Characterization of tynorphin, a potent endogenous inhibitor of dipeptidyl peptidaseIII.
To find a more effective inhibitor than spinorphin (LVVYPWT), an endogenous factor derived from bovine spinal cord, we synthesized spinorphin analogues and assayed their inhibitory activity toward DPPIII among enkephalin-degrading enzymes. Tynorphin (VVYPW), an N-terminal and C-terminal truncated form of spinorphin, exhibited more potent inhibitory activity and an IC50 value of 0.086 +/- 0.05 microg/ml (n = 4), whereas structures smaller than four amino acid residues exhibited almost no or less activity, suggesting that a five amino acid structure containing a Tyr-Pro residue is essential for the inhibition. The inhibition of DPPIII by tynorphin was predominantly competitive and the Ki value was found to be 7. 50 +/- 1.19 x 10(-8) M on Lineweaver-Burk plotting. The inhibitory activity of tynorphin toward other enkephalin-degrading enzymes such as neutral endopeptidase, aminopeptidase, and angiotensin-converting enzyme was not as high as that toward DPPIII, suggesting that tynorphin is a specific inhibitor of DPPIII. In HPLC analysis, human serum cleaved tynorphin rapidly (38% of control at 2 h and background level at 4 h), but in the presence of leuhisitin, an aminopeptidase inhibitor, tynorphin was maintained at the original level for 24 h. These results indicated that tynorphin had a more effective structure for expression of inhibitory activity toward DPPIII.
Yamamoto
Y
Y
Department of Molecular Oncology, The Tokyo Metropolitan Institute of Medical Science, Honkomagome 3-18-22, Bunkyo-ku, Tokyo 113-0021, Japan. yyama@rinshoken.or.jp
Hashimoto
J
J
Shimamura
M
M
Yamaguchi
T
T
Hazato
T
T
D000818:Animals; D001921:Brain / Q000201:enzymology; D002417:Cattle; D004152:Dipeptidyl-Peptidases and Tripeptidyl-Peptidases / Q000037:antagonists & inhibitors*; D004355:Drug Stability; D000882:Haplorhini; D006801:Humans; D009842:Oligopeptides / Q000097:blood / Q000737:chemistry*; D011480:Protease Inhibitors / Q000097:blood / Q000737:chemistry*; D013329:Structure-Activity Relationship
D009842:Oligopeptides; D011480:Protease Inhibitors; C412863:tynorphin; C082392:spinorphin; D004152:Dipeptidyl-Peptidases and Tripeptidyl-Peptidases; C100152:dipeptidyl peptidase III