Strauss A W, Zimmerman M, Boime I, Ashe B, Mumford R A, Alberts A W
Proc Natl Acad Sci U S A. 1979 Sep;76(9):4225-9. doi: 10.1073/pnas.76.9.4225.
Proteolytic removal of the pre-segment from growing nascent chains of pre-human placental lactogen (hPL) occurred during in vitro translation of placental mRNA if crude membranes derived from ascites lysates, dog pancreas, or rat liver rough endoplasmic reticulum were added to the translation mixtures. The cotranslational proteolytic event was inhibited by the peptide protease inhibitor, chymostatin, but not by leupeptin, antipain, or elastatinal. The proteases involved in cleavage were solubilized with detergent and converted completed pre-hPL to hPL (post-translational processing). Direct assay of the solubilized membranes, with synthetic fluorogenic aminocoumarin peptide substrates, revealed no significant tryptic or elastase-like activity, but activity against a chymotrypsin substrate [(succinyl-Ala-Ala-Phe)-7-amino-4-methyl-coumarin] was found. This activity was dependent upon both an endopeptidase and an aminopeptidase. Although bestatin inhibited the aminopeptidase activity, it had no effect on the endopeptidase or on post-translational cleavage. Although this endopeptidase cleaved on the COOH side of an alanine residue, it was not inhibited by elastatinal. However, it was inhibited by high levels of chymostatin and by some serine protease inhibitors.
如果将源自腹水裂解物、狗胰腺或大鼠肝脏粗面内质网的粗制膜添加到翻译混合物中，在体外翻译胎盘mRNA的过程中，前体人胎盘催乳素（hPL）新生链上的前肽段会发生蛋白水解去除。共翻译蛋白水解事件受到肽蛋白酶抑制剂抑肽酶的抑制，但不受亮抑酶肽、抗蛋白酶或弹性蛋白酶抑制剂的抑制。参与切割的蛋白酶用去污剂溶解，并将完整的前体hPL转化为hPL（翻译后加工）。用合成荧光氨基香豆素肽底物对溶解的膜进行直接检测，未发现明显的胰蛋白酶或类弹性蛋白酶活性，但发现了针对胰凝乳蛋白酶底物[（琥珀酰 - 丙氨酸 - 丙氨酸 - 苯丙氨酸）-7-氨基-4-甲基香豆素]的活性。这种活性依赖于一种内肽酶和一种氨肽酶。虽然贝司他汀抑制氨肽酶活性，但它对内肽酶或翻译后切割没有影响。虽然这种内肽酶在丙氨酸残基的COOH侧切割，但它不受弹性蛋白酶抑制剂的抑制。然而，它受到高浓度抑肽酶和一些丝氨酸蛋白酶抑制剂的抑制。
