Kato H, Adachi N, Iwanaga S, Abe K, Takada K, Kimura T, Sakakibara S
J Biochem. 1980 Apr;87(4):1127-32.
Kallikrein in human urine was measured using a fluorogenic peptide substrate, proly-phenyl-alanyl-arginine-4-methylcoumaryl-7-amide (Pro-Phe-Arg-MCA). Using 20 microliters of normal urine, kallikrein activity was quantitatively assayed by incubation with a final concentration of 10(-4) M Pro-Phe-Arg-MCA at 37 degrees C for 90 min. The reaction was terminated by adding 50 units of Trasylolr or 20 microliters of 10% acetic acid. Using this method, kallikrein activity was measured in urine from normal subjects and patients with glomerulonephitis and Bartter's syndrome. These values were comparable with the values obtained by radioimmunoassay using bovine low-molecular-weight kininogen. When normal urine was applied to a column of arginine-Sepharose 4B, two activities, Pro-Phe-Arg-MCA hydrolyzing activity and kinin-releasing activity toward bovine low-molecular-weight kininogen, were eluted in the same fraction. These results indicate that the present method is useful for the estimation of kallikrein in urine, in terms of specificity, sensitivity, and simplicity.
使用荧光肽底物脯氨酰-苯丙氨酰-精氨酸-4-甲基香豆素-7-酰胺（Pro-Phe-Arg-MCA）测定人尿中的激肽释放酶。取20微升正常尿液，通过在37℃下与终浓度为10⁻⁴M的Pro-Phe-Arg-MCA孵育90分钟来定量测定激肽释放酶活性。通过加入50单位的抑肽酶或20微升10%的乙酸终止反应。使用该方法，测定了正常受试者以及肾小球肾炎和巴特综合征患者尿液中的激肽释放酶活性。这些值与使用牛低分子量激肽原通过放射免疫测定法获得的值相当。当将正常尿液应用于精氨酸-琼脂糖4B柱时，两种活性，即Pro-Phe-Arg-MCA水解活性和对牛低分子量激肽原的激肽释放活性，在同一馏分中被洗脱。这些结果表明，就特异性、敏感性和简便性而言，本方法可用于尿液中激肽释放酶的测定。
