from a monkey kidney cell culture.
Culture fluid of a monkey kidney cell culture was harvested every two days, for a two week period, in order to obtain urokinase in the zymogen form. Pro-urokinase was isolated by immunoadsorption chromatography and gel filtered on Sephadex G-150, which resulted in three peaks with pro-urokinase activity. SDS-polyacrylamide gel electrophoresis showed that the first peak contained 55 kd pro-urokinase, aggregated with high molecular weight contaminants, whereas the second and third peaks consisted of almost pure 55 kd and 30 kd pro-urokinase, respectively. The latter form represented a relatively unknown and inactive precursor of low molecular weight urokinase, which was, like 55 kd pro-urokinase, activatable with plasmin. In comparison with tissue-type plasminogen activator, 55 kd and 30 kd pro-urokinase only bound weakly to purified fibrin clots and fibrin-sepharose columns. The extent of binding of the two pro-urokinases and their plasmin-activated forms to fibrin-sepharose decreased in the following order: 55 kd pro-urokinase 30 kd pro-urokinase 55 kd urokinase 30 kd urokinase. These results indicate that the two precursors exhibited stronger binding to fibrin-sepharose than the corresponding active enzymes, and the two 55 kd forms exhibited stronger binding than the corresponding 30 kd forms. This indicates the importance of both the zymogen nature and an intact NH2-terminal part of the molecules for binding to fibrin.
D000818:Animals; D002478:Cells, Cultured; D004792:Enzyme Precursors / Q000302:isolation & purification / Q000378:metabolism; D005337:Fibrin / Q000378:metabolism*; D007668:Kidney / Q000201:enzymology; D008252:Macaca fascicularis; D008970:Molecular Weight; D010960:Plasminogen Activators / Q000302:isolation & purification* / Q000378:metabolism; D011485:Protein Binding; D014568:Urokinase-Type Plasminogen Activator / Q000302:isolation & purification* / Q000378:metabolism
D004792:Enzyme Precursors; D005337:Fibrin; D010960:Plasminogen Activators; D014568:Urokinase-Type Plasminogen Activator