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.

A new feature of angiotensin-converting enzyme in the brain: Hydrolysis of substance P

1983 ◽  
Vol 116 (2) ◽  
pp. 735-742 ◽  
Author(s):  
Hideyoshi Yokosawa ◽  
Shogo Endo ◽  
Yasuhiko Ogura ◽  
Shin-ichi Ishii
Keyword(s):  
Substance P ◽  
Angiotensin Converting Enzyme ◽  
Converting Enzyme ◽  
New Feature ◽  
Hydrolysis Of ◽  
The Brain

scholarly journals Novel activity of angiotensin-converting enzyme. Hydrolysis of cholecystokinin and gastrin analogues with release of the amidated C-terminal dipeptide

1989 ◽  
Vol 262 (1) ◽  
pp. 125-130 ◽  
Author(s):  
P Dubreuil ◽  
P Fulcrand ◽  
M Rodriguez ◽  
H Fulcrand ◽  
J Laur ◽  
...  
Keyword(s):  
Angiotensin Converting Enzyme ◽  
Chloride Ion ◽  
Peptide Bond ◽  
Major Product ◽  
Enzyme Hydrolysis ◽  
Ion Concentration ◽  
Converting Enzyme ◽  
Rabbit Lung ◽  
Hydrolysis Of

ACE (angiotensin-converting enzyme; peptidyl dipeptidase A; EC 3.4.15.1), cleaves C-terminal dipeptides from active peptides containing a free C-terminus. We investigated the hydrolysis of cholecystokinin-8 [CCK-8; Asp-Tyr(SO3H)-Met-Gly-Trp-Met-Asp-Phe-NH2] and of various gastrin analogues by purified rabbit lung ACE. Although these peptides are amidated at their C-terminal end, they were metabolized by ACE to several peptide fragments. These fragments were analysed by h.p.l.c., isolated and identified by comparison with synthetic fragments, and by amino acid analysis. The initial and major site of hydrolysis was the penultimate peptide bond, which generated a major product, the C-terminal amidated dipeptide Asp-Phe-NH2. As a secondary cleavage, ACE subsequently released di- or tri-peptides from the C-terminal end of the remaining N-terminal fragments. The cleavage of CCK-8 and gastrin analogues was inhibited by ACE inhibitors (Captopril and EDTA), but not by other enzyme inhibitors (phosphoramidon, thiorphan, bestatin etc.). Hydrolysis of [Leu15]gastrin-(14-17)-peptide [Boc (t-butoxycarbonyl)-Trp-Leu-Asp-Phe-NH2] in the presence of ACE was found to be dependent on the chloride-ion concentration. Km values for the hydrolysis of CCK-8, [Leu15]gastrin-(11-17)-peptide and Boc-[Leu15]gastrin-(14-17)-peptide at an NaCl concentration of 300 mM were respectively 115, 420 and 3280 microM, and the catalytic constants were about 33, 115 and 885 min-1. The kcat/Km for the reactions at 37 degrees C was approx. 0.28 microM-1.min-1, which is approx. 35 times less than that reported for the cleavage of angiotensin I. These results suggest that ACE might be involved in the metabolism in vivo of CCK and gastrin short fragments.

scholarly journals Kinetic probes for inter-domain co-operation in human somatic angiotensin-converting enzyme

2005 ◽  
Vol 391 (3) ◽  
pp. 641-647 ◽  
Author(s):  
Olga E. Skirgello ◽  
Peter V. Binevski ◽  
Vladimir F. Pozdnev ◽  
Olga A. Kost
Keyword(s):  
Angiotensin Converting Enzyme ◽  
Enzymatic Activity ◽  
Active Site ◽  
The Other ◽  
Converting Enzyme ◽  
Human Enzyme ◽  
Independent Functions ◽  
The Common ◽  
The Individual ◽  
Hydrolysis Of

