Fluorescence polarization for mycotoxin determination

Over the last few years several laboratories have reported fluorescence polarization (FP) immunoassays for mycotoxins. These have included assays for fumonisins, deoxynivalenol and acetylated derivatives, aflatoxins, ochratoxin A, and zearalenone and related metabolites. Sensitivity in the FP assays may change dramatically over time, depending upon the antibody/tracer combination used. An important aspect of these homogeneous assays is the time required to reach an equilibrium endpoint. Although it is counterintuitive, the sensitivity of FP assays can actually be improved with shorter incubation times. However, the need for sensitivity must often be balanced against the need for the analyst to reproducibly time the incubation. The technical acumen of the analyst would be relatively more important in assays where measurements are taken before the system reaches equilibrium. In many cases the desired assays are those which reach equilibrium (and therefore give a stable endpoint) quickly, which may occur at the expense of sensitivity. It is for this reason the FP immunoassays are frequently not as sensitive as traditional ELISAs. Nevertheless, for many of the major mycotoxins rapid FP immunoassays can be developed, provided the appropriate combinations of antibody and tracer are used. Presented at the EU-USA Bilateral Workshop on Toxigenic Fungi & Mycotoxins, New Orleans, USA, July 5–7, 2005 Financial support: United States Department of Agriculture     

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

Highly purified rat brain angiotensin-converting enzyme hydrolyzes substance P which contains a C-terminal amino acid with an amidated carboxyl group. The hydrolysis of substance P verified by amino-group fluorometry and by high-performance liquid chromatography is inhibited by captopril, but not by phosphoramidon. The presence of sodium chloride is essential for the hydrolysis. The analyses of cleavage products indicate that the enzyme hydrolyzes substance P between Phe7-Phe8 and Phe8-Gly9 by an endopeptidase action, followed by successive release of dipeptides by a dipeptidyl carboxypeptidase action.     

Comparison of angiotensin-converting enzyme, malonaldehyde, zinc, and copper levels in preeclampsia

Preeclampsia is a syndrome of unknown etiopathogenesis. Recent studies carried out on preeclampsia have focused on the increase in free radicals in the feto-placental unit with poor perfusion. It is believed that the renin-angiotensin system (RAS) has a role in the poor perfusion of the placenta. It is uncertain whether there is a pre-existing impairment in RAS in pre-eclamptic pregnant women or not. In the present study, we measured angiotensin-converting enzyme (ACE), malonaldehyde (MDA), zinc, and copper levels in the placental tissue of 16 pre-eclamptic pregnant women and compared them with those in 20 healthy pregnant women. Whereas ACE activity and MDA were found to be high in the placentas of pre-eclamptic patients, zinc and copper levels were low and there was a negative correlation between ACE activity and zinc concentration. These findings suggest that high ACE activity might play a role in the increase in tissue hypoxia and consequent lipid peroxidation through vasoconstriction; zinc deficiency in the placental tissue might cause insufficiency of superoxide dismutase, an antioxidant enzyme. Furthermore, deficiency in placental zinc also plays a role in the biosynthesis of connective tissue, maintaining its integrity, which might have an impact on the structure of the spiral arteries     

Fluorescence polarization studies of the interactions between protamine and enzymes in aqueous solutions

Fluorescence polarization has been used to study the interaction of dansylated protamine with the enzymes: pepsin, α-chymotrypsin, alkaline phosphatase and invertase. These interactions have been compared with those between dansylated protamine and polyacrylate, or polyvinylsulphate. Each of the various complexes was found to be dissociated by the addition of sodium nitrate and a critical electrolyte concentration (CEC) was determined for each system, to allow assessment of the relative order of binding to the dansylated protamine. This order was: polyvinylsulphate >pepsin >polyacrylate >alkaline phosphatase >α-chymotrypsin. The strength of binding was also assessed by determination of a binding constant, K_a. The values of K_a showed the same relative order of binding as the CEC values. Invertase behaved similarly to the other enzymes, but it was not possible to obtain an unambiguous assessment of the comparative strength of binding. In each case, the stoichiometry of the complex was also determined.     

