Inhibition of substance P degradation in rat brain preparations by peptide hydroxamic acids.
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. [Eur. J. Biochem. 114, 315-327 (1981)]. 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 microM. 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 microM. The degradation of substance P by rat diencephalon slices was inhibited to the same extent (40-50%) by IBH-Phe-Phe-Gly-NHOH (20 microM) and by phosphoramidon (1 microM). 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.
R
R
Laufer
A
A
Ewenson
C
C
Gilon
M
M
Chorev
Z
Z
Selinger
D000818:Animals; D001921:Brain / Q000201:enzymology*; D004027:Diencephalon / Q000201:enzymology; D006877:Hydroxamic Acids / Q000138:chemical synthesis / Q000494:pharmacology*; D066298:In Vitro Techniques; D008297:Male; D008666:Metalloendopeptidases; D009842:Oligopeptides / Q000494:pharmacology; D010446:Peptide Fragments / Q000378:metabolism; D010455:Peptides / Q000138:chemical synthesis / Q000494:pharmacology*; D011480:Protease Inhibitors; D011863:Radioimmunoassay; D051381:Rats; D013373:Substance P / Q000031:analogs & derivatives / Q000378:metabolism*
D006877:Hydroxamic Acids; D009842:Oligopeptides; D010446:Peptide Fragments; D010455:Peptides; D011480:Protease Inhibitors; D013373:Substance P; C046092:3-iododesaminotyrosyl-phenylalanyl-phenylalanyl-glycine hydroxamic acid; C046093:substance P (6-11), N(alpha)-(3-iododesaminotyrosyl)-; D008666:Metalloendopeptidases; C029610:substance P degrading enzyme