Purification of a specific inhibitor of pyroglutamyl aminopeptidase II from the marine annelide Hermodice carunculata. in vivo effects in rodent brain.
An inhibitor of the metallo-ectoenzyme, pyroglutamyl aminopeptidase II (PPII), a thyrotropin releasing hormone-specific peptidase, was identified by screening extracts from marine species of the Cuban coast-line belonging to the phylla Chordata, Echinodermata, Annelida, Mollusca, Cnidaria, Porifera, Chlorophyta and Magnoliophyta. Isolation of the inhibitor (HcPI), from the marine annelide Hermodice carunculata, was achieved by trichloroacetic acid treatment of the aqueous extract, followed by ion-exchange chromatography on DEAE Sephacel, gel filtration on Sephadex G-25 and reverse phase-HPLC. HcPI had a small apparent molecular weight (below 1000 Da) and was not a peptide. It inhibited rat PPII (a membrane preparation with 8.5mg protein/ml) with an apparent K(i) of 51 nM. HcPI did not inhibit serine (trypsin, chymotrypsin, elastase and dipeptidyl aminopeptidase IV), cysteine (papain, bromelain and pyroglutamyl aminopeptidase I), aspartic (pepsin and recombinant human immunodeficiency virus 1 protease (HIV1-PR)) nor other metallo proteinases (collagenase, gelatinase, angiotensin converting enzyme, aminopeptidase N and carboxypeptidase A). HcPI was non-toxic and active in vivo. Intraperitoneal injection of HcPI reduced mouse pituitary and brain PPII activity. Potency of the effect was higher in hypophysis and hypothalamus than in other brain regions. Intrathecal administration to male rats reduced PPII activity in the spinal cord. In conclusion we have identified a specific inhibitor of PPII that is the first M1 family zinc metallo-peptidase inhibitor isolated from marine invertebrates. It may be useful for elucidating the in vivo role of PPII in the pituitary and central nervous system.
Centro de Estudios de Proteínas, Facultad de Biología, Universidad de la Habana, Calle 25, Plaza de la Revolución, La Habana 10400, Cuba.
Isel
I
Pascual
Shirley
S
Gil-Parrado
Miguel
M
Cisneros
Patricia
P
Joseph-Bravo
Joaquín
J
Díaz
Lourival D
LD
Possani
Jean Louis
JL
Charli
María
M
Chávez
D000626:Aminopeptidases / Q000037:antagonists & inhibitors*; D000818:Animals; D001921:Brain / Q000187:drug effects*; D005591:Chemical Fractionation; D007274:Injections, Intraperitoneal; D007276:Injections, Intraventricular; D008297:Male; D051379:Mice; D008807:Mice, Inbred BALB C; D008970:Molecular Weight; D011077:Polychaeta / Q000737:chemistry*; D011480:Protease Inhibitors / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D011761:Pyrrolidonecarboxylic Acid / Q000031:analogs & derivatives; D051381:Rats; D017208:Rats, Wistar; D013116:Spinal Cord / Q000187:drug effects*; D013997:Time Factors
D011480:Protease Inhibitors; D000626:Aminopeptidases; C023166:pyroglutamyl-peptidase II; D011761:Pyrrolidonecarboxylic Acid