Neuropeptide specificity and inhibition of recombinant isoforms of the endopeptidase 3.4.24.16 family: comparison with the related recombinant endopeptidase 3.4.24.15.
Endopeptidase EC 3.4.24.16 (EP24.16c, neurolysin) and thimet oligopeptidase EC 3.4.24.15 are close related members of a large family of metalloproteases. Besides their cytosolic and membrane bound form, endopeptidase EC 3.4.24.16 appears to be present in the inner membrane of the mitochondria (EP24.16m). We have overexpressed two porcine EP24.16 isoforms in E. coli and purified the recombinant proteins to homogeneity. We show here that these peptidases hydrolyse a series of neuropeptides with similar rates and at sites reminiscent of those elicited by classically purified human brain EP24.16c. All neuropeptides, except neurotensin, were similarly cleaved by recombinant endopeptidase 3.4.24.15 (EP24.15, thimet oligopeptidase), another zinc-containing metalloenzyme structurally related to EP24.16. These two EP24.16 isoforms were drastically inhibited by Pro-Ile and dithiothreitol and remained unaffected by a specific carboalkyl inhibitor (CFP-AAY-pAb) directed toward the related EP24.15. The present purification procedure of EP24.16 should allow to establish, by mutagenesis analysis, the mechanistic properties of the enzyme.
Rioli
V
V
Biomedical Science Institute, University of São Paulo, 05508-900, Brazil.
Kato
A
A
Portaro
F C
FC
Cury
G K
GK
te Kaat
K
K
Vincent
B
B
Checler
F
F
Camargo
A C
AC
Glucksman
M J
MJ
Roberts
J L
JL
Hirose
S
S
Ferro
E S
ES
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D003600:Cytosol / Q000201:enzymology; D018076:DNA, Complementary; D004789:Enzyme Activation; D006801:Humans; D006868:Hydrolysis; D007527:Isoenzymes / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D008666:Metalloendopeptidases / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D008928:Mitochondria / Q000201:enzymology; D008969:Molecular Sequence Data; D009479:Neuropeptides / Q000378:metabolism*; D011994:Recombinant Proteins / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism; D013379:Substrate Specificity; D013552:Swine
D018076:DNA, Complementary; D007527:Isoenzymes; D009479:Neuropeptides; D011994:Recombinant Proteins; D008666:Metalloendopeptidases; C022510:thimet oligopeptidase; C062316:neurolysin