Screening for Rabbit Brain Neuropeptide‐metabolizing Peptidases. Inhibition of Endopeptidase B by Bradykinin Potentiating Peptide 9a (SQ 20881)

Abstract

Neutral thiol-activated peptidases present in the pH 5-soluble fraction of rabbit brain (separated by step-elution chromatography on diethylaminoethyl cellulose) were screened for the hydrolysis of bradykinin. Lys-bradykinin, Met-Lys-bradykinin, angiotensin I, angiotensin II, substance P, luteinizing hormone-releasing hormone (LH-RH), and neurotensin by bioassay. The column effluent was monitored for bradykinin inactivation and arylamidase activity and combined in six pools on the basis of bradykinin inactivation. The pools were characterized by determining the peptide fragments and amino acids released from bradykinin with an amino acid analyzer. Pools 1 through 3 contained 80% of the kininase activity and essentially all of the endopeptidase A and B activity, whereas pools 4 through 6 accounted for 98% of the recovered arylamidase activity. Bradykinin, angiotensin I, angiotensin II, and substance P were inactivated by all the pools, whereas LH-RH and neurotensin were inactivated by pools 3 and 4, and pools 3, 4, and 5, respectively. These data show that rabbit brain contains peptidases having some selectivity for the inactivation of neuropeptides. Endopeptidase B purified from pool 3 is inhibited by bradykinin-potentiating peptide 9a (BPP9a, SQ 20881) (< Glu-Trp-Pro-Arg-Pro-Gln-Ile-Pro-Pro), a competitive inhibitor of the hydrolysis of bradykinin (Km = 3.5 X 10(-5) M, Ki = 3 X 10(-6) M) which also completely inhibits the inactivation of LH-RH.

Brain endo-oligopeptidase B: a post-proline cleaving enzyme that inactivates angiotensin I and II.

“…The I M , 5 X 10" 8 M, for BPP te inhibition of the hydrolysis of 2 X 10" 4 M All is near the Ki, 3 X 10"* M, (competitive inhibition) determined using bradykinin as the substrate. 7 In contrast, captopril does not appreciably inhibit endo-oligopeptidase B. Although the inhibition of the enzyme by.…”

“…The enzyme was prepared from the supernatant fraction of rabbit brain by step elution chromatography on DEAE-cellulose, gel filtration on Sephadex G-100, and isoelectric focusing as described by Oliveira et al' with the modifications in DEAEcellulose chromatography described by Martins et al 7 The specific activity of the enzyme was 0.78 Mmole/min~1«mg protein" 1 for the hydrolysis of 8 X 10" 8 M bradykinin in 0.05 M sodium phosphate buffer, pH 7.5, containing 0.1 M NaCl, 5 X 10" 4 M DTT at 37°C. 7 The enzyme hydrolyzed only the Pro 7 -Phe a bond of bradykinin 7 and gave only one band after polyacrylamide gel electrophoresis at pH 8.9.…”

“…7 The enzyme hydrolyzed only the Pro 7 -Phe a bond of bradykinin 7 and gave only one band after polyacrylamide gel electrophoresis at pH 8.9. "…”

“…6 Endo-oligopeptidase B also inactivates luliberin and is inhibited by the nonapeptide BPP M (SQ 20,881, <Gln-Trp-Pro-Arg-Pro-Gln-IlePro-Pro). 7 BPP,,, some related peptides, and amino acid derivatives are potent inhibitors of angiotensinconverting enzyme that have been used to show the participation of the renin-angiotensin system in blood pressure regulation. 8 "…”

Peptidases in dog-ileum circular and longitudinal smooth-muscle plasma membranes. Their relative contribution to the metabolism of neurotensin

“…Thus, proline endopeptidasc and carboxypeptidase were never detected in the brain. Since these enzymes were previously shown to behave as cytosoluble peptidases [39], the question arises as to whether they represent genuinely membrane-associated proteins or result from minor contaminations of peripheral membranes by soluble proteins.…”