Studies on plasminogen activating enzyme (Urokinase)

Abstract

Urokinase, from human urine, has the ability to catalyze the activation of plasminogen to plasmin. Urokinase was purified by conventional method and affinity chromatography. Adsorbents used in affinity chromatography were Sepharose derivatives which contained substrate analogues, lysine, arginine and agmatine as ligands. Of these, agmatine adsorbent attached covalently to Sepharose 4B-$\varepsilon$-aminocaproic acid was effective and urokinase purified by using this column had a specific activity of 466.5 units per mg protein, which is considerably higher than that obtained by conventional method, DEAE-Cellulose and Sephadex G-100 column. chromatography. Recovery of affinity chromatography was 90\%, but that of conventional method was only 20\%. The general characteristics was also shown; optimum pH, 7.5-8.0, optimum temperature, 30$^\circ$C, molecular weight, 31,000, and 58,000, and effect of several metal ions, $Zn^{++},\; Hg^+$, and $Fe^{++}$ showed the strong inhibition of esterase activity of urokinase, but the inhibition of $Mg^{++},\; Cu^{++},\; Na^+$ and $K^+$ were negligible. By studying the inhibition mode of substrate analogues, it was known that esterase activity of urokinase has broad specificity. The enzyme affinity to plasminogen as a native substrate, was about 3 times, higher than that to the artificial substrate, CT. With regard to the proteolytic activity, UK, plasmin and trypsin showed a very close relationship, but they can be differentiated by their specific inhibition of $\varepsilon$-ACA and soybean trypsin inhibitor. Therefore, it can be suggested that the nature of three enzymes may be different physiologi cally. Studies on the mechanism of the enzyme action was carried out. Vm, Vm/Km and Km plotting against pH indicated that pKa and pKb values of the ionizing groups which were essential in the free enzyme were 6.7-6.8 and 8.9-9.0, and that in the intermediate pKa`` was 6.4-6.5. These pK values obtained may suggest that a histidine imi...