Modulation of angiotensin-converting enzyme in cultured human vascular endothelial cells

Summary Previous work has suggested that not all immunoreactive angiotensin-converting enzyme (ACE) in tissues or cells is in a biologically active state. We have explored this possibility in cultured human umbilical vein endothelial cells (HUVEC), one of the most widely studied in vitro endothelial cell systems. Our approach included characterization of the effect of increasing passage number on ACE activity and expression of immunoreactive ACE at the single cell level, the subcellular compartmentalization of active ACE, and the effect of phorbol ester (PMA) treatment. We found that both ACE activity and expression of ACE antigen were downregulated by cultivation (30% of ACE-positive cells at seventh passage vs. 90% in primary culture). ACE downregulation is specific (number of CD31-positive cells did not change with cultivation) and correlated with downregulation of factor VIII-antigen. The percentage of ACE-positive cells in permeabilized HUVEC at third passage was almost twice that in nonpermeabilized HUVEC (90% vs. 50%), indicating that HUVEC contain intracellular immunoreactive ACE. ACE activity, however, was similar when measured in intact cells and in cell lysates. Moreover, diazonium salt of sulfanilic acid (DASA), a membrane-impermeable ACE inhibitor, inhibited ACE activity in intact cells and in cell lysates at the same extent, thus implying that intracellular ACE is inactive. PMA (100 nM) treatment increased the percentage of ACE-positive cells at third passage from 57 to 96%. ACE activity was increased 3-fold in cell and 1.5-fold in the culture medium of PMA-treated cells. Analysis of ACE activity in intact monolayers and cell lysates of control and PMA-treated cells revealed that all enzymatically active ACE in PMA-treated cells is localized on the plasma membrane and acts as an ectoenzyme. We conclude that expression of ACE by HUVEC is downregulated by repeated passage in culture but can be restored by PMA treatment. In addition, ACE expression is heterogeneous between neighboring cells, and total immunoreactive ACE protein associated with HUVEC includes an inactive pool of the enzyme.     

Fluorescence polarization of trypsin digested photosystem II membranes

Fluorescence polarization of photosystem II particles treated with trypsin and incubated with high salt-medium (2M NaCl) was investigated. The presence of atrazine and TMPD in normal and salt-washed particles induced a decrease in the polarization ratios. Similar results were obtained at low concentrations of trypsin. On the basis of our observations we suggest that the presence of these perturbing agents causes a reorganisation of the membrane components and alters pigment-pigment and pigment-protein interactions. The results of fluorescence polarization demonstrate trypsin entry into the membrane after the digestion of the peripheral proteins.Abbreviations Chl chlorophyll - Mes 2-(N-morpholino)-ethanesulfonic acid - OEC oxygen evolving complex - PS II photosystem II - TMPD N, N, N - N tetramethyl-p-phenyl-enediamine - DPH 1, 6-diphenyl-1, 3, 5-hexatriene     

Insight into the binding of ACE-inhibitory peptides to angiotensin-converting enzyme: a molecular simulation

Angiotensin-I-converting enzyme (EC 3.4.15.1; ACE) plays an important physiological role in the regulation of blood pressure by converting angiotensin I to angiotensin II, a potent vasoconstrictor. Therefore, ACE inhibition has become a major target control for hypertension. Pipefish, or hailong, is an essential traditional Chinese medicine that is widely used in anti-fatigue and anti-cancer. A recent study has found two ACE-inhibitory peptides (TFPHGP and HWTTQR) purified from the seaweed pipefish by Alcalase enzymatic hydrolysis. Two peptides exhibited different ACE-inhibitory activities; however, the molecular mechanism involved is poorly understood. Further investigations are necessary to elucidate the relationship between the inhibition mechanism and the peptides. The current study is focused on investigating the interactions between each ACE-inhibitory peptide and ACE by performing molecular docking and molecular dynamics (MD) simulations. ACE protein remained stable throughout the simulations. Furthermore, ACE-TFPHGP complex showed lower binding energy as compared to ACE-HWTTQR complex, which is in good agreement with the experimental results. Glu384 and Glu411 of ACE are key residues upon the ACE-inhibitory peptides binding. Molecular basis generated by this attempt could provide valuable information towards designing new medicine for ACE inhibitor.     

Testicular atrophy, zinc concentration, and angiotensin-converting enzyme activity in the testes of vitamin a-deficient rats

Angiotensin-converting enzyme (ACE) as a part of the renin angiotensin system (RES) regulates blood pressure and fluid and electrolyte homeostasis, and the enzyme is considered to have a function in reproduction. Reduced enzyme activities have been observed in atrophied testes as a results of zinc and pituitary deficiencies. Vitamin A deficiency causes atrophy of testes. The present study was conducted on three groups of male, 3-wk-old, Wistar rats. After 54 d of the experimental period, testicular weights of the vitamin A-deficient rats (Agroup, allowed free access to vitamin Adeficient diet) was significantly lower than its pair-fed, PF (given restricted amount control diet) and A+ (allowed free access to control diet) groups. Zinc concentrations and both soluble and particulate ACE activities in the testes of vitamin A-deficient rats (Agroup) were significantly lower than the other two groups. No significant differences were observed regarding zinc concentration, particulate ACE, and total ACE activities in the testes of PF and A+ groups. Vitamin A deficiency did not significantly affect the enzyme activity in the lung. From the observations of the present study, we speculate that testicular atrophy in vitamin A deficiency may have resulted from lower zinc concentration and decreased ACE activity in that organ.     

