New fluorogenic peptide substrates for plasmin.
New fluorogenic peptide substrates for plasmin.
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纤溶酶的新荧光肽底物。
DOI：
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发表时间：
1980
期刊：
影响因子：
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通讯作者：
S. Sakakibara
中科院分区：
文献类型：
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作者：
H. Kato;N. Adachi;Y. Ohno;S. Iwanaga;K. Takada;S. Sakakibara
新合成荧光肽，肽基-4-甲基香豆酰-7-酰胺 (MCA)，含有 COOH 末端赖氨酸残基，并作为纤溶酶底物进行测试。在六种肽基-MCA 中，Boc-Val-Leu-Lys-MCA 和 Boc-Glu-Lys-Lys-MCA 被发现可用于纤溶酶的特异性和灵敏测定。使用人和牛纤溶酶根据这些底物的 Line-weaver-Burk 图估计的 Km 值在 10(-4) M 范围内。Boc-Glu-Lys-Lys-MCA 被牛血浆激肽释放酶轻微水解，Boc-Val-Leu-Lys-MCA 被人和猪尿激肽释放酶和猪胰激肽释放酶轻微水解。然而，两种荧光肽基本上不受尿激酶、α-凝血酶、因子Xa、因子IXa、因子XIa和因子XIIa的影响。已证实纤溶酶水解Boc-Glu-Lys-Lys-MCA，裂解赖氨酰-MCA键，但不裂解赖氨酰-赖氨酰键。这些荧光肽对链激酶激活的人纤溶酶具有抗性。在超过 5 倍摩尔过量的链激酶存在下，Boc-Glu-Lys-Lys-MCA 不会被人纤溶酶或纤溶酶原水解。 Boc-Val-Leu-Lys- 的敏感性超过链激酶 5 倍摩尔过量。 Boc-Val-Leu-Lys-MCA 对人纤溶酶的敏感性也降低，但即使存在 20 倍摩尔过量的链激酶，纤溶酶仍保留 35% 的最大活性。这些结果表明链激酶-纤溶酶复合物对Boc-Glu-Lys-Lys-MCA基本上没有活性。
Fluorogenic peptides, peptidyl-4-methylcoumaryl-7-amides (MCA), containing COOH-terminal lysine residues, were newly synthesized and tested as substrates for plasmin. Among six peptidyl-MCA's, Boc-Val-Leu-Lys-MCA and Boc-Glu-Lys-Lys-MCA were found to be useful for the specific and sensitive assay of plasmin. The Km values estimated from Line-weaver-Burk plots for these substrates using human and bovine plasmins were in the region of 10(-4) M. Boc-Glu-Lys-Lys-MCA was slightly hydrolyzed by bovine plasma kallikrein, and Boc-Val-Leu-Lys-MCA was slightly hydrolyzed by human and hog urinary kallikreins and hog pancreatic kallikrein. However, both of the fluorogenic peptides were essentially unaffected by urokinase, alpha-thrombin, Factor Xa, Factor IXa, Factor XIa, and Factor XIIa. It was confirmed that plasmin hydrolyzed Boc-Glu-Lys-Lys-MCA, cleaving the lysyl-MCA bond, but not the lysyl-lysyl bond. These fluorogenic peptides were resistant to human plasmin activated by streptokinase. Boc-Glu-Lys-Lys-MCA was not hydrolyzed by human plasmin or plasminogen in the presence of more than a 5-fold molar excess of streptokinase. The sensitivity of Boc-Val-Leu-Lys- of more than a 5-fold molar excess of streptokinase. The sensitivity of Boc-Val-Leu-Lys-MCA to human plasmin was also reduced, but plasmin retained 35% of the maximum activity even in the presence of a 20-fold molar excess of streptokinase. These results suggest that streptokinase-plasmin complex has essentially no activity towards Boc-Glu-Lys-Lys-MCA.