Inhibition of substance P degradation in rat brain preparations by peptide hydroxamic acids

Abstract

A peptidase activity of rat diencephalon membranes, which acts on the C-terminal hexapeptide sequence of substance P, was characterized using the radiolabeled substrate N.alpha.-([125I]iododesaminotyrosyl)-substance P (6-11)-hexapeptide. This activity presents certain characteristics similar to those of the substance P-degrading enzyme purified from human brain by Lee et al. It is inhibited by metal chelators and some thiol reagents, but is insensitive to inhibitors of serine proteases and aminopeptidases. The activity is different from angiotensin-converting enzyme and enkephalinase, since it is not affected by specific inhibitors of these enzymes. Substance P and substance P C-terminal fragments longer than the pentapeptide inhibited the degradation of the radiolabeled substrate with inhibition constants around 200 .mu.M. Short fragments of the substance P sequence, such as Boc-Phe-Phe-OMe and Boc-Phe-Phe-Gly-OEt, were also found to inhibit the degradation of the substrate. When the metal-chelating hydroxamic acid moiety was attached to the carboxyl terminus of these short peptides, potent inhibitors of the substance-P-degrading activity were obtained, with inhibition constants in the micromolar range. The most potent of these compounds, iododesaminotyrosyl-Phe-Phe-Gly-NHOH (IBH-Phe-Phe-Gly-NHOH), is a competitive inhibitor, with a Ki value of 1.9 .mu.M. The degradation of substance P by rat diencephalon slices was inhibited to the same extent (40-50%) by IBH-Phe-Phe-Gly NHOH (20 .mu.M) and by phosphoramidon (1 .mu.M). A combination of both reagents reduced the degradation rate by 75-80%, suggesting that both enkephalinase and the substance-P-degrading activity are involved in the metabolism of substance P in this preparation. IBH-Phe-Phe-Gly-NHOH seems to be quite specific for the latter enzyme, since at a high concentration (0.1 mM) it did not affect the degradation of the radiolabeled substrate by .alpha.-chymotrypsin, papain or thermolysin.