共查询到20条相似文献,搜索用时 31 毫秒
1.
Pedersen KB Sriramula S Chhabra KH Xia H Lazartigues E 《American journal of physiology. Regulatory, integrative and comparative physiology》2011,301(5):R1293-R1299
Angiotensin-converting enzyme 2 (ACE2) is a component of the renin-angiotensin system, and its expression and activity have been shown to be reduced in cardiovascular diseases. Enzymatic activity of ACE2 is commonly measured by hydrolysis of quenched fluorescent substrates in the absence or presence of an ACE2-specific inhibitor, such as the commercially available inhibitor DX600. Whereas recombinant human ACE2 is readily detected in mouse tissues using 1 μM DX600 at pH 7.5, the endogenous ACE2 activity in mouse tissues is barely detectable. We compared human, mouse, and rat ACE2 overexpressed in cell lines for their sensitivity to inhibition by DX600. ACE2 from all three species could be inhibited by DX600, but the half maximal inhibitory concentration (IC(50)) for human ACE2 was much lower (78-fold) than for rodent ACE2. Following optimization of pH, substrate concentration, and antagonist concentration, rat and mouse ACE2 expressed in a cell line could be accurately quantified with 10 μM DX600 (>95% inhibition) but not with 1 μM DX600 (<75% inhibition). Validation that the optimized method robustly quantifies ACE2 in mouse tissues (kidney, brain, heart, and plasma) was performed using wild-type and ACE2 knockout mice. This study provides a reliable method for measuring human, as well as endogenous ACE2 activity in rodents. Our data underscore the importance of validating the effect of DX600 on ACE2 from each particular species at the experimental conditions employed. 相似文献
2.
Sabatini RA Bersanetti PA Farias SL Juliano L Juliano MA Casarini DE Carmona AK Paiva AC Pesquero JB 《Analytical biochemistry》2007,363(2):255-262
An assay using fluorescence resonance energy transfer peptides was developed to assess angiotensin I-converting enzyme (ACE) activity directly on the membrane of transfected Chinese hamster ovary cells (CHO) stably expressing the full-length somatic form of the enzyme. The advantage of the new method is the possibility of using selective substrates for the two active sites of the enzyme, namely Abz-FRK(Dnp)P-OH for somatic ACE, Abz-SDK(Dnp)P-OH for the N domain, and Abz-LFK(Dnp)-OH for the C domain. Hydrolysis of a peptide bond between the donor/acceptor pair (Abz/Dnp) generates detectable fluorescence, allowing quantitative measurement of the enzymatic activity. The kinetic parameter K(m) for the hydrolysis of the three substrates by ACE in this system was also determined and the values are comparable to those obtained using the purified enzyme in solution. The specificity of the activity was demonstrated by the complete inhibition of the hydrolysis by the ACE inhibitor lisinopril. Therefore, the results presented in this work show for the first time that determination of ACE activity directly on the surface of intact CHO cells is feasible and that the method is reliable and sensitive. In conclusion, we describe a methodology that may represent a new tool for the assessment of ACE activity which will open the possibility to study protein interactions in cells in culture. 相似文献
3.
Organ-specific distribution of ACE2 mRNA and correlating peptidase activity in rodents 总被引:7,自引:0,他引:7
Gembardt F Sterner-Kock A Imboden H Spalteholz M Reibitz F Schultheiss HP Siems WE Walther T 《Peptides》2005,26(7):1270-1277
Biochemical analysis revealed that angiotensin-converting enzyme related carboxy-peptidase (ACE2) cleaves angiotensin (Ang) II to Ang-(1-7), a heptapeptide identified as an endogenous ligand for the G protein-coupled receptor Mas. No data are currently available that systematically describe ACE2 distribution and activity in rodents. Therefore, we analyzed the ACE2 expression in different tissues of mice and rats on mRNA (RNase protection assay) and protein levels (immunohistochemistry, ACE2 activity, western blot). Although ACE2 mRNA in both investigated species showed the highest expression in the ileum, the mouse organ exceeded rat ACE2, as also demonstrated in the kidney and colon. Corresponding to mRNA, ACE2 activity was highest in the ileum and mouse kidney but weak in the rat kidney, which was also confirmed by immunohistochemistry. Contrary to mRNA, we found weak activity in the lung of both species. Our data demonstrate a tissue- and species-specific pattern for ACE2 under physiological conditions. 相似文献
4.
Immobilized angiotensin-converting enzyme (ACE) was utilized as an affinity ligand to isolate a naturally occurring ACE binding protein from normal human serum. The enzyme was isolated from solubilized bovine lung membrane preparations by lisinopril affinity chromatography. It had an estimated molecular weight of 180 000 and was recognized by the anti-ACE antibody for the rabbit testicular ACE in immunoblots. ACE was immobilized onto epoxy Sepharose as well as Affi-Gel 15. Immobilized ACE on Affi-Gel 15 had higher catalytic activity (0.176 U/mL) compared with the enzyme immobilized on epoxy Sepharose (0.00005 U/mL). Immobilized ACE served as the affinity ligand for the identification of the ACE binding protein in human serum with an estimated molecular weight of 14 000 as observed by SDS polyacrylamide gel electrophoresis. The identification and further characterization of ACE binding proteins in serum and tissues may facilitate the greater understanding of the endogenous regulation of this key enzyme, which is involved in blood pressure homeostasis. 相似文献
5.
Angiotensin I-converting enzyme (ACE) is involved in various physiological and physiopathological conditions; therefore, the measurement of its catalytic activity may provide essential clinical information. This protocol describes a sensitive and rapid procedure for determination of ACE activity using fluorescence resonance energy transfer (FRET) substrates containing o-aminobenzoic acid (Abz) as the fluorescent group and 2,4-dinitrophenyl (Dnp) as the quencher acceptor. Hydrolysis of a peptide bond between the donor/acceptor pair generates fluorescence that can be detected continuously, allowing quantitative measurement of the enzyme activity. The FRET substrates provide a useful tool for kinetic studies and for ACE determination in biological fluids and crude tissue extracts. An important benefit of this method is the use of substrates selective for the two active sites of the enzyme, namely Abz-SDK(Dnp)P-OH for N-domain, Abz-LFK(Dnp)-OH for C-domain and Abz-FRK(Dnp)P-OH for somatic ACE. This methodology can be adapted for determinations using a 96-well fluorescence plate reader. 相似文献
6.
Vandingenen A Hens K Macours N Schoofs L De Loof A Huybrechts R 《Comparative biochemistry and physiology. Part B, Biochemistry & molecular biology》2002,132(1):27-35
Angiotensin converting enzyme (ACE) activity, defined as a captopril-inhibitable dipeptidyl carboxypeptidase activity towards 3H-hippurylglycylglycine, was demonstrated in haemolymph, testes and ovaries of the grey fleshfly Neobellieria bullata, hereby suggesting a physiological role for ACE in these particular tissues. While the ACE activity in haemolymph and testes reached relatively high levels, only minute ACE activity could be detected in ovaries throughout the entire vitellogenic cycle. Ovarian extracts of Neobellieria bullata do contain, however, in addition to Neb-TMOF, the Neobellieria bullata trypsin modulating oostatic factor which is an in vitro and a putative in vivo substrate of ACE in circulation, several other heat-stable molecules which individually function either as an ACE substrate or ACE inhibitor. Presumably these ACE interactive factors mask ACE activity in the fly ovaries, as measured by a classic substrate-binding assay. Purification and characterisation of these ACE substrates/inhibitors is in progress and is likely to facilitate the elucidation of the enigmatic physiological relevance of ACE in insects. 相似文献
7.
A new fluorogenic acceptor for sialyltransferase, 2-[(2-pyridyl)amino]ethyl O-beta-D-galactopyranosyl-(1----4)-beta-D-glucopyranoside, was prepared from lactose as a starting material. Sialyltransferase activity was assayed by incubation of the enzyme with the acceptor and CMP-N-acetylneuraminic acid, separation of the fluorogenic sialylated product from the enzymatic reaction mixture by HPLC, and measurement of the product. Compared to assays so far reported that use radioactive substrates, this assay is simple and rapid. This method was used to assay sialyltransferase activity in human serum. 相似文献
8.
Ken-Ichi Iijima Tatsuo Takei Masami Hino Taro Hayakawa 《Journal of biochemical and biophysical methods》1980,3(2):89-96
We have developed a new fluorescence assay for dipeptidylpeptidase IV using a tripeptide, l-prolyl-l-prolyl-l-alanine, which might be one of the potential natural substrates. The principle of the assay is based on the measurement of fluorescent adduct between alanine liberated from the tripeptide by enzymatic hydrolosis and o-phthaldialdehyde in the presence of 2-mercaptoethanol in aqueous alkaline medium. This new assay is sensitive enough to measure the enzyme activity in as little as 0.01 μl of human serum and in crevicular fluid obtained from human gingival sulcus. The Km value for the tripeptide was 1.7 · 10?5 M which is less than one-tenth of that obtained with other chromogenic or fluorogenic substrates. The interference by serum was overcome by simply incorporating the same amount of serum in the standards. 相似文献
9.
Cell associated elastase activities of rat mammary tumour cells 总被引:1,自引:0,他引:1
As part of our studies into the role of tumour cell proteinases in cancer invasion, we have adapted a fluorogenic assay to measure the elastase activities of intact rat mammary adenocarcinoma cells using the elastase specific substrates Cbz-Ala-Ala-Pro-Val-6-aminoquinoline and Ac-Ala-Ala-Pro-Ala-7-amino-4-methylcoumarin. This is a sensitive assay which enables rapid (30-120 min) measurement of enzyme activities under conditions of physiological pH and ionic strength and can differentiate between cell-associated and secreted enzyme activities. As the substrates are non-toxic and the method is non-invasive, cells can be reclaimed for further studies. This method thus provides a useful means for screening intact cells for elastase activity. Cell-surface elastase extracts were inhibited by phenylmethylsulphonyl fluoride but not by EDTA, indicating that they are serine proteinases. Extracts also degraded insoluble elastin confirming that these rat mammary adenocarcinoma cells produce elastase. 相似文献
10.
Total serum angiotensin converting enzyme activity in rats and dogs after enalapril maleate (MK-421)
A centrifugal gel filtration separation of serum angiotensin converting enzyme (ACE) from a potent stable inhibitor is described. This, together with a 20 hr assay incubation of very dilute enzyme, permitted the assessment of the effects of enalapril maleate treatment on total serum ACE in rats and dogs. Total serum ACE increased in both species after 1 or 2 weeks at 10 mg/kg/day. Serum ACE in rats was more than doubled; whereas the increase was modest in dogs (48 +/- 9% minimum). The effect of the drug on serum ACE combined with inherent variability of ACE precluded use of serum ACE activity as an accurate measure of inhibitor concentration in animals receiving enalapril maleate. 相似文献
11.
Deubiquitinating enzymes (DUBs) regulate diverse cellular functions by their activity of cleaving ubiquitin from specific protein substrates. Ubiquitin-Specific Protease 46 (USP46) has recently been identified as a quantitative trait gene responsible for immobility in the tail suspension test and forced swimming test in mice. Mice with a lysine codon (Lys 92) deletion in USP46 exhibited loss of 'behavioral despair' under inescapable stresses in addition to abnormalities in circadian behavioral rhythms and the GABAergic system. However, whether this deletion affects enzyme activity is unknown. Here we show that USP46 has deubiquitinating enzyme activity detected by USP cleavage assay using GST-Ub52 as a model substrate. Interestingly, compared to wild type, the Lys 92 deletion mutant resulted in a decreased deubiquitinating enzyme activity of 27.04%. We also determined the relative expression levels of Usp46 in rat tissues using real-time RT-PCR. Usp46 mRNA was expressed in various tissues examined including brain, with the highest expression in spleen. In addition, like rat USP46, both human and mouse USP46 are active toward to the model substrate, indicating the USP cleavage assay is a simple method for testing the deubiquitinating enzyme activity of USP46. These results suggest that the Lys 92 deletion of USP46 could influence enzyme activity and thereby provide a molecular clue how the enzyme regulating the pathogenesis of mental illnesses. 相似文献
12.
A competitive enzyme-linked immunoassay (CELIA) for human serum angiotensin-1-converting enzyme (ACE) was developed. The sensitivity was amplified by using a secondary antibody and an avidin biotin-conjugated horseradish peroxidase complex as the enzyme-labeled reagent. This configuration was compared to three other configurations for an indirect CELIA, and was found to be the most sensitive. A sensitivity of 39 ng/ml ACE was achieved with intraassay and interassay coefficients of variation of 6.3 and 8%, respectively. CELIA will detect ACE in human serum without interference from either pharmacological or endogenous ACE inhibitors. In normal human volunteers, ACE values obtained using CELIA correlated well with values obtained by enzymatic assay. 相似文献
13.
Angiotensin I-converting enzyme (ACE) is a dipeptidyl carboxypeptidase that removes C-terminal dipeptides from relatively short oligopeptides, usually smaller than 15 amino acids. In mammals, the enzyme has several important roles in the metabolism of vasoactive peptides, but its physiological role in insects is not fully understood. We now report the properties of an ACE in a lepidopteran species (the tomato moth, Lacanobia oleracea) and suggest new physiological roles for the enzyme in this insect. ACE activity increases four-fold during the last stadium and in early pupae, a rise which, in its timing, is similar to what has been observed previously in the transition of larva to pupa in Drosophila melanogaster. This suggests that the increase in ACE activity might be of general importance for peptide metabolism during metamorphosis in holometabolous insects. High levels of ACE activity were found in the haemolymph of sixth stadium larvae and adult insects, and in the reproductive tissues of both male and female adults. Almost all of the ACE activity in the reproductive tissues was found in the accessory glands of the male and the spermatheca and bursa copulatrix of the female. The decline in accessory gland ACE in mated males and the concomitant rise in ACE activity in the spermatheca and bursa copulatrix of the female suggested the transfer of ACE from the male to the female during copulation. Using several convenient peptides as substrates, we have shown that the spermatophore/bursa copulatrix taken from mated female insects possess an aminopeptidase, a carboxypeptidase and a dipeptidase, in addition to high levels of ACE. These peptidases might be involved in the breakdown of proteins to peptides and eventually to amino acids in the spermatophore. Evidence for such a proteolytic pathway and its role in providing substrates for the TCA cycle has been obtained previously in a study of reproduction in Bombyx mori. 相似文献
14.
Ryuichi Yajima Toshiyuki Chikuma Takeshi Kato 《Journal of chromatography. B, Analytical technologies in the biomedical and life sciences》1995,667(2)
A rapid and sensitive high-performance liquid chromatographic (HPLC)-fluorimetric assay method has been developed for the determination of carboxypeptidase H activity based on the measurement of N-(5-dimethyl-aminonaphthalene-1-sulfonyl)glycine (dansyl-Gly) formed enzymatically from dansyl-Gly-L-Lys or dansyl-Gly-L-Arg. Dansyl-Gly is eluted faster than the substrates with an N-2-hydroxyethylpiperazine-N′-2-ethanesulfonic acid (Hepes) buffer at pH 7.0 containing methanol, but eluted slower with an acidic buffer at pH 4.6. The new HPLC method separates the product and substrate in less than 5 min using an elution buffer at pH 7.0 containing 60% methanol. Using this method carboxypeptidase H activity has been detected in rat sciatic nerves. This HPLC method facilitates the assay of carboxypeptidase H activity in the enzyme samples from various tissues. 相似文献
15.
Angiotensin converting enzyme (ACE; EC 3. 4. 15. 1) activities were compared in the serum of various mammals. Cattle, human, monkey, and swine serum showed enzyme levels from 3.7 to 67 mU/ml. Relatively high enzyme activity was observed in rodents, the rat (Wistar) and mouse (BALB/c) showing levels of 93 +/- 7 and 1052 +/- 165 mU/ml, respectively. Among the mammalian sera examined, that of the guinea pig contained the highest ACE level, 2262 +/- 574 mU/ml. No age-related difference in enzyme activity was observed in 10-day-old to 1-year-old guinea pigs. 相似文献
16.
A simple chromogenic assay for human alpha thrombin is developed through aptamer affinity capture and a subsequent enzyme reaction. Thrombin is captured on the aptamer-modified magnetic beads, and catalyzes the conversion of chromogenic substrates to optically measured products. The measurement of the generated products by an absorbance spectrometer allows for the final quantification of thrombin. This assay shows high sensitivity by taking advantage of sample enrichment and enzyme amplification, and exhibits good specificity by involving the selective aptamer binding and the specific enzyme reaction. A concentration detection limit of 40 fM can be reached when the tripeptide substrate of tosyl-Gly-Pro-Arg-p-nitroanilide is used in a 24 h enzyme reaction, and the use of 2h enzyme reaction in the assay enables the detection of 400 fM thrombin for a rapid analysis. This assay can be applied to detect thrombin in dilute human serum. 相似文献
17.
New Insights into Butyrylcholinesterase Activity Assay: Serum Dilution Factor as a Crucial Parameter
Joanna Jońca Monika ?uk Bartosz Was?g Anna Janaszak-Jasiecka Krzysztof Lewandowski Bartosz Wielgomas Krzysztof Waleron Jacek Jasiecki 《PloS one》2015,10(10)
Butyrylcholinesterase (BChE) activity assay and inhibitor phenotyping can help to identify patients at risk of prolonged paralysis following the administration of neuromuscular blocking agents. The assay plays an important role in clinical chemistry as a good diagnostic marker for intoxication with pesticides and nerve agents. Furthermore, the assay is also commonly used for in vitro characterization of cholinesterases, their toxins and drugs. There is still lack of standardized procedure for measurement of BChE activity and many laboratories use different substrates at various concentrations. The purpose of this study was to validate the BChE activity assay to determine the best dilution of human serum and the most optimal concentration of substrates and inhibitors. Serum BChE activity was measured using modified Ellman’s method applicable for a microplate reader. We present our experience and new insights into the protocol for high-throughput routine assays of human plasma cholinesterase activities adapted to a microplate reader. During our routine assays used for the determination of BChE activity, we have observed that serum dilution factor influences the results obtained. We show that a 400-fold dilution of serum and 5mM S-butyrylthiocholine iodide can be successfully used for the accurate measurement of BChE activity in human serum. We also discuss usage of various concentrations of dibucaine and fluoride in BChE phenotyping. This study indicates that some factors of such a multicomponent clinical material like serum can influence kinetic parameters of the BChE. The observed inhibitory effect is dependent on serum dilution factor used in the assay. 相似文献
18.
Somatic angiotensin-converting enzyme (ACE) contains two homologous domains, each bearing a functional active site. Studies on the selectivity of these ACE domains towards either substrates or inhibitors have mostly relied on the use of mutants or isolated domains of ACE. To determine directly the selectivity properties of each ACE domain, working with wild-type enzyme, we developed an approach based on the combined use of N-domain-selective and C-domain-selective ACE inhibitors and fluorogenic substrates. With this approach, marked differences in substrate selectivity were revealed between rat, mouse and human somatic ACE. In particular, the fluorogenic substrate Mca-Ala-Ser-Asp-Lys-DpaOH was shown to be a strict N-domain-selective substrate of mouse ACE, whereas with rat ACE it displayed marked C-domain selectivity. Similar differences in selectivity between these ACE species were also observed with a new fluorogenic substrate of ACE, Mca-Arg-Pro-Pro-Gly-Phe-Ser-Pro-DpaOH. In support of these results, changes in amino-acid composition in the binding site of these three ACE species were pinpointed. Together these data demonstrate that the substrate selectivity of the N-domain and C-domain depends on the ACE species. These results raise concerns about the interpretation of functional studies performed in animals using N-domain and C-domain substrate selectivity data derived only from human ACE. 相似文献
19.
Angiotensin-converting enzyme (ACE) activity was found increased in serum of patients with chronic alcoholism. We studied this enzymatic activity in serum and liver tissue of rats with alcoholic fatty liver due to prolonged intake of ethanol with a liquid diet, according to De Carli and Lieber. Serum and liver ACE activity did not show any significant increase in rats with alcoholic fatty liver when compared with controls, whereas gamma-glutamyltransferase activity exhibited a striking enhancement in serum and liver. Our data suggest that ACE is not an alcohol-induced enzyme in the experimental rat model. 相似文献
20.
《Comp. Biochem. Physiol. C, Comp. Pharmacol. Toxicol.》1993,106(2):395-401
1. Angiotensin I-converting enzyme (ACE) activity was determined in serum and nine tissues from control and captopril-treated rats, mice, guinea pigs and rabbits.2. ACE activity was determined with and without sample pretreatment with diamide (total and basal activity, respectively).3. A very different pattern of response to captopril was observed among the different species.4. There was no relationship between serum ACE activity and the response to captopril.5. There were important differences in the determinations of total or basal ACE activities.6. Endogenous ACE inhibitors were found in some tissues from mouse and rabbit. 相似文献