Enzymatic inactivation of bradykinin by rat brain neuronal perikarya.
1. Bradykinin (Bk; Arg1-Pro2-Pro3-Gly4-Phe5-Ser6-Pro7-Phe8-Arg8) inactivation by bulk isolated neurons from rat brain is described. 2. Bk is rapidly inactivated by neuronal perikarya (4.2 +/- 0.6 fmol/min/cell body). 3. Sites of inactivating cleavages, determined by a kininase bioassay combined with a time-course Bk-product analysis, were the Phe5-Ser6, Pro7-Phe8, Gly4-Phe5, and Pro3-Gly4 peptide bonds. The cleavage of the Phe5-Ser6 bond inactivated Bk at least five fold faster than the other observed cleavages. 4. Inactivating peptidases were identified by the effect of inhibitors on Bk-product formation. The Phe5-Ser6 bond cleavage is attributed mainly to a calcium-activated thiol-endopeptidase, a predominantly soluble enzyme which did not behave as a metalloenzyme upon dialysis and was strongly inhibited by N-[1(R,S)-carboxy-2-phenylethyl]-Ala-Ala-Phe-p-aminobenzoate and endo-oligopeptidase A antiserum. Thus, neuronal perikarya thiol-endopeptidase seems to differ from endo-oligopeptidase A and endopeptidase 24.15. 5. Endopeptidase 24.11 cleaves Bk at the Gly4-Phe5 and, to a larger extent, at the Pro7-Phe8 bond. The latter bond is also cleaved by angiotensin-converting enzyme (ACE) and prolyl endopeptidase (PE). PE also hydrolyzes Bk at the Pro3-Gly4 bond. 6. Secondary processing of Bk inactivation products occurs by (1) a rapid cleavage of Ser6-Pro7-Phe8-Arg8 at the Pro7-Phe8 bond by endopeptidase 24.11, 3820ACE, and PE; (2) a bestatin-sensitive breakdown of Phe8-Arg9; and (3) conversion of Arg1-Pro7 to Arg1-Phe5, of Gly4-Arg9 to both Gly4-Pro7 and Ser6-Arg9, and of Phe5-Arg9 to Ser6-Arg9, Phe8-Arg9, and Ser6-Pro7, by unidentified peptidases. 7. A model for the enzymatic inactivation of bradykinin by rat brain neuronal perikarya is proposed.
DelBel
E A
EA
Departamento de Farmacologia, Faculdade de Medicina de Ribeirão Prêto, Universidade de São Paulo, Brasil.
Padovan
A P
AP
Padovan
G J
GJ
Sellinger
O Z
OZ
Martins
A R
AR
D000596:Amino Acids / Q000032:analysis; D000818:Animals; D001681:Biological Assay; D001920:Bradykinin / Q000378:metabolism*; D001921:Brain / Q000166:cytology / Q000187:drug effects / Q000201:enzymology*; D004247:DNA / Q000032:analysis; D003956:Dialysis; D010450:Endopeptidases / Q000032:analysis; D006868:Hydrolysis; D008670:Metals / Q000494:pharmacology; D009419:Nerve Tissue Proteins / Q000032:analysis; D009474:Neurons / Q000187:drug effects / Q000201:enzymology* / Q000648:ultrastructure; D010455:Peptides / Q000378:metabolism; D007703:Peptidyl-Dipeptidase A / Q000032:analysis; D000085822:Prolyl Oligopeptidases; D011480:Protease Inhibitors / Q000494:pharmacology; D051381:Rats; D011919:Rats, Inbred Strains; D012697:Serine Endopeptidases
D000596:Amino Acids; D008670:Metals; D009419:Nerve Tissue Proteins; D010455:Peptides; D011480:Protease Inhibitors; D004247:DNA; D010450:Endopeptidases; D007703:Peptidyl-Dipeptidase A; D012697:Serine Endopeptidases; D000085822:Prolyl Oligopeptidases; D001920:Bradykinin