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At least 19 recordsLinked to original sources
PubMed2005

Metal ion modulation of cystinyl aminopeptidase.

Cystinyl aminopeptidase has one Zn2+-binding motif and is a member of the M1 aminopeptidase family. Ion modulation of its catalytic activity was studied in membranes of CHO-K1 cells (Chinese-hamster ovary K1 cells) using L-leucine-p-nitroanilide as substrate. The planar bidentate chelators 1,10-phenanthroline and 2,2'-bipyridine inhibited the activity in a concentration-dependent manner with Hill slopes of 3.32+/-1.78 and 2.10+/-0.26 respectively. The acetic acid-containing chelators EDTA, EGTA and DTPA (diethylenetriamine-N,N,N',N'',N''-penta-acetic acid) weakly affected the activity, but they increased the potency of the planar chelators up to a limit, at which Hill slopes became close to unity. Moreover, competition between 1,10-phenanthroline and the substrate only took place in the presence of EDTA. These findings are compatible with a model in which the bidentate chelators inhibit enzyme activity by decreasing the free Zn2+ concentration. By removing a modulatory ion from an allosteric site at the enzyme, the acetic acid-containing chelators facilitate the direct interaction between the bidentate chelators and the catalytic Zn2+. The inhibitory effect of EDTA plus 1,10-phenanthroline could be completely reversed by Zn2+. Ca2+ and Mg2+ increased the potency of Zn2+ for this process. This is expected if they interact with the modulatory site to decrease the sensitivity of the enzyme towards 1,10-phenanthroline. Conversely, the bidendate chelators increased the high-affinity [125I]angiotensin IV binding to the membranes and this was potentiated by the acetic acid-containing chelators. These findings support the concept that high-affinity [125I]angiotensin IV binding, previously referred to as 'AT4 receptor binding', only occurs for the cystinyl aminopeptidase apoenzyme.

2,2'-Dipyridyl↗
PubMed2006

Angiotensin AT4 receptor ligand interaction with cystinyl aminopeptidase and aminopeptidase N: [125I]Angiotensin IV only binds to the cystinyl aminopeptidase apo-enzyme.

Due to its high affinity for [(125)I]Angiotensin IV, cystinyl aminopeptidase (CAP) has recently been assigned as the 'angiotensin AT(4) receptor'. Since the aminopeptidase N (AP-N) activity is also susceptible to inhibition by Angiotensin IV, it might represent an additional target for this peptide. Based on [(125)I]Angiotensin IV binding and catalytic activity measurements, we compared the ligand interaction properties of recombinant human CAP and human AP-N. Both enzymes displayed distinct pharmacological profiles. Although their activity is inhibited by Angiotensin IV and LVV-hemorphin 7, both peptides are more potent CAP-inhibitors. On the other hand, substance P and l-methionine have a higher potency for AP-N. High affinity binding of [(125)I]Angiotensin IV to CAP occurs in the presence of chelators but not to AP-N in either the absence or presence of chelators. These differences were exploited to determine whether CAP and/or AP-N are present in different cell lines (CHO-K1, COS-7, HEK293, SK-N-MC and MDBK). We provide evidence that CAP predominates in these cell lines and that, comparatively, CHO-K1 cells display the highest level of this enzyme.

Angiotensin II↗
PubMed2002

Ectodomain shedding of cystinyl aminopeptidase from human placental membranes.

Cystinyl aminopeptidase (CAP; EC 3.4.11.3) is an integral protein of the placental membrane that is also found in a soluble form in maternal serum during pregnancy. CAP was found to be shed from human placental membranes in a temperature- and time-dependent process. The released form of CAP was hydrophilic as assessed by phase separation in Triton X-114 and high-speed centrifugation. This ectodomain shedding of CAP was inhibited by the hydroxamic acid-based compounds marimastat and BB3103. The inhibition profile for the shedding of CAP was distinct to that for the release of angiotensin converting enzyme, implicating the involvement of distinct zinc metallosecretases in the shedding of these two proteins. These results have implications for our understanding of the mechanism underlying the reduction in serum levels of CAP observed in certain pregnancy-related disorders, such as pre-eclampsia.

Adult↗
PubMed2004

Synergistic modulation of cystinyl aminopeptidase by divalent cation chelators.

Membranes of Chinese hamster ovary (CHO-K1) cells were used to study the opposite modulation of enzyme activity and [125I]Ang IV binding to cystinyl aminopeptidase (EC 3.4.11.3) by divalent cation chelators. Whereas ethylene diamine tetraacetic acid (EDTA) or ethylene glycol-bis(2-aminoethylether)-N,N,N',N'-tetraacetic acid (EGTA) alone only slightly affected the enzyme activity, 1,10-phenanthrolin (1,10-PHE) produced a complete and concentration-dependent inhibition. Interestingly EDTA (> or =0.05 mM) or EGTA (> or =0.15 mM) enhanced the inhibitory effect of 1,10-PHE. Two-site analysis of the corresponding inhibition curves revealed that EDTA and EGTA converted enzymes with low sensitivity towards 1,10-PHE into enzymes with high sensitivity. The combined inhibition by EDTA (0.1 mM) and 1,10-PHE (0.1 mM) could be prevented and reversed by addition of Zn2+ (at about 0.04-0.1 mM). In contrast, specific binding of [125I]Ang IV was enhanced in the presence of 1,10-PHE. Binding was only slightly affected by EDTA or EGTA alone. Furthermore, the stimulatory effect of 1,10-PHE was potentiated by EDTA (> or =0.05 mM) as well as EGTA (> or =0.15 mM). In the presence of EDTA (0.1 mM) and 1,10-PHE (0.1 mM), specific [125I]Ang IV binding was completely inhibited by Zn2+ (IC50= 39.7 +/- 6.2 microM). The present data show that divalent cations such as Zn2+ are essential for the enzyme activity of cystinyl aminopeptidase and inhibitory for [125I]Ang IV binding. Modulation of the effects of 1,10-PHE by other chelators such as EDTA or EGTA, suggests that, in addition to the binding site for zinc in the catalytic site, cystinyl aminopeptidase also bears a regulatory divalent cation binding site.

Angiotensin II↗
PubMed2004

Endogenous cystinyl aminopeptidase in Chinese hamster ovary cells: characterization by [125I]Ang IV binding and catalytic activity.

The angiotensin II C-terminal hexapeptide fragment angiotensin IV (Ang IV) exerts central and cardiovascular effects. Cystinyl aminopeptidase (EC 3.4.11.3), a membrane-associated zinc-dependent metallopeptidase of the M1 family, has recently been found to display high affinity for Ang IV and it was proposed to represent the AT4 receptor. We present evidence for the presence of endogenous cystinyl aminopeptidase in membranes from Chinese hamster ovary (CHO-K1) cells by binding studies with [125I]Ang IV and by measuring the cleavage of L-leucine-p-nitroanilide. The equilibrium dissociation constant of [125I]Ang IV in saturation binding studies (KD= 0.90 nM) was similar to the value (KD= 0.70 nM) calculated from the association and dissociation rates. Binding was displaced with high potency by the "AT4 receptor" ligands (Ang IV > divalinal1-Ang IV approximately LVV-hemorphin-7 approximately LVV-hemorphin-6 > Ang (3-7) > Ang III > Ang (4-8)) but not by AT1/AT2 receptor antagonists. Enzymatic activity in CHO-K1 cell membranes was competitively inhibited upto 94% by Ang IV and other "AT4 receptor" ligands (Ang IV > Ang III approximately divalinal1-Ang IV approximately Ang (3-7) approximately LVV-hemorphin-7 > Ang (4-8) approximately LVV-hemorphin-6). High affinity binding of [125I]Ang IV required the presence of metal chelators and the ligands such as Ang IV and LVV-hemorphin-7 displayed higher potency in the binding studies as in the enzyme assay. This difference in potency varied from one peptide to another. These pharmacological properties match those previously reported for the recombinantly-expressed human cystinyl aminopeptidase in embryonal kidney cells.

Angiotensin II↗
PubMed2001

Pituitary aminopeptidase activities involved in blood-pressure regulation are modified by dietary cholesterol: sex differences.

Given that the existence of a local renin-angiotensin system (RAS) in the pituitary and its participation in the regulation of blood pressure and other biological functions are widely accepted, the aim of this work is to analyze the influence of dietary cholesterol on the activity of the enzymes involved in the metabolism of the effector peptides of the renin-angiotensin system (angiotensin II and III) and vasopressin, in the pituitary of male and female mice fed on a cholesterol-enriched diet (1% cholesterol and 0.5% cholic acid). Soluble and membrane-bound pituitary aminopeptidase A (aspartyl- and glutamyl-aminopeptidase), aminopeptidase M (alanyl-aminopeptidase), aminopeptidase B (arginyl-aminopeptidase) and cystinyl-aminopeptidase activities were fluorimetrically measured. In female mice, cholesterol-enriched diet produced a significant increase in soluble aspartyl- and membrane-bound aspartyl- and glutamyl-aminopeptidase activities, and a significant decrease in membrane-bound alanyl-, arginyl- and cystinyl-aminopeptidase activities. In male mice, after feeding the diet, a significant increase in soluble glutamyl- and membrane-bound arginyl-aminopeptidase activities was observed. Our results indicate differential effects of dietary cholesterol on the metabolism of angiotensin II and III and vasopressin in the pituitary of male and female mice.

Animals↗
PubMed1998

Activities of aminopeptidases in a rat saline model of volume hypertension.

Aminopeptidase activity plays a role in the metabolism of several peptides that could be involved in blood pressure control. This activity has been implicated in the pathogenesis of hypertension, essentially in spontaneously hypertensive rats. However, few studies have examined aminopeptidase activities in animal models other than genetic hypertension. To analyze the aminopeptidase response to the specific conditions of the reduced renal mass saline model of arterial hypertension, aminopeptidase A activity (glutamyl- and aspartyl-aminopeptidase), aminopeptidase M activity (alanyl-aminopeptidase), aminopeptidase B activity (arginyl-aminopeptidase), pyroglutamyl-aminopeptidase and cystinyl-aminopeptidase were measured in the neurohypophysis, in the adrenal gland and in serum of this model of hypertension. In the neurohypophysis, there was a significant increase of soluble cystinyl-, alanyl-, arginyl-, pyroglutamyl- and membrane-bound aspartyl-aminopeptidase activities in hypertensive animals. In the adrenal gland, soluble cystinyl-, alanyl-, arginyl- and pyroglutamyl-aminopeptidase activities were also higher in hypertensive rats than in normotensive controls. No differences were found in serum levels of aminopeptidase activities between both groups of animals. A highly significant positive correlation between the neurohypophysis and the adrenal gland was observed for soluble cystinyl- and alanyl-aminopeptidase activities in the model of hypertension, whereas no correlation was observed in normotensive rats. Our results suggest that aminopeptidase activities could be involved in the regulatory response to the reduced renal mass hypertension and also suggest a coordinate response between the adrenal gland and the neurohypophysis, to the specific metabolic conditions of this model of hypertension.

Adrenal Glands↗
PubMed1995

An aminopeptidase activity from porcine kidney that hydrolyzes oxytocin and vasopressin: purification and partial characterization.

An aminopeptidase from porcine kidney, hydrolyzing oxytocin and vasopressin in vitro, was purified by chromatography on hydroxyapatite, DEAE-cellulose and nickel ion chelate gel and gel filtration on Sephadex G-100. The enzyme appeared to be a high molecular mass (M(r) 105,000) monomeric protein. It was sensitive to inhibition by metal chelator, o-phenanthroline. Cobalt ion and sulfhydryl activator, 2-mercaptoethanol, had activating effects, while p-chloromercuribenzoate, amino acids with large hydrophobic side chains, L-cystine and aminopeptidase inhibitors, bestatin and amastatin, had inhibitory effects on the enzyme activity. The enzyme hydrolyzed several aminoacyl p-nitroanilides, and had the highest specificity against S-benzyl-L-cysteine p-nitroanilide. The properties of the enzyme were distinct from those of well-characterized leucyl aminopeptidase (EC 3.4.11.1), membrane alanyl aminopeptidase (EC 3.4.11.2) and primate placental cystinyl aminopeptidase (EC 3.4.11.3).

Aminopeptidases↗
PubMedDate not supplied

Effects of dietary supplementation with fish oil, lard, or coconut oil on oxytocinase activity in the testis of mice.

Oxytocin (OT), locally synthesized in the testis, is involved in androgen biosynthesis. The use of polyunsaturated fatty acids (e.g., fish oil) in the diet may improve the fertilizing ability in mammals. Cystinyl aminopeptidase (oxytocinase) activity plays a major role regulating the functional status of OT. Sex steroids and the type of the fatty acid used in the diet modify aminopeptidase activities in serum. In the present study, the authors compared the effect of a fish oil supplemented diet with two other diets supplemented with saturated oils (lard and coconut) on oxytocinase activity in the testis of mice. The enzymatic activity was determined fluorometrically using cystinyl-beta-naphthylamide as substrate. The results demonstrated higher levels of oxytocinase activity in mice fed the diet supplemented with fish oil than in those that were fed diets containing lard or coconut oils. The testicular functions in which OT is involved may be attenuated by the use of fish oil in the diet.

Animals↗
PubMed2001

Bestatin as an experimental tool in mammals.

Bestatin, an antibiotic of microbial origin, is a potent inhibitor of some, but not all aminopeptidases. It can be administered, with low toxicity, to cultured cells, intact animals and humans. It has become a useful tool in elucidating the physiological role of some mammalian exopeptidases in the regulation of the immune system, in the growth of tumors and their invasion of surrounding tissues, and in the degradation of cellular proteins. Bestatin-sensitive enzymes play important roles in the digestion and absorption of peptides in the brush border of the intestine and the kidney, in the reproductive system, and in the metabolism of opioid peptides and leukotrienes. Aminopeptidase N emerges as the major target for the effects of bestatin on the immune system and some of its effects on tumor growth and the endometrium. It is also the major bestatin-sensitive enzyme involved in the degradation of oligopeptides on the surface of intestine and kidney brush borders, and the inactivation of enkephalins in the brain. Bestatin-sensitive cytosolic exopeptidases are important in the degradation to amino acids of di- and tripeptides generated in most cells by cellular protein degradation, as well as those absorbed through the brush border of intestine and kidney. Inhibition of one of these exopeptidases, cytosol alanine aminopeptidase, results in apoptosis. Bestatin-sensitive cystinyl aminopeptidase is abundant in placenta. Two bestatin-sensitive enzymes, aminopeptidase B and nardilysin, are particularly abundant in late spermatids. Finally bestatin-sensitive LTA4 hydrolase generates the potent chemotactic agent, LTB4.

Aminopeptidases↗
PubMed2006

Synergistic inhibition of the enzymatic activity of aminopeptidase N by divalent metal ion chelators.

Membranes of HEK293 cells that were transfected with human aminopeptidase N (AP-N, CD13, EC 3.4.11.2) and purified soluble porcine kidney AP-N were used to study inhibition of its enzyme activity by divalent cation chelators. Whereas pre-incubation for 10 min with ethylenediaminetetraacetic acid (EDTA), did not or only weakly affected the enzyme activity, the bidentate chelator 1,10-phenanthroline produced a complete and concentration-dependent inhibition of AP-N. The corresponding curves had Hill slopes of 2.50 +/- 0.23 and 2.73 +/- 0.01 for soluble and recombinant AP-N respectively. EDTA increased the potency of 1,10-phenanthroline till a limit, at which Hill slopes became close to unity. In the absence of EDTA, the inhibition by 1,10-phenanthroline was only weakly affected by the substrate concentration. On the other hand, competition between 1,10-phenanthroline and the substrate took place in the presence of EDTA. Similar findings were reported for the related metallopeptidase cystinyl aminopeptidase and point towards a model in which 1,10-phenanthroline inhibit enzyme activity by decreasing the free Zn2+ concentration. Moreover, EDTA is capable of removing a modulatory ion from an allosteric site at the enzyme, facilitating the direct interaction between 1,10-phenanthroline and the catalytic Zn2+. Compatible with this model, Ca2+ may bind to this allosteric site resulting in the potentiation of Zn2+-mediated re-activation of the enzyme activity in the presence of EDTA and 1,10-phenanthroline.

Animals↗
PubMed1987

[A new criminologic procedure: study of L-cystine-aminopeptidase activity for the detection of seminal stains].

L-Cystine-aminopeptidase (CAP) enzyme cleaves L-cystine-di-beta-naphthylamide substrate to beta-naphthylamine. The final product of this process is a violet azostain. CAP enzyme determination was developed for criminological procedures. CAP shows almost 100 times greater activity in human semen than in other body fluids. Samples taken at different time intervals (1 week, 1-3-6 months, 1 year, and 5-10 years) were investigated; they were dried on cloth and filter paper and then stored at room temperature for various time periods. The enzyme was also shown to be specific in the comparative studies performed with other human secretions (nasal discharge, saliva, vaginal secretion, feces, urine, breast milk) and fruit juices (raspberry, strawberry, apple, pear, plum, tomato, etc.).

Aminopeptidases↗
PubMed1977

Oxytocinase and human placental lactogen for the prediction of intrauterine growth retardation.

Oxytocinase and human placental lactogen (hPL) were measured in 705 serial maternal plasma samples collected from 74 patients whose pregnancies were complicated by hypertension. The trend and absolute levels of each substance were evaluated in a search for reliable antepartum indicators of intrauterine growth retardation (IUGR). There were 21 patients who were delivered of growth-retarded infants and the oxytocinase values were abnormal in 16 cases (75 per cent), while hPL levels were abnormal in 15 cases (71 per cent). There were 24 patients with subnormal placental weights. Abnormal oxytocinase values were observed in 17 (71 per cent) and abnormal hPL in 19 cases (79 per cent). The remaining 37 patients had "normal" fetal and placental weights. Abnormal oxytocinase values were observed in 15 patients (41 per cent) and abnormal hPL in 19 (52 per cent). In this group of pregnant mothers thought to be at risk for IUGR due to pregnancy hypertension, either test alone predicted subnormal neonatal or placental weight in about three fourths of cases. However, in the mothers with hypertension, who had no evidence of IUGR, false abnormal values of oxytocinase and hPL were found in nearly half the patients. No advantage was gained in the predictive accuracy when both tests were used.

Aminopeptidases↗