Characterization, localization and quantification of neutral metalloendopeptidase, the degradative enzyme for atrial natriuretic peptide

心房利钠肽（ANP）被中性金属内肽酶（NEP）迅速降解，肾脏是ANP清除的主要部位。NEP已通过体外放射自显影术在大鼠肾脏中解剖定位，并通过使用新开发的放射抑制剂作为放射配体的放射抑制结合试验（RIBA）研究了其活性位点。SCH47896是SCH39370的酚类衍生物，SCH39370是一种有效的NEP特异性抑制剂，可以用^<125>I进行放射性碘化。SCH47896及其二碘类似物SCH48446可抑制NEP在大鼠肾脏中的催化活性。[^<125>I]SCH47896特异性结合肾质膜符合单位点模型，Kd = 43.3 nM，最大结合位点密度= 13.8 pmol/mg蛋白。因此，[^<125>I]SCH47896保留了完全的酶抑制活性和与NEP活性位点的完全结合。使用[^<125>I]SCH47896的x线自摄影显示与肾近端小管深层结合最大。这种结合被NEP抑制剂以剂量依赖的方式取代。目前的研究表明，NEP对ANP的降解主要发生在近端小管深部，而外皮层的近曲小管不是NEP的主要定位部位。这些特性使得[^<125>I]SCH47896可以用作RIBA和体外放射自显影的放射配体。利用这些方法，研究不同生理和病理生理条件下NEP的调节是可能的。此外，这种结合放射抑制剂的方法适用于任何酶，只要可以构建合适的放射配体。

Atrial natriuretic peptide (ANP) is rapidly degraded by neutral metalloendopeptidase (NEP), with the kidney being a major site of ANP clearance. NEP has been anatomically localized in the rat kidney by in vitro autoradiography and the active site studied by a radioinhibitory binding assay (RIBA) using a newly developed radioinhibitor as a radioligand. SCH47896 is a phenolic derivative of SCH39370, a potent specific inhibitor of NEP, which can be radioiodinated with ^<125>I.NEP catalytic activity in the rat kidney was inhibited by SCH47896 and its di-iodo analog SCH48446. Specific binding of [^<125>I]SCH47896 to renal plasma membranes fitted a single-site model with Kd = 43.3 nM and maximal binding site density = 13.8 pmol/mg protein. Thus, [^<125>I]SCH47896 retains full enzymatic inhibitory activity and full binding to the active site of the NEP.Autoradiographs using [^<125>I]SCH47896 demonstrated maximal binding to deep proximal renal tubules. This binding was displaced in a dose-dependent manner by NEP inhibitors. The present studies suggest that degradation of ANP by NEP occurs mainly in the deep proximal tubules, and that the proximal convoluted tubule in the outer cortex is not a major site of location of NEP.These properties enable [^<125>I]SCH47896 to be used as a radioligand for RIBA and in vitro autoradiography. Using these methods, investigation of the regulation of NEP under different physiological and pathophysiological conditions is possible. Furthermore, this method of radioinhibitor binding is applicable to any enzyme, provided a suitable radioligand can be constructed.