DEVELOPMENT OF [I-125] RB104, A POTENT INHIBITOR OF NEUTRAL ENDOPEPTIDASE-24.11, AND ITS USE IN DETECTING NANOGRAM QUANTITIES OF THE ENZYME BY INHIBITOR GEL-ELECTROPHORESIS

Neutral endopeptidase 24.11, also known as the common acute lymphoblastic leukemia antigen, is a zinc metallopeptidase involved in the inactivation of biologically active peptides, such as the enkephalins and atrial natriuretic peptide. The highly potent radiolabeled inhibitor 2-{(3-[I-125]iodo-4-hydroxy)phenylmethyl}-4-N-[3-(hydroxyamino-3-oxo-1-phenylmethyl)propyl]amino-4-oxobutanoic acid ([I-125]RB104; K(i) = 30 pM) has been developed for the enzyme. [I-125]RB104 is highly specific, its K(i) for another widely distributed zinc peptidase, angiotensin-converting enzyme, being 15-mu-M. In binding studies using rat brain slices, [I-125]RB104 was shown to have a high affinity (K(d) = 300 +/- 20 pM) and high specific binding at the K(d) concentration (90%). With rat brain homogenates the K(d) of [I-125]RB104 was 26.8 +/- 0.9 pM, close to the kinetically derived K(d), 7.0 +/- 0.8 pM. Using the inhibitor, we have developed a simple, rapid, and quantitative technique to detect low nanogram quantities of the endopeptidase directly from tissue extracts after SDS/PAGE. The method has been used to show the presence of low quantities of the enzyme in rabbit bone marrow. Apart from its sensitivity, "inhibitor gel electrophoresis" using [I-125]RB104 has the advantage over immunohistochemical methods of being able to label the enzyme in all tissues and species. It will therefore be of great value in determining the exact role of this important regulatory peptidase in a number of biological systems. Moreover, this one-step characterization of neutral endopeptidase 24.11 could be extended to other zinc metallopeptidases such as angiotensin-converting enzyme or collagenases, and inhibitors with affinities as high as RB104 could open the way to visualization of zinc metallopeptidases in different tissues by electron microscopy.