Molecular cloning and expression of rat brain endopeptidase 3.4.24.16.
We have isolated by immunological screening of a lambda ZAPII cDNA library constructed from rat brain mRNAs a cDNA clone encoding endopeptidase 3.4.24.16. The longest open reading frame encodes a 704-amino acid protein with a theoretical molecular mass of 80,202 daltons and bears the consensus sequence of the zinc metalloprotease family. The sequence exhibits a 60.2% homology with those of another zinc metallopeptidase, endopeptidase 3.4.24.15. Northern blot analysis reveals two mRNA species of about 3 and 5 kilobases in rat brain, ileum, kidney, and testis. We have transiently transfected COS-7 cells with pcDNA3 containing the cloned cDNA and established the overexpression of a 70-75-kDa immunoreactive protein. This protein hydrolyzes QFS, a quenched fluorimetric substrate of endopeptidase 3.4.24.16, and cleaves neurotensin at a single peptide bond, leading to the formation of neurotensin (1-10) and neurotensin (11-13). QFS and neurotensin hydrolysis are potently inhibited by the selective endopeptidase 3.4.24.16 dipeptide blocker Pro-Ile and by dithiothreitol, while the enzymatic activity remains unaffected by phosphoramidon and captopril, the specific inhibitors of endopeptidase 3.4.24.11 and angiotensin-converting enzyme, respectively. Altogether, these physicochemical, biochemical, and immunological properties unambiguously identify endopeptidase 3.4.24.16 as the protein encoded by the isolated cDNA clone.
Dauch
P
P
Institut de Pharmacologie Moléculaire et Cellulaire, Valbonne, France.
Vincent
J P
JP
Checler
F
F
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001921:Brain / Q000201:enzymology*; D002460:Cell Line; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000235:genetics; D015870:Gene Expression; D008297:Male; D008666:Metalloendopeptidases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009496:Neurotensin / Q000378:metabolism; D009842:Oligopeptides / Q000737:chemistry / Q000378:metabolism; D016366:Open Reading Frames; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D051381:Rats; D013379:Substrate Specificity; D014018:Tissue Distribution; D014162:Transfection
D018076:DNA, Complementary; D009842:Oligopeptides; D012333:RNA, Messenger; C064546:7-methoxycoumarin-3-carboxylyl-prolyl-leucyl-glycyl-prolyl-lysyl-2,4-dinitrophenyl; D009496:Neurotensin; D008666:Metalloendopeptidases; C062316:neurolysin