scholarly journals The metabolism of neuropeptides. Neurokinin A (substance K) is a substrate for endopeptidase-24.11 but not for peptidyl dipeptidase A (angiotensin-converting enzyme)

1985 ◽  
Vol 231 (2) ◽  
pp. 357-361 ◽  
Author(s):  
N M Hooper ◽  
A J Kenny ◽  
A J Turner
Keyword(s):  
Substance P ◽  
Selective Inhibitor ◽  
Synaptic Membranes ◽  
Neurokinin A ◽  
Pig Kidney ◽  
Endopeptidase 24.11 ◽  
The Family ◽  
General Capacity ◽  
Substance K ◽  
Hydrolysis Of

Both endopeptidase-24.11 and peptidyl dipeptidase A have previously been shown to hydrolyse the neuropeptide substance P. The structurally related peptide neurokinin A is also shown to be hydrolysed by pig kidney endopeptidase-24.11. The identified products indicated hydrolysis at two sites, Ser5-Phe6 and Gly8-Leu9, consistent with the known specificity of the enzyme. The pattern of hydrolysis of neurokinin A by synaptic membranes prepared from pig striatum was similar to that observed with purified endopeptidase-24.11, and hydrolysis was substantially abolished by the selective inhibitor phosphoramidon. Peptidyl dipeptidase A purified from pig kidney was shown to hydrolyse substance P but not neurokinin A. It is concluded that endopeptidase-24.11 has the general capacity to hydrolyse and inactivate the family of tachykinin peptides, including substance P and neurokinin A.

scholarly journals Metabolism of neuropeptides. Hydrolysis of the angiotensins, bradykinin, substance P and oxytocin by pig kidney microvillar membranes

1987 ◽  
Vol 241 (1) ◽  
pp. 237-247 ◽  
Author(s):  
S L Stephenson ◽  
A J Kenny
Keyword(s):  
Substance P ◽  
Membrane Fraction ◽  
Angiotensin I ◽  
Peptide Bonds ◽  
Prolonged Incubation ◽  
Pig Kidney ◽  
Angiotensin I Converting Enzyme ◽  
Endopeptidase 24.11 ◽  
20 Nm ◽  
Hydrolysis Of

Microvillar membranes derived from the brush border of the renal proximal tubule are very rich in peptidases. Pig kidney microvilli contain endopeptidase-24.11 associated with a battery of exopeptidases. The manner by which some neuropeptides are degraded by the combined attack of the peptidases of this membrane has been investigated. The contribution of individual peptidases was assessed by including inhibitors (phosphoramidon, captopril, amastatin and di-isopropyl fluorophosphate) with the membrane fraction when incubated with the peptides. Substance P, bradykinin and angiotensins I, II and III and insulin B-chain were rapidly hydrolysed by kidney microvilli. Oxytocin was hydrolysed much more slowly, but no products were detected from [Arg8]vasopressin or insulin under the conditions used for other peptides. The peptide bonds hydrolysed were identified and the contributions of the different peptidases were quantified. For each of the susceptible peptides, the main contribution came from endopeptidase-24.11 (inhibited by phosphoramidon). Peptidyl dipeptidase A (angiotensin-I-converting enzyme) was of less importance, even in respect of angiotensin I and bradykinin. When [2,3-Pro3,4-3H]bradykinin was also investigated at a lower concentration (20 nM), the conclusions in regard to the contributions of the two peptidases were unchanged. The possibility that endopeptidase-24.11 might attack within the six-residue disulphide-bridged rings of oxytocin and vasopressin was examined by dansyl(5-dimethylaminonaphthalene-1-sulphonyl)ation and by reduction and carboxymethylation of the products after incubation. Additional peptides were only observed after prolonged incubation, consistent with hydrolysis at the Tyr2-Ile3 and Tyr2-Phe3 bonds respectively. These results show that a range of neuropeptides are efficiently degraded by microvillar membranes and that endopeptidase-24.11 plays a key role in this process.

scholarly journals The hydrolysis of α-human atrial natriuretic peptide by pig kidney microvillar membranes is initiated by endopeptidase-24.11

1987 ◽  
Vol 243 (1) ◽  
pp. 183-187 ◽  
Author(s):  
S L Stephenson ◽  
A J Kenny
Keyword(s):  
Atrial Natriuretic Peptide ◽  
Natriuretic Peptide ◽  
Angiotensin I ◽  
Human Atrial Natriuretic Peptide ◽  
Pig Kidney ◽  
Angiotensin I Converting Enzyme ◽  
Endopeptidase 24.11 ◽  
Hydrolysis Of ◽  
Atrial Natriuretic

alpha-Human atrial natriuretic peptide, a 28-amino-acid-residue peptide, was rapidly hydrolysed by pig kidney microvillar membranes in vitro, with a t1/2 of 8 min, comparable with the rate observed with angiotensins II and III. The products of hydrolysis were analysed by h.p.l.c., the pattern obtained with membranes being similar to that with purified endopeptidase-24.11 (EC 3.4.24.11). No hydrolysis by peptidyl dipeptidase A (angiotensin I converting enzyme, EC 3.4.15.1) was observed. The contribution of the various microvillar membrane peptidases was assessed by including specific inhibitors. Phosphoramidon, an inhibitor of endopeptidase-24.11, caused 80-100% suppression of the products. Captopril and amastatin (inhibitors of peptidyl dipeptidase A and aminopeptidases respectively) had no significant effect. Hydrolysis at an undefined site within the disulphide-linked ring occurred rapidly, followed by hydrolysis at other sites, including the Ser25--Phe26 bond.

Distinct binding sites for substance P and neurokinin A (substance K): Co-transmitters in rat brain

Peptides ◽  
1985 ◽  
Vol 6 (2) ◽  
pp. 343-345 ◽  
Author(s):  
C.W. Shults ◽  
S.H. Buck ◽  
E. Burcher ◽  
T.N. Chase ◽  
T.L. O'Donohue
Keyword(s):  
Substance P ◽  
Rat Brain ◽  
Binding Sites ◽  
Neurokinin A ◽  
Substance K

scholarly journals Kidney neutral endopeptidase and the hydrolysis of enkephalin by synaptic membranes show similar sensitivity to inhibitors

1982 ◽  
Vol 203 (2) ◽  
pp. 519-522 ◽  
Author(s):  
I S Fulcher ◽  
R Matsas ◽  
A J Turner ◽  
A J Kenny
Keyword(s):  
Neutral Endopeptidase ◽  
Synaptic Membranes ◽  
General Role ◽  
Leucine Enkephalin ◽  
Pig Kidney ◽  
Pig Brain ◽  
Endopeptidase Activity ◽  
Hydrolysis Of

Neutral endopeptidase (EC 3.4.24.11) from pig kidney hydrolyses [125I]iodo-insulin B-chain and leucine-enkephalin. Both activities were equally sensitive to inhibition by phosphoramidon [N-(alpha-L-rhamnopyranosyloxyhydroxyphosphinyl)-L-leucyl-L-tryptophan] and thiorphan [N-(DL-2-benzyl-3-mercaptopropionyl)glycine]. Thermolysin hydrolysis of insulin B-chain was also sensitive to both inhibitors. The hydrolysis of the Gly3-Phe4 bond of Leu-enkephalin by synaptic membranes prepared from pig brain was partially inhibited by phosphoramidon and thiorphan. Synaptic membranes appear to contain another endopeptidase activity that is insensitive to these reagents. These observations suggest that enzymes similar to the kidney endopeptidase may play a general role in neuropeptide metabolism.

scholarly journals A comparison of the zinc contents and substrate specificities of the endothelial and testicular forms of porcine angiotensin converting enzyme and the preparation of isoenzyme-specific antisera

1992 ◽  
Vol 288 (3) ◽  
pp. 875-881 ◽  
Author(s):  
T A Williams ◽  
K Barnes ◽  
A J Kenny ◽  
A J Turner ◽  
N M Hooper
Keyword(s):  
Substance P ◽  
Angiotensin Converting Enzyme ◽  
Catalytic Site ◽  
Immunofluorescent Staining ◽  
Seminiferous Tubules ◽  
Converting Enzyme ◽  
Pig Kidney ◽  
Specific Antisera ◽  
Lh Rh ◽  
Hydrolysis Of

