Anthocyanidin synthase from Gerbera hybrida catalyzes the conversion of (+)-catechin to cyanidin and a novel procyanidin

Anthocyanidins were proposed to derive from (+)-naringenin via (2R,3R)-dihydroflavonol(s) and (2R,3S,4S)-leucocyanidin(s) which are eventually oxidized by anthocyanidin synthase (ANS). Recently, the role of ANS has been put into question, because the recombinant enzyme from Arabidopsis exhibited primarily flavonol synthase (FLS) activity with negligible ANS activity. This and other studies led to the proposal that ANS as well as FLS may select for dihydroflavonoid substrates carrying a "beta-face" C-3 hydroxyl group and initially form the 3-geminal diol by "alpha-face" hydroxylation. Assays with recombinant ANS from Gerbera hybrida fully supported the proposal and were extended to catechin and epicatechin isomers as potential substrates to delineate the enzyme specificity. Gerbera ANS converted (+)-catechin to two major and one minor product, whereas ent(-)-catechin (2S,3R-trans-catechin), (-)-epicatechin, ent(+)-epicatechin (2S,3S-cis-epicatechin) and (-)-gallocatechin were not accepted. The K(m) value for (+)-catechin was determined at 175 microM, and the products were identified by LC-MS(n) and NMR as the 4,4-dimer of oxidized (+)-catechin (93%), cyanidin (7%) and quercetin (trace). When these incubations were repeated in the presence of UDP-glucose:flavonoid 3-O-glucosyltransferase from Fragariaxananassa (FaGT1), the product ratio shifted to cyanidin 3-O-glucoside (60%), cyanidin (14%) and dimeric oxidized (+)-catechin (26%) at an overall equivalent rate of conversion. The data appear to identify (+)-catechin as another substrate of ANS in vivo and shed new light on the mechanism of its catalysis. Moreover, the enzymatic dimerization of catechin monomers is reported for the first time suggesting a role for ANS beyond the oxidation of leucocyanidins.     

Mechanistic studies on three 2-oxoglutarate-dependent oxygenases of flavonoid biosynthesis: anthocyanidin synthase, flavonol synthase, and flavanone 3beta-hydroxylase

Anthocyanidin synthase (ANS), flavonol synthase (FLS), and flavanone 3beta-hydroxylase (FHT) are involved in the biosynthesis of flavonoids in plants and are all members of the family of 2-oxoglutarate- and ferrous iron-dependent oxygenases. ANS, FLS, and FHT are closely related by sequence and catalyze oxidation of the flavonoid "C ring"; they have been shown to have overlapping substrate and product selectivities. In the initial steps of catalysis, 2-oxoglutarate and dioxygen are thought to react at the ferrous iron center producing succinate, carbon dioxide, and a reactive ferryl intermediate, the latter of which can then affect oxidation of the flavonoid substrate. Here we describe work on ANS, FLS, and FHT utilizing several different substrates carried out in 18O2/16OH2, 16O2/18OH2, and 18O2/18OH2 atmospheres. In the 18O2/16OH2 atmosphere close to complete incorporation of a single 18O label was observed in the dihydroflavonol products (e.g. (2R,3R)-trans-dihydrokaempferol) from incubations of flavanones (e.g. (2S)naringenin) with FHT, ANS, and FLS. This and other evidence supports the intermediacy of a reactive oxidizing species, the oxygen of which does not exchange with that of water. In the case of products formed by oxidation of flavonoid substrates with a C-3 hydroxyl group (e.g. (2R,3R)-trans-dihydroquercetin), the results imply that oxygen exchange can occur at a stage subsequent to initial oxidation of the C-ring, probably via an enzyme-bound C-3 ketone/3,3-gem-diol intermediate.     

Isolation of acein-2, a novel angiotensin-I-converting enzyme inhibitory peptide derived from a tryptic hydrolysate of human plasma

There are two forms of rabbit muscle adenylate kinase (AK) with different 8-anilino-1-naphthalenesulfonic acid (ANS) binding properties in equilibrium solution. One form (about 70%, denoted N₁) binds rapidly with ANS, whereas the other (about 30%, denoted N₂) does not. Furthermore, native forms of AK should adopt different conformations for binding with substrates and products, which should be pre-existing for performing its catalytic function. The present experiments demonstrate both forms of AK distinguished by ANS probe are active. The activity of N₂ is about 0.8 fold higher than N₁ and shows higher susceptibility to proteolysis by trypsin. This means that the native state of AK might be an ensemble of kinetically attainable conformers and the energy landscapes of AK folding should be rugged with more than one local minimum.     

Atrial natriuretic peptides stimulate renal gluconeogenesis

Atrial natriuretic peptide (5-28AA; ANP) and atrial extract (ANS) stimulated rat renal gluconeogenesis in cortical tubule suspension in a dose dependent fashion only from substrates that enter gluconeogenesis via phosphoenol-pyruvate carboxylase. The effects of ANP and ANS were significantly potentiated by cAMP and cGMP, whereas methoxamine showed no effect. Extracellular calcium revealed a key role for ANP and ANS response to gluconeogenesis: a concentration of calcium higher than 1 mM was essential. Isolated cells from cortex which lost cell membrane polarity by warming but responded solely to cAMP and cGMP showed no effect by ANP nor ANS. These data suggest that ANP or ANS may act mainly from the basolateral site in the proximal tubule cell and promote gluconeogenesis through cAMP and/or cGMP system.     

Interaction of native and modified Naja melanoleuca phospholipases A2 with the fluorescent probe 8-anilinonaphtalene-1-sulfonate

The fluorescent probe 8-anilinonaphtalene-1-sulfonate (ANS) binds at the active site of the Naja melanoleuca snake venom phospholipase A2, thus protecting the enzyme against active-site-directed chemical modification. Both hydrophobic and electrostatic interactions are involved in the binding. At pH 7.5, a binding constant of 100 microM was determined, which improved twofold upon addition of the enzymatic cofactor Ca2+. The pH dependence of the ANS binding in the absence and presence of Ca2+ ions showed a perturbation of a group with a pKa value of 5.2, which could be assigned to the carboxylate group of the Ca2+-binding ligand Asp49 at the active site of the protein. Monomeric concentrations of the substrate analog n-decylphosphocholine displace ANS from the protein, indicating again that both ligands bind at the active site. Binding studies with several modified N. melanoleuca enzymes showed that a loss of enzymatic activity on aggregated substrates was correlated with a loss of affinity for the active site bound ANS molecule. It is suggested therefore, that the fluorescent ANS probe can detect structural rearrangements at the active site, which are important for enzymatic activity.     

Substrate binding to phosphoglycerate kinase monitored by 1-anilino-8-naphthalenesulfonate

The interaction between 1-anilino-8-naphthalenesulfonate (ANS) and yeast phosphoglycerate kinase (ATP:3-phospho-D-glycerate 1-phosphotransferase, EC 2.7.2.3) and the use of ANS as a probe for studying the structure and function of phosphoglycerate kinase has been investigated. The interaction has been studied by kinetic methods, equilibrium dialysis, and fluorometric titrations. ANS inhibits the activity of the enzyme. More than one inhibitor site exists. ANS is competitive with MgATP and noncompetitive with 3-phosphoglycerate at the first detected inhibitor binding site. The Ki value is 1-2 mM. Several ANS molecules bind to the enzyme. By fluorometric titrations the first detected site has a dissociation constant that is in the same range as Ki or bigger. When ANS interacts with phosphoglycerate kinase its fluorescence is increased and a blue shift occurs. ANS appears to bind to a strongly hydrophobic site. The fluorescence is sensitive to the addition of substrates. ADP, ATP, or combinations of Mg2+ and nucleotide decreases the fluorescence as does free Mg2+. 3-Phosphoglycerate, on the other hand, increases the fluorescence giving evidence for conformational changes upon 3-phosphoglycerate binding.     

1H and 13C NMR spectra,protonation, deprotonation,and host-guest complexation-induced shifts of some fluorescence dyes

¹H and ¹³C NMR signal assignments for 8-anilinonaphthalenesulfonic acid (ANS) and dansyl amide (DNSA) are achieved using high-field spectra, decoupling, and two-dimensional NMR techniques as well as shift differences between conjugate acid and bases. Complexation of ANS and DNSA with a macrocyclic azoniacyclophane is measured by fluorescence and by NMR shift titration, furnishing an independent check for the equilibrium constant determination. The complexation-induced shifts (CIS) for ANS and DNSA are analyzed on the basis of aromatic ring current and linear electric field effect models. Comparison of equilibrium constants of the cyclophane and different substrates shows that, e.g., for ANS, lipophilic/hydrophobic binding dominates over electrostatic effects despite the presence of charges and the absence of a lipophilic cap or bottom on the receptor molecule.     

Rapid methods for differentiating gram-positive from gram-negative aerobic and facultative anaerobic bacteria

Different tests based on lysis by KOH and on reaction with fluorogenic and chromogenic substrates, L-alanine-4-nitroanilide (LANA); L-alanine-4-methoxy- beta-naphthylamide (MNA); 4-alanine-2-amidoacridone (AAA); L-alanine-7-amido- 4-methylcoumarin (AAMC); 8-anilino-1-naphthalene-sulphonic acid (ANS) were compared for their suitability to distinguish Gram-positive from Gram-negative bacteria. A concentration of 100 micrograms/ml was chosen for incorporating LANA, AAA, AAMC and ANS into the growth medium, based on sensitivity tests. MNA did not show any detectable reaction over a concentration range from 50 to 200 micrograms/ml, and led to inhibition of all bacteria at 200 micrograms/ml. In the examination of a total of 146 bacterial strains, including Yersinia enterocolitica, Bacillus cereus, and B. subtilis the KOH test was not comparable with the Gram staining. A good correlation with Gram staining was found between LANA, AAA and AAMC added to plate count agar on one hand, and LANA and AAMC impregnated paper strips on the other hand, thereby utilizing the aminopeptidase activity. Agar containing ANS showed detectable fluorescence with all Gram-negative strains, but with Staphylococcus aureus and Staph. epidermidis a weak reaction was also observed. AAMC was selected for a rapid paper strip test. With this substrate a pronounced blue fluorescence was obtained with Gram-negative colonies.     

Comparison of the effects of bile acids and GSH on the fluorescence of bound 1-anilino-8-naphthalene sulfonate and the enzymatic activity of cationic and neutral human hepatic GSH S-transferases

Three cationic (C1, C2, A1) and a neutral (N1) glutathione (GSH) S-transferase were purified to homogeneity from human liver, as we have previously reported. GSH had no effect on the fluorescence of 1-anilino-8-naphthalene sulfonate (ANS) bound by transferase C1 and N1, but markedly enhanced the fluorescence with C2 and A1 without changing the affinity for ANS. This effect of GSH was saturable and with C2 was intermediate between A1 and C1. Bile acids inhibited the fluorescence of ANS bound to C1 and C2. GSH in the presence of bile acids further decreased the fluorescence of ANS bound to C1 and increased the fluorescence with C2. Transferase A1 showed decreased fluorescence in the presence of lithocholic acid and increased fluorescence in the presence of cholic acid; both changes were reversed by GSH. Transferase N1 showed increased fluorescence of bound ANS in the presence of various bile acids and this effect was diminished in the presence of GSH. Enzyme activity of the transferase was inhibited by bile acids with the exception of transferase A1. All the proteins bound lithocholic acid. The inhibition of C1 and N1 was greater at pH 6.5 than 7.4 and the order of addition of substrates and inhibitor made no difference.     

Structure and mechanism of anthocyanidin synthase from Arabidopsis thaliana.

Flavonoids are common colorants in plants and have long-established biomedicinal properties. Anthocyanidin synthase (ANS), a 2-oxoglutarate iron-dependent oxygenase, catalyzes the penultimate step in the biosynthesis of the anthocyanin class of flavonoids. The crystal structure of ANS reveals a multicomponent active site containing metal, cosubstrate, and two molecules of a substrate analog (dihydroquercetin). An additional structure obtained after 30 min exposure to dioxygen is consistent with the oxidation of the dihydroquercetin to quercetin and the concomitant decarboxylation of 2-oxoglutarate to succinate. Together with in vitro studies, the crystal structures suggest a mechanism for ANS-catalyzed anthocyanidin formation from the natural leucoanthocyanidin substrates involving stereoselective C-3 hydroxylation. The structure of ANS provides a template for the ubiquitous family of plant nonhaem oxygenases for future engineering and inhibition studies.     

Gluconeogenesis and the peroxisome

The interaction between hydroperoxides, cytochrome P450 and 8-anilino-1-naphthalenesulfonic acid (ANS) has been investigated. The addition of ANS to the cytochrome P450 solution did not effect the P450 Soret absorption peak or the reduced CO difference spectrum, suggesting that ANS may not bind to P450 heme directly. H2O2 or CuOOH alone did not effect ANS fluorescence and absorption spectra indicating that no detectable reaction occurs between hydroperoxide and ANS in the absence of P450. The reconstituted system of cytochrome P450, P450 reductase, lipid and NADPH did not mediate ANS metabolism. In the presence of P450, the addition of either H2O2 or CuOOH, however, leads to a decrease in ANS absorption around 258 nm and 350 nm indicating possible destruction of ANS. ANS destruction was confirmed with the disappearance of the ANS elution peak in the reverse phase HPLC profiles and with the changes in P450-bound ANS fluorescence intensity and the shift of max of ANS. Moreover , a very sensitive method to detect trace fluorescent products of ANS by thin layer chromatography has been developed based on the fact that ANS fluorescence is enhanced more than 1000-fold by the organic solvent butanol. A UV-sