Inactivation of endothelin I by deamidase (lysosomal protective protein).
Inactivation of endothelin I by deamidase (lysosomal protective protein).
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DOI：
10.1016/s0021-9258(19)50665-2
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发表时间：
1992-02
期刊：
影响因子：
--
通讯作者：
H. Jackman;P. Morris;P. Deddish;R. Skidgel;E. G. Erdös
中科院分区：
文献类型：
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作者：
H. Jackman;P. Morris;P. Deddish;R. Skidgel;E. G. Erdös
脱酰胺酶可裂解各种底物中的酯键和肽键，并使受保护的 COOH 末端氨基酸脱酰胺。它优先水解 P1' 和/或 P1 位含有疏水性氨基酸的肽。由于内皮素 I 的 COOH 末端含有疏水序列 -Ile19-Ile20-Trp21-OH，因此我们研究了从血小板中纯化的人脱酰胺酶是否可以使该肽失活。我们发现脱酰胺酶很容易在酸性 pH 最适条件下裂解 Trp21，Km = 22 microM，kcat 为 1454 min-1，kcat/Km 为 68 microM-1 min-1。我们还发现这种酶存在于血管平滑肌细胞的内皮素靶细胞中。培养的血管平滑肌细胞的提取物通过释放 COOH 末端氨基酸来裂解合成荧光底物 5-二甲氨基萘-1-磺酰基 (Dns)-Phe-Leu-Arg 和内皮素 I。该反应受到氟磷酸二异丙酯、苄氧羰基-Gly-Leu-Phe-CH2Cl 和对氯汞苯磺酸盐的抑制，它们会抑制纯化的脱酰胺酶，但不会被其他一些肽酶的抑制剂抑制。对于Dns-Phe-Leu-Arg，可溶性100,000×g最终上清液中内皮素I的水解速率为2.1μmol/h/mg和3.1μmol/h/mg。因此，平滑肌、血小板和许多其他含有脱酰胺酶的组织可以通过裂解COOH末端色氨酸来灭活内皮素。
Deamidase cleaves ester and peptide bonds in various substrates and deamidates protected COOH-terminal amino acids. It preferentially hydrolyzes peptides which contain hydrophobic amino acids in the P1' and/or P1 position. Because the COOH-terminal end of endothelin I contains the hydrophobic sequence -Ile19-Ile20-Trp21-OH, we investigated whether human deamidase, purified from platelets, could inactivate this peptide. We found that deamidase readily cleaved off Trp21 with an acid pH optimum, a Km = 22 microM, a kcat of 1454 min-1, and a kcat/Km of 68 microM-1 min-1. We also found the enzyme to be present in target cells of endothelin, in vascular smooth muscle cells. Extracts of cultured vascular smooth muscle cells cleave both the synthetic fluorescent substrate 5-dimethylaminonaphthalene-1-sulfonyl(Dns)-Phe-Leu-Arg and endothelin I by releasing the COOH-terminal amino acid. The reaction was inhibited by diisopropyl fluorophosphate, benzyloxycarbonyl-Gly-Leu-Phe-CH2Cl, and p-chloromercuribenzenesulfonate, which inhibit the purified deamidase, but not by inhibitors of some other peptidases. The rate of hydrolysis of endothelin I in the soluble, 100,000 x g final supernatant of the homogenized smooth muscle cells was 2.1 mumol/h/mg and 3.1 mumol/h/mg for Dns-Phe-Leu-Arg. Thus, smooth muscles, platelets, and many other tissues which contain the deamidase can inactivate endothelin by cleaving the COOH-terminal tryptophan.