Angiotensin converting enzyme (ACE; EC 3.4.15.1) was purified from porcine kidney and lung (endothelial isoenzyme) and testis (testicular isoenzyme) by affinity chromatography on lisinopril-2.8 nm-Sepharose. Atomic-absorption spectroscopy revealed that ACE purified from kidney and lung contained 2.58 and 2.35 atoms of zinc per molecule of enzyme (M(r) 147,000) respectively. In contrast, ACE purified from testis contained only 1.58 atoms of zinc per molecule of enzyme (M(r) 80,000). Thus it would appear that both putative zinc-binding sites in endothelial ACE contain zinc and may therefore be catalytically active. No differences were observed in the pattern of products generated on hydrolysis of benzoyl (Bz)-Gly-His-Leu, substance P, luteinizing-hormone-releasing hormone (LH-RH) and its analogue, des-Gly10-LH-RH-ethylamide, by kidney and testicular ACE. There was also no difference in the initial rates of hydrolysis of Bz-Gly-His-Leu or substance P by the two isoenzymes, although LH-RH and its analogue were hydrolysed twice as rapidly by kidney ACE. It is therefore unlikely that the N-terminal catalytic site in porcine endothelial ACE is predominantly responsible for the atypical cleavage of LH-RH generating the N-terminal tripeptide. Two polyclonal antisera were raised to the affinity-purified forms of pig kidney and testicular ACE. Isoenzyme-specific antisera were then isolated from these by absorbing out those antibodies recognizing determinants on the other isoenzyme. Immunoelectrophoretic blot analyses and immunofluorescent staining of sections of pig kidney were used to demonstrate the specificity of the antisera. Immunofluorescent staining of sections of pig testis with the antiserum specific to testicular ACE localized testicular ACE solely to the lumen of the seminiferous tubules, whereas the antiserum specific to endothelial ACE revealed the presence of this isoenzyme only in blood vessels. The antiserum to endothelial ACE, which recognizes determinants in the unique N-terminal domain, was investigated as a possible specific inhibitor of the N-terminal catalytic site. Although this antiserum failed to inhibit testicular ACE, the effect on the activity of endothelial ACE appeared to be due to inhibition of both the N- and C-terminal catalytic sites.

scholarly journals The metabolism of neuropeptides. The hydrolysis of peptides, including enkephalins, tachykinins and their analogues, by endopeptidase-24.11

1984 ◽  
Vol 223 (2) ◽  
pp. 433-440 ◽  
Author(s):  
R Matsas ◽  
A J Kenny ◽  
A J Turner
Keyword(s):  
Amino Acid ◽  
Amino Acid Residues ◽  
Peptide Substrate ◽  
Peptide Substrates ◽  
Pig Kidney ◽  
Endopeptidase 24.11 ◽  
C Terminus ◽  
Wide Range ◽  
Amidated Peptide ◽  
Hydrolysis Of

Endopeptidase-24.11 (EC 3.4.24.11), purified to homogeneity from pig kidney, was shown to hydrolyse a wide range of neuropeptides, including enkephalins, tachykinins, bradykinin, neurotensin, luliberin and cholecystokinin. The sites of hydrolysis of peptides were identified, indicating that the primary specificity is consistent with hydrolysis occurring at bonds involving the amino group of hydrophobic amino acid residues. Of the substrates tested, the amidated peptide substance P is hydrolysed the most efficiently (Km = 31.9 microM; kcat. = 5062 min-1). A free alpha-carboxy group at the C-terminus of a peptide substrate is therefore not essential for efficient hydrolysis by the endopeptidase. A large variation in kcat./Km values was observed among the peptide substrates studied, a finding that reflects a significant influence of amino acid residues, remote from the scissile bond, on the efficiency of hydrolysis. These subsite interactions between peptide substrate and enzyme thus confer some degree of functional specificity on the endopeptidase. The inhibition of endopeptidase-24.11 by several compounds was compared with that of pig kidney peptidyldipeptidase A (EC 3.4.15.1). Of the inhibitors examined, only N-[1(R,S)-carboxy-2-phenylethyl]-Phe-p-aminobenzoate inhibited endopeptidase-24.11 but not peptidyldipeptidase. Captopril (D-3-mercapto-2-methylpropanoyl-L-proline), Teprotide (pGlu-Trp-Pro-Arg-Pro-Gln-Ile-Pro-Pro) and MK422 [N-[(S)-1-carboxy-3-phenylpropyl]-L-Ala-L-Pro] were highly selective as inhibitors of peptidyldipeptidase. Although not wholly specific, phosphoramidon was a more potent inhibitor of endopeptidase-24.11 than were any of the synthetic compounds tested.

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