NEUTRAL ENDOPEPTIDASE 24.11 IN HUMAN-NEUTROPHILS - CLEAVAGE OF CHEMOTACTIC PEPTIDE
NEUTRAL ENDOPEPTIDASE 24.11 IN HUMAN-NEUTROPHILS - CLEAVAGE OF CHEMOTACTIC PEPTIDE
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DOI：
10.1073/pnas.82.24.8737
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发表时间：
1985-12-01
影响因子：
11.1
通讯作者：
ERDOS, EG
中科院分区：
文献类型：
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作者：
CONNELLY, JC;SKIDGEL, RA;ERDOS, EG
膜金属内肽酶（NEP;中性内肽酶，肾刷状缘中性蛋白酶，脑啡肽酶，EC 3.4.24.11）在疏水性氨基酸的氨基侧切割肽。虽然已知这种酶存在于肾脏和大脑等器官中，但我们在人类中性粒细胞中发现了它。这些细胞以9.5nmol·cm-1的速率切割NEP底物戊二酰（Glut）-Ala-Ala-Phe-（4-甲氧基萘胺）（Glut-Ala-Ala-Phe-MNA）。hr-1/106细胞，而phosphoramidon（1 μ M）抑制水解90%。来自吸烟者的完整中性粒细胞的NEP活性约为不吸烟者的两倍。亚细胞分级和蔗糖密度梯度离心的中性粒细胞裂解表明，大部分的NEP活性的膜结合。来自人嗜中性粒细胞的洗涤的膜级分快速切割0.5mM Glut-Ala-Ala-Phe-MNA 996 nmol. min-1. mg ~（-1），磷酸酰胺和人肾NEP特异性抗血清均能抑制NEP的水解。经洗涤的膜级分也快速裂解0.1 mM缓激肽（34 nmol. min-1 mg-1）和0.1 mM fMet-Leu-Phe（49 nmol. min-1. mg ~（-1）。膜结合酶裂解的肽底物在相同的网站作为均匀的人肾NEP，和phosphoramidon和thiorphan抑制水解。用纯的人肾NEP进行的动力学研究表明，趋化肽fMet-Leu-Phe是最好的生物活性底物之一（Km，59 × 106）。10-6 M; kcat，3654 min-1）。免疫细胞化学在光镜水平显示高浓度的NeP的细胞膜上的中性粒细胞。这是证实了电子显微镜使用免疫金技术的冷冻切片。这些研究表明，中性粒细胞的NEP可能在炎症和趋化中具有重要的功能。
Membrane metallo-endopeptidase (NEP; neutral endopeptidase, kidney-brush-border neutral proteinase, enkephalinase, EC 3.4.24.11) cleaves peptides at the amino side of hydrophobic amino acids. While the enzyme is known to be in organs such as kidney and brain, we found it in human neutrophils. These cells cleaved the NEP substrate glutaryl (Glut)-Ala-Ala-Phe-(4-methoxynaphthylamine) (Glut-Ala-Ala-Phe-MNA) at a rate of 9.5 nmol .cntdot. hr-1 per 106 cells, and phosphoramidon (1 .mu.M) inhibited the hydrolysis by 90%. Intact neutrophils from donors who smoked had NEP activities about twice that of nonsmokers. Subcellular fractionation and sucrose density gradient centrifugation of lysed neutrophils showed that most of the NEP activity was membrane bound. A washed membrane fraction from human neutrophils rapidly cleaved 0.5 mM Glut-Ala-Ala-Phe-MNA 996 nmol .cntdot. min-1 .cntdot. mg-1) and the hydrolysis was inhibited by phosphoroamidon and by specific antiserum to human renal NEP. The washed membrane fraction also rapidly cleaved 0.1 mM bradykinin (34 nmol .cntdot. min-1 mg-1) and 0.1 mM fMet-Leu-Phe (49 nmol .cntdot. min-1 .cntdot. mg-1). The membrane-bound enzyme cleaved the peptide substrates at the same site as the homogeneous human renal NEP, and phosphoramidon and thiorphan inhibited the hydrolysis. Kinetic studies with pure human renal NEP showed that the chemotactic peptide fMet-Leu-Phe was one of the best biologically active substrates (Km, 59 .times. 10-6 M; kcat, 3654 min-1). Immunocytochemistry at the light microscopic level revealed a high concentration of NeP on the cell membrane of neutrophils. This was confirmed with electron microscopy using the immunogold technique on ultrathin cryosections. These studies indicate that NEP in neutrophils may have important functions in inflammation and chemotaxis.