Effects of three peptidase inhibitors, amastatin, captopril and phosphoramidon, on the hydrolysis of [Met5]-enkephalin-Arg6-Phe7 and other opioid peptides.
The contents of [Met5]-enkephalin-Arg6-Phe7 (met-enk-RF) and its six hydrolysis products: Y, YG, YGG, YGGF, YGGFM, and YGGFMR were estimated after incubating met-enk-RF with either a guinea-pig ileal or striatal membrane fraction for various times at 37 degrees C. After 45 min incubation with either ileal or striatal membranes, met-enk-RF was completely hydrolyzed, yielding Y as the major product. Incubation with either membrane preparation for 60 min in the presence of the aminopeptidase inhibitor amastatin hydrolyzed 90 or 92% of met-enk-RF, respectively, with YGG being the major product. If the dipeptidyl carboxypeptidase I inhibitor captopril is also included in the incubation, met-enk-RF hydrolysis decreases by about half for both membranes, with YGG remaining the major product. Inclusion of three peptidase inhibitors, amastatin, captopril, and phosphoramidon (inhibition of endopeptidase-24.11) further reduced met-enk-hydrolysis, with 87% or more remaining intact. This shows that met-enk-RF was mainly hydrolyzed by three enzymes, amastatin-sensitive aminopeptidase, captopril-sensitive dipeptidyl carboxypeptidase I and phosphoramidon-sensitive endopeptidase-24.11, in both ileal and striatal membranes. Additionally, estimations of [Leu5]-enkephalin (leu-enk), alpha- and beta-neoendorphins (alpha- and beta-neoends), and dynorphin B (dyn B) contents after incubating the individual peptides with striatal membrane for 60 min in the presence of the three peptidase inhibitors showed that 98, 32, 5, and 23%, respectively, remained intact. Our previous studies together with the data obtained here show that one group of endogenous opioid peptides: met-enk, leu-enk, met-enk-RF, met-enk-RGL, and dyn A-(1-8) are largely or almost exclusively hydrolyzed by the three enzymes, amastatin-sensitive aminopeptidase, captopril-sensitive dipeptidyl carboxypeptidase I, and phosphoramidon-sensitive endopeptidase-24.11, and indicate that an unidentified fourth enzyme(s) is involved in the hydrolysis of another group of peptides: alpha-neoend, beta-neoend, and dyn B.
Hiranuma
T
T
Department of Pharmacology, School of Medicine, Tokai University, Isehara, Japan.
Kitamura
K
K
Taniguchi
T
T
Kobayashi
T
T
Tamaki
R
R
Kanai
M
M
Akahori
K
K
Iwao
K
K
Oka
T
T
D000806:Angiotensin-Converting Enzyme Inhibitors / Q000494:pharmacology*; D000818:Animals; D000900:Anti-Bacterial Agents / Q000494:pharmacology*; D002216:Captopril / Q000494:pharmacology*; D004745:Enkephalins / Q000187:drug effects* / Q000378:metabolism; D006020:Glycopeptides / Q000494:pharmacology*; D006168:Guinea Pigs; D006868:Hydrolysis / Q000187:drug effects; D007082:Ileum / Q000187:drug effects / Q000378:metabolism; D008297:Male; D008566:Membranes / Q000187:drug effects / Q000378:metabolism; D018847:Opioid Peptides / Q000187:drug effects* / Q000378:metabolism; D010455:Peptides; D011480:Protease Inhibitors / Q000494:pharmacology*; D014793:Visual Cortex / Q000187:drug effects / Q000378:metabolism
D000806:Angiotensin-Converting Enzyme Inhibitors; D000900:Anti-Bacterial Agents; D004745:Enkephalins; D006020:Glycopeptides; D018847:Opioid Peptides; D010455:Peptides; D011480:Protease Inhibitors; C016977:amastatin; D002216:Captopril; C008890:phosphoramidon