Urokinase, which is excreted from human urine, has the ability to convert the precursor of the fibrinolytic enzyme of blood plasma, plasminogen, into the active enzyme plasmin. The recognition of the physiological significance of the fibrinolytic system during thrombotic conditions has promoted the clinical investigation of urokinase as fibrinolytic agent in man. The activity of urokinase has been usually determined by the indirect, fibrinolytic assay method. In this experiment, the enzyme activity was measured by the method of hydrolysis of p-nitrophenylester, N-t-BOCO-benzyl-L-tyrosine p-nitrophenylester(BBTN) or N-carbo-benzoxy-L-phenuylalanine p-nitrophenylester(CPN), which was very simple, rapid, sensitive and direct method.
As results, urokinase preparation showed a constant relationship between fibrinolysis-promoting and ester-hydrolysis activities. The apparent Michaelis constant of BBTN was 0.58mM and Vmax was 292.4nmoles/min/mg protein, in case of CPN, Km was 2.0mM and Vmax was 213.67nmoles/min/mg protein. N-acetyl-L-tyrosine ethyl ester(ATEe) competitively inhibited the hydrolysis of the nitrophenylester by urokinase. Nitrophenylester hydrolysis was affected by parathion or iodoacetamide, but, not affected by soybean trypsin inhibitor. The optimum pH of urokinase was 7.5 and urokinase was shown heat stable enzyme.
urokinase는 fibrin 용해능력을 가지고 있을뿐만 아니라 nitrophenyl ester도 가수분해 시킬수 있음을 이용하여 N-t-BOC-obenzyl-L-tyrosine p-nitrophenyl ester (BBTN) 또는 Ncarbobenzoxy-L-phenylalanine p-nitrophenyl ester (CPN)을 기질로하여 다음과 같은 결과를 얻었다.
urokinase의 fibrinolytic activity가 증가할수록 esterolytic activity 도 비례하여 증가하였고, BBTN을 기질로 사용하였을 때 Km 0.58mM, Vmax 292.4 nmoles/min/mg protein 이었고, CPN의 경우 Km은 2.0mM Vmax는 213.67 nmoles/min/mg protein 이었다. urokinase에 의한 BBTN 가수분해는 ATEe, CHPMe, BABe 등을 addition 하였을때 competitive inhibition 받았고 GMe에 의해서는 영향이 없었다. parathion,iodoacetamide에 의해 urokinase는 저해작용을 받았으며 soybean trypsin inhibitor에 의해서는 전혀 영향을 받지 않았다.
CPN에 대한 urokinase의 최적 pH는 7.5이었고, BBTN의 경우 60℃에서 활성이 가장 높았으며 activation energy는 11.1 Kcal /mole 이었고 그 밖에 본 실험에서는 urokinase의 활성에 대한 금속 이온 효과를 보았다.
