Norma G. Delaney

Atrial natriuretic peptides cleaved by endopeptidase are inactive in conscious spontaneously hypertensive rats

The dose-related natriuretic and depressor responses to atrial natriuretic peptides (ANP) 99-126, 103-126 and 103-123 were determined in unanesthetized spontaneously hypertensive rats (SHR) and were compared to the activities of their Cys105-Phe106 ring-opened metabolites. These metabolites were previously identified as the major initial products formed by incubation of the intact peptides with neutral endopeptidase (NEP). The areas over the curves (AOC) of the depressor responses to the intact peptides were dose-related and, at 30 nmole/kg, iv were greatest for ANP 99-126 and 103-126 (833 +/- 241 and 1157 +/- 221 mm Hg x min). Thirty nmole/kg of ANP 103-123, a possible product of NEP cleavage of ANP 103-126, produced a lesser AOC (442 +/- 152 mm Hg x min) than did either of the longer peptides. The AOC responses to 100 nmole/kg of the ring-opened metabolites of ANP 99-126, 103-126 and 103-123 (105 +/- 80, 153 +/- 43 and 148 +/- 64 mm Hg x min) were not significantly different from the effect of vehicle treatment (84 +/- 23 mm Hg x min). Although the natriuretic responses to increasing doses of the intact peptides did not occur in a linear fashion, sodium excretion was maximally elevated by 24 +/- 4, 16 +/- 3 and 10 +/- 3 microEq/kg/min by 3 nmole/kg of ANP 99-126, 30 nmole/kg of ANP 103-126 and 10 nmole/kg of ANP 103-123, respectively. In contrast, the natriuretic responses to 100 nmole/kg of the ring-opened metabolites of ANP 99-126, 103-126 and 103-123 (1 +/- 0, 5 +/- 2 and 2 +/- 1 microEq/kg/min, respectively) were not significantly different from the response to vehicle treatment (3 +/- 1 microEq/kg/min). In conclusion, three ring-opened products of NEP cleavage of ANP 99-126, 103-126 and 103-123 were inactive in conscious SHR.

Rat brain enkephalinase: Characteristion of the active site using mercaptopropanoyl amino acid inhibitors, and comparison with angiotensin-converting enzyme

Over fifty mercaptopropanoyl amino acids and related derivatives were synthesized to define the steric, electronic and stereochemical requirements for binding to the active site of enkephalinase (ENKASE), and also for their ability to inhibit angiotensin-converting enzyme (ACE). In this way the character of ENKASE and ACE active sites were compared.

Purification and characterization of enkephalinase, angiotensin converting enzyme, and a third peptidyldipeptidase from rat brain

Three distinct peptidyldipeptidases (exopeptidases releasing carboxyl terminal dipeptide residues) can be solubilized from nerve terminal membrane fractions from whole rat brain or striatum, and separated by ion exchange chromatography. Brain angiotensin-converting enzyme (PDP-1) cleaves Hip-His-Leu, but not 80 nM [3H-Tyr1, Leu5]-enkephalin, and is markedly inhibited by several specific inhibitors such as captopril, teprotide, and MK-422. Enkephalinase (PDP-2) cleaves 80 nM [3H-Tyr1, Leu5]-enkephalin, but not Hip-His-Leu; it is not inhibited by any of the standard competitive inhibitors of angiotensin-converting enzyme (all analogs of carboxyl-terminal peptide sequences Phe-Ala-Pro or Ala-Pro), but is strongly inhibited by captopril analogs such as thiorphan (Phe-Gly analog). A third peptidyldipeptidase (PDP-3) cleaves Hip-His-Leu, but not 80 nM [3H-Tyr1, Leu5]-enkephalin; it is inhibited by dipeptide analog inhibitors such as captopril and thiorphan, but not by longer peptides such as teprotide or tripeptide analog inhibitors such as MK-422. Both PDP-2 (enkephalinase) and PDP-3 are apparently present in nerve terminal membranes predominantly as inactive proenzyme precursors, which elute from DEAE-cellulose at high salt concentration, and are activated very slowly by a process involving one or more trypsin-like enzymes. Rechromatography of activated PDP-2 and PDP-3 achieves a nearly complete separation of the two enzymes, both markedly purified, since each is much less acidic than its proenzyme precursor. Purified enkephalinase does not appear to have any significant endopeptidase activity. It cleaves Hip-Phe-Arg 200 times more effectively than Hip-Phe-Arg-NH2, and appears to be quite selective for cleaving the terminal dipeptide residue, Phe-Arg, from bradykinin, with no release of the second dipeptide and no cleavage of the Gly4-Phe5 interior peptide bond.