Induction of granuloma-dependent angiotensin-converting enzyme and eosinophil chemotactic factor in the skin of athymic nude mice

Activities of angiotensin-converting enzyme (ACE), other proteinases, and eosinophil chemotactic factor (ECF-G) are known to be elevated in hepatic hypersensitivity granulomas of thymus intact (nu/+) mice after Schistosoma mansoni infection. The enzyme activities also increase, but to a lesser degree in hepatic granulomas of athymic nude (nu/nu) mice, and ECF-G is not detectable. In this study isolated hepatic granulomas from nu/+ mice were grafted into the skin of uninfected nu/nu mice, and changes in those cellular functions were determined to examine whether the newly formed granulomas by recipient nu/nu cells acquire the functional activities as well as the histological appearance of nu/+ granulomas. ACE and ECF-G rapidly disappeared from grafted sites during the first 5 days, corresponding to loss of nu/+ cells from the graft. Reduction in activities of arylsulfatases, lysozyme, and acid phosphatase also occurred, but to a lesser extent. Recovery of ACE and ECF-G activities to the levels seen in nu/+ hepatic granulomas was observed by 14 days after grafting when nu/nu cells had accumulated in the grafts and formed new granulomas. Other enzymes increased to approximately half the levels seen in grafted donor granulomas. Circulating eosinophilia also increased. The findings indicate that nu/nu cells that accumulated in the skin grafts not only morphologically mimicked nu/+ type granulomas but also demonstrated nu/+ levels of cellular function. Analysis of skin granulomas developing in nu/+ mice after grafting of nu/+ hepatic granulomas showed the similar histology and enzymatic changes, whereas the skin sites inoculated with purified schistosome eggs alone caused neither significant histological changes nor elevation of ACE activity.     

Real-time fluorescence polarization measurements: interaction of phospholipase A2 with a fluorescent lecithin derivative

We have modified an SLM 4800 fluorometer to allow continuous measurement of fluorescence polarization. The modifications included construction of simple mechanical and electronic accessories which allowed polarizer position to be program-controlled by an HP 9815 calculator. With these modifications we were able to follow the interaction of the fluorescent lipid analog 1-acyl-2-(N-4-nitrobenzo-2-oxa-1,3-diazole)-aminododecanoyl phosphatidylcholine (I) with Naja-naja venom phospholipase A2 (PLA2) (E.C.3.1.1.4). Upon addition of aliquots of PLA2 containing up to 85 ng protein to a cuvette containing 1-2 microM I, the total measured polarization decreased linearly with time for at least 1000 s. Concomitant analyses of equivalent incubation mixtures and analyses of the contents of the cuvette after incubation and collection of fluorescence data revealed time-dependent formation of N-4-nitrobenzo-2-oxa-1,3-diazole-aminododecanoic acid (II). The decrease in total measured polarization was accelerated by Ca2+ and inhibited by EDTA. These data suggest that PLA2 activity in Naja-naja venom can be measured rapidly at low concentrations of both enzyme (0.01 microgram protein) and substrate (1 microM). Since this technique can be used to collect polarization data over time intervals as short as 4 s, it should be possible to measure the early changes in polarization during the interaction of fluorescent probes with proteins or membranes.     

Metallopeptide-promoted inactivation of angiotensin-converting enzyme and endothelin-converting enzyme 1: toward dual-action therapeutics

A series of metallopeptides based on the amino terminal copper/nickel (ATCUN) binding motif have been evaluated as classical inhibitors and catalytic inactivators of both rabbit and human angiotensin-converting enzyme (hACE), and human endothelin-converting enzyme 1 (hECE-1). The cobalt complex [KGHK–Co(NH₃)₂]²⁺, where KGHK is lysylglycylhistidyllysine, displayed similar K _I and IC₅₀ values to those found for [KGHK–Cu]⁺, in spite of the enhanced charge, and so either the influence of charge is offset by the steric influence of the axially coordinated ammine ligands, or binding is dominated by contributions from the amino acid side chains, especially the C-terminal lysine that mimics the binding pattern observed for lisinopril. Moreover, the inhibition observed for [KGHK–Co(NH₃)₂]²⁺ contrasts with the activation of hACE by Co²⁺(aq), reflecting the stimulation of enzyme activity following replacement of the catalytic zinc cofactor by cobalt ion at each of the two active sites. Quantitative analysis of the dose-dependent stimulation of activity by Co²⁺(aq) yielded apparent affinities of 1.3 ± 0.2 and 56 ± 8 μM for the two sites in the presence of saturating Zn²⁺ (10 μM). Catalytic inactivation of hACE by [KGHK–Cu] ⁺ at subsaturating concentrations had previously been characterized, with k _obs = 2.9 ± 0.5 × 10⁻² min⁻¹. Under similar conditions, the same complex is found to catalytically inactivate hECE-1, with k _obs = 2.12 ± 0.16 × 10⁻² min⁻¹, demonstrating the potential for dual-action activity against two key drug targets in cardiovascular disease. Irreversible inactivation of a drug target represents a novel mechanism of drug action that complements existing classical inhibitor strategies that underlie current drug discovery efforts.Electronic Supplementary Material Supplementary material is available to authorized users in the online version of this article at .     

Variations in angiotensin-converting enzyme gene insertion/deletion polymorphism in Indian populations of different ethnic origins

The pattern of angiotensin-converting enzyme (ACE) gene insertion/deletion (I/D) polymorphism in the Indian population is poorly known. In order to determine the status of the polymorphism, young unrelated male army recruits were screened. The population had cultural and linguistic differences and lived in an environment that varied significantly from one region to another. Analysis of the genotype, showed higher frequency of the insertion allele in four of the five groups i.e. I allele frequency was significantly higher (P< 005) in Dogras, Assamese and Kumaonese. The deletion allele frequency was comparatively higher in the fifth group that belonged to Punjab. A correlation was observed between the genotype and enzyme activity. Involvement of a single D allele in the genotype enhanced the activity up to 37.56 ± 313%. The results suggested ethnic heterogeneity with a significant gene cline with higher insertion allele frequency. Such population-based data on various polymorphisms can ultimately be exploited in pharmacogenomics.     

A fluorometric assay for angiotensin-converting enzyme activity

A simple and sensitive assay for angiotensin-converting enzyme (ACE; EC 3.4.15.1) activity has been developed which employs fluorescently labeled tripeptides. ACE hydrolyzes dansylphenylalanyl-arginyl-tryptophan or dansyl-phenylalanyl-arginyl-phenylalanine, liberating dansyl-phenylalanine and a dipeptide. Dansyl-phenylalanine partitions quantitatively into chloroform, whereas the substrates are virtually insoluble in chloroform. This allows rapid measurement of ACE activity with high signal-to-noise ratios even when microliter aliquots of human serum are assayed. Inhibition studies of the dansyl-tripeptide cleaving activity of human serum and rat lung, the identity of the products of enzyme action, and the regional distribution of enzyme activity among rat tissues demonstrate that only ACE cleaves these substrates under the conditions employed here. This assay may be useful for the clinical measurement of human serum ACE activity and for research investigations of ACE from a variety of tissues.     

Fluorescence polarization of stretched polytene chromosomes stained with acridine orange

The molecules of the fluorescent dye acridine orange (AO) bind to DNA in such a way that the absorption and emission dipoles lie on a plane perpendicular to the DNA axis. For this reason, definite fluorescence polarization should correspond to each mode of spatial DNA packing. A chromosome, considered as an axially symmetrical ensemble of DNA, was characterized by two experimental parameters, P and P , i.e., by polarizations of fluorescence excited by light polarized parallel and perpendicular to the symmetry axis. In view of the sequential order in the packing levels of DNA fiber in a chromosome, it was suggested that, under mechanical stretching, the highest level is disrupted first, then the others, in the order of their sequence.Isolated chromosomes of Chironomus thummi were stained with AO and stretched with needles of a micromanipulator. From the changes of P and P measured during stretching it was concluded the polytene chromosome bands have three, at least, DNA packing levels, tentatively described as 100 å fiber, 250 å coil and chromomere.     

Fluorescence polarization: a novel indicator of cardiomyocyte contraction

The changes measured in intracellular fluorescein fluorescence polarization (IFFP) are used as a new tool for tracing cytoplasmic effects during contractile cycles of cardiac myocytes (1-2-day-old rat hearts), in addition to the established Ca(2+) monitoring and/or videometric methods of tracking cell-shortening. This novel method was found to be non-intrusive to the contraction cycles. The decay of the transient IFFP signal (from 0.220+/-0.01 to 0.170+/-0.013) seems to be closely related to the extended phase of contractile activation. This fact was further supported when Ca(2+) exchanger inhibitor was introduced and significantly decreased (90%) the rate of beats of contraction and IFFP, but not the Ca(2+) beat rate changes. This result suggests that the IFFP indicator is probably associated with the physiological activation, rather than with Ca(2+) alterations. The IFFP measure monitors the average of effective changes in the micro-viscosity of the cytoplasm protein matrix, associated with cellular activation.     

The study of substrate specificity of a new peptide substrate of endothelin-converting enzyme

A new hexapeptide CMC-Ala-Gly-Gly-Trp-Phe-Arg was used as a substrate for assay of endothelin-converting (ECE; EC 3.4.24.71) and angiotensin-converting (ACE; EC 3.4.15.1) enzymes and of neutral endopeptidase (NEP; EC 3.4.24.11). The specific inhibitors lisinopril (for ACE) and thiorphan (for NEP) were used for discrimination between activities of these enzymes.     

Fluorescence polarization as an analytical tool to evaluate galectin-ligand interactions

Galectins are a family of beta-galactose binding lectins associated with functions such as immunological and malignant events. To study the binding affinity of galectins for natural and artificial saccharides and glycoconjugates we have developed an assay using fluorescence polarization. A collection of fluorescein-conjugated saccharides was synthesized and used as probes with galectins-1 and -3 and the two carbohydrate recognition domains of galectin-4. Direct binding of a fixed probe amount with different amounts of each galectin defined specificity and selectivity and permitted selection of the optimal probe for inhibition studies. Then fixed amounts of galectin and selected probe were used to screen the inhibitory potency of a library of nonfluorescent compounds. As the assay is in solution and does not require separation of free and bound probe, it is simple and rapid and can easily be applied to different unlabeled galectins. As all interaction components are known, K(d) values for galectin-inhibitor interaction can be directly calculated without approximation other than the assumption of a simple one-site competition.     

Screening of angiotensin-converting enzyme inhibitory activities in microalgae

As part of the investigation on useful constituents in microalgae, we searched for angiotensin converting enzyme inhibitory activities in water-soluble and fat-soluble fractions of sixteen species and strains. Water-soluble fractions of eleven species and fat-soluble fractions of eight species showed inhibitory activities. In particular, the water-soluble fractions of the freshwater cyanophytesMicrocystis spp. showed inhibition at a concentration as low as 0.25 mg ml⁻¹, and that of the halotolerant chlorophyteDunaliella bardawil showed moderate inhibition. The active principles were suggested to be low molecular peptides.     

Fluorescence polarization studies and biochemical properties of membranes exfoliated from the cell surface of rabbit thymocytes in situ

In the course of our work on membrane phenomena related to the differentiation of lymphocytes in the rabbit thymus, we isolated membranous material from the extracellular compartment of this organ. With respect to their ultra-structural appearance, enzyme activity, lipid composition (cholesterol/phospholipid molar ratio, fatty acid composition of total phospholipids, phospholipid composition) and lipid fluidity, these membranes were shown to exhibit characteristics similar to those of purified plasma membranes isolated from disrupted thymocytes. Moreover, their antigenic specificity as determined in a cytotoxicity adsorption test was identical. From our experiments, we hypothesize that the extracellular membrane fragments found in the rabbit thymus are derived mainly from material shed by immature thymocytes.     

Fluorescence polarization assay for calmodulin binding to plasma membrane Ca-ATPase: Dependence on enzyme and Ca concentrations

Calmodulin (CaM) is a Ca²⁺ signaling protein that binds to a wide variety of target proteins, and it is important to establish methods for rapid characterization of these interactions. Here we report the use of fluorescence polarization (FP) to measure the K_d for the interaction of CaM with the plasma membrane Ca²⁺-ATPase (PMCA), a Ca²⁺ pump regulated by binding of CaM. Previous assays of PMCA-CaM interactions were indirect, based on activity or kinetics measurements. We also investigated the Ca²⁺ dependence of CaM binding to PMCA. FP assays directly detect CaM-target interactions and are rapid, sensitive, and suitable for high-throughput screening assay formats. Values for the dissociation constant K_d in the nanomolar range are readily measured. We measured the changes in anisotropy of CaM labeled with Oregon Green 488 on titration with PMCA, yielding a K_d value of CaM with PMCA (5.8 ± 0.5 nM) consistent with previous indirect measurements. We also report the binding affinity of CaM with oxidatively modified PMCA (K_d = 9.8 ± 2.0 nM), indicating that the previously reported loss in CaM-stimulated activity for oxidatively modified PMCA is not a result of reduced CaM binding. The Ca²⁺ dependence follows a simple Hill plot demonstrating cooperative binding of Ca²⁺ to the binding sites in CaM.