s-ACE (the somatic form of angiotensin-converting enzyme) consists of two homologous domains (N- and C-domains), each bearing a catalytic site. Negative co-operativity between the two domains has been demonstrated for cow and pig ACEs. However, for the human enzyme there are conflicting reports in the literature: some suggest possible negative co-operativity between the domains, whereas others indicate independent functions of the domains within s-ACE. We demonstrate here that a 1:1 stoichiometry for the binding of the common ACE inhibitors, captopril and lisinopril, to human s-ACE is enough to abolish enzymatic activity towards FA {N-[3-(2-furyl)acryloyl]}-Phe-GlyGly, Cbz (benzyloxycarbonyl)-Phe-His-Leu or Hip (N-benzoylglycyl)-His-Leu. The kinetic parameters for the hydrolysis of seven tripeptide substrates by human s-ACE appeared to represent average values for parameters obtained for the individual N- and C-domains. Kinetic analysis of the simultaneous hydrolysis of two substrates, Hip-His-Leu (S1) and Cbz-Phe-His-Leu (S2), with a common product (His-Leu) by s-ACE at different values for the ratio of the initial concentrations of these substrates (i.e. σ=[S2]0/[S1]0) demonstrated competition of these substrates for binding to the s-ACE molecule, i.e. binding of a substrate at one active site makes the other site unavailable for either the same or a different substrate. Thus the two domains within human s-ACE exhibit strong negative co-operativity upon binding of common inhibitors and in the hydrolysis reactions of tripeptide substrates.

scholarly journals Hydrolysis of Biological Peptides by Human Angiotensin-converting Enzyme-related Carboxypeptidase

2002 ◽  
Vol 277 (17) ◽  
pp. 14838-14843 ◽  
Author(s):  
Chad Vickers ◽  
Paul Hales ◽  
Virendar Kaushik ◽  
Larry Dick ◽  
James Gavin ◽  
...  
Keyword(s):  
Angiotensin Converting Enzyme ◽  
Converting Enzyme ◽  
Hydrolysis Of

Angiotensin converting enzyme in brush-border membranes of avian small intestine

1988 ◽  
Vol 135 (1) ◽  
pp. 1-8
Author(s):  
B. R. Stevens ◽  
A. Fernandez ◽  
C. del Rio Martinez
Keyword(s):  
Angiotensin Converting Enzyme ◽  
Brush Border ◽  
Physiological Role ◽  
Intestinal Epithelial ◽  
Converting Enzyme ◽  
Brush Border Membranes ◽  
Fluid Uptake ◽  
The Common ◽  
Kinetics Of ◽  
Hydrolysis Of

Angiotensin converting enzyme activity was identified in brush-border membranes purified from the small intestinal epithelium of the common grackle, Quiscalus quiscula. Angiotensin converting enzyme was enriched 20-fold in the membrane preparation, compared with intestinal epithelial cell scrapes, and was coenriched with the brush-border markers, alkaline phosphatase and aminopeptidase N. The kinetics of hydrolysis of N-[3-(2-furyl)acryloyl]-L-phenylalanylglycylglycine (FAPGG) gave a Vmax of 907 +/− 41 units g-1 and a Km of 55 +/− 6 mumol l-1. The avian intestinal angiotensin converting enzyme was inhibited by the antihypertensive drug, Ramipril, with a median inhibitory concentration (IC50) of 1 nmol l-1. In the light of previous studies on angiotensin converting enzyme in mammalian epithelia, these results may implicate a physiological role for angiotensin converting enzyme in regulating electrolyte and fluid uptake in bird small intestines.

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.

Effects of gas composition and pH on kinetics of lung angiotensin-converting enzyme

1987 ◽  
Vol 62 (3) ◽  
pp. 1216-1221 ◽  
Author(s):  
D. A. Rickaby ◽  
R. D. Bongard ◽  
M. J. Tristani ◽  
J. H. Linehan ◽  
C. A. Dawson
Keyword(s):  
Angiotensin Converting Enzyme ◽  
Surface Area ◽  
Ph Dependence ◽  
Gas Composition ◽  
Converting Enzyme ◽  
Hydrolysis Process ◽  
Hypoxic Vasoconstriction ◽  
Ace Activity ◽  
Kinetics Of ◽  
Hydrolysis Of

Given the pH dependence of enzymes in general and the potential importance of a blood and alveolar gas composition dependency on the interpretation of changes in the hydrolysis of angiotensin-converting enzyme (ACE) substrates by pulmonary endothelial ACE, we examined the influence of Pco2 and Po2 on the hydrolysis of a synthetic ACE substrate (benzoyl-phenylalanyl-alanyl-proline, BPAP) on passage through isolated rabbit lungs. Perfusate pH values of about 7.1, 7.4, and 7.9 were obtained by ventilating the lungs with gas containing different CO2 concentrations and Po2 values of approximately 110 and approximately 10 Torr were obtained by varying the concentration of O2 in the ventilating gas mixture. In the range studied neither acidosis nor alkalosis produced any significant changes in BPAP hydrolysis or in the kinetic parameters, Vmax and Km, for the hydrolysis process. On the other hand, a reduction in BPAP hydrolysis was detected when the Po2 was reduced from 110 to 10 Torr. The Vmax for BPAP hydrolysis by the lung was inversely correlated with the magnitude of the hypoxic vasoconstriction that occurred, suggesting that the reduced BPAP hydrolysis with hypoxia was due to the loss of perfused surface area due to the vasoconstriction. The results suggest that correlations between Pco2 and/or pH and whole-lung ACE activity that might occur in diseased lungs do not imply causalty. The hemodynamic consequences of changing Po2 (i.e., hypoxic vasoconstriction) may alter whole-organ ACE activity in the sense of changing the perfused surface area (i.e., the amount of ACE in contact with flowing perfusate).

Hydrolysis of a synthetic angiotensin-converting enzyme substrate in dog lungs

1989 ◽  
Vol 257 (6) ◽  
pp. H2006-H2016
Author(s):  
J. H. Linehan ◽  
T. A. Bronikowski ◽  
D. A. Rickaby ◽  
C. A. Dawson
Keyword(s):  
Angiotensin Converting Enzyme ◽  
Flow Rate ◽  
Dilution Method ◽  
Converting Enzyme ◽  
Pulmonary Endothelium ◽  
Multiple Indicator Dilution ◽  
Enzyme Substrate ◽  
Multiple Indicator ◽  
Pass Through ◽  
Hydrolysis Of

The present study was carried out to begin to evaluate the saturable kinetics of the hydrolysis of a synthetic substrate, benzoyl-phenylalanyl-alanyl-proline (BPAP), for angiotensin-converting enzyme (ACE), by the pulmonary endothelium of the dog using a multiple indicator dilution method. In the experiments, isolated dog lung lobes were perfused with a salt solution containing 5% bovine serum albumin. Boluses containing [3H]BPAP, and various amounts of unlabeled BPAP were injected into the lobar artery, and timed samples of venous effluent were collected. The samples were analyzed to determine the fractional hydrolysis of the injected BPAP. The BPAP hydrolysis on passage through the lungs exhibited the saturable behavior and the relative insensitivity to changing flow rate previously described. Since we have described previously that BPAP behaves as if it exists in two forms, one of which is virtually unhydrolyzable on a single pass through the lungs, a model was formulated to include the influence of the unhydrolyzable form, as well as the saturable hydrolysis of the hydrolyzable form, on the fractional hydrolysis of the injected BPAP. This model provides a new method for estimating the kinetic parameters of BPAP hydrolysis by pulmonary endothelial ACE, and it explains the observation that the fractional BPAP hydrolysis does not vary with flow rate and transit time to the extent predicted by previous models.

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