Dauch P, Vincent J P, Checler F
Institut de Pharmacologie Moléculaire et Cellulaire, Valbonne, France.
J Biol Chem. 1995 Nov 10;270(45):27266-71. doi: 10.1074/jbc.270.45.27266.
We have isolated by immunological screening of a lambda ZAPII cDNA library constructed from rat brain mRNAs a cDNA clone encoding endopeptidase 3.4.24.16. The longest open reading frame encodes a 704-amino acid protein with a theoretical molecular mass of 80,202 daltons and bears the consensus sequence of the zinc metalloprotease family. The sequence exhibits a 60.2% homology with those of another zinc metallopeptidase, endopeptidase 3.4.24.15. Northern blot analysis reveals two mRNA species of about 3 and 5 kilobases in rat brain, ileum, kidney, and testis. We have transiently transfected COS-7 cells with pcDNA3 containing the cloned cDNA and established the overexpression of a 70-75-kDa immunoreactive protein. This protein hydrolyzes QFS, a quenched fluorimetric substrate of endopeptidase 3.4.24.16, and cleaves neurotensin at a single peptide bond, leading to the formation of neurotensin (1-10) and neurotensin (11-13). QFS and neurotensin hydrolysis are potently inhibited by the selective endopeptidase 3.4.24.16 dipeptide blocker Pro-Ile and by dithiothreitol, while the enzymatic activity remains unaffected by phosphoramidon and captopril, the specific inhibitors of endopeptidase 3.4.24.11 and angiotensin-converting enzyme, respectively. Altogether, these physicochemical, biochemical, and immunological properties unambiguously identify endopeptidase 3.4.24.16 as the protein encoded by the isolated cDNA clone.
我们通过对从大鼠脑信使核糖核酸构建的λZAPII互补脱氧核糖核酸文库进行免疫筛选，分离出了一个编码内肽酶3.4.24.16的互补脱氧核糖核酸克隆。最长的开放阅读框编码一个704个氨基酸的蛋白质，理论分子量为80,202道尔顿，并具有锌金属蛋白酶家族的共有序列。该序列与另一种锌金属肽酶内肽酶3.4.24.15的序列具有60.2%的同源性。Northern印迹分析显示，在大鼠脑、回肠、肾脏和睾丸中存在约3和5千碱基的两种信使核糖核酸。我们用含有克隆互补脱氧核糖核酸的pcDNA3瞬时转染了COS-7细胞，并实现了一种70 - 75千道尔顿免疫反应性蛋白质的过表达。这种蛋白质水解内肽酶3.4.24.16的淬灭荧光底物QFS，并在一个肽键处切割神经降压素，导致神经降压素(1 - 10)和神经降压素(11 - 13)的形成。QFS和神经降压素的水解受到选择性内肽酶3.4.24.16二肽阻断剂Pro-Ile和二硫苏糖醇的强烈抑制，而酶活性不受内肽酶3.4.24.11的特异性抑制剂磷酰胺脒和血管紧张素转换酶的特异性抑制剂卡托普利的影响。总之，这些物理化学、生物化学和免疫学特性明确地将内肽酶3.4.24.16鉴定为分离的互补脱氧核糖核酸克隆所编码的蛋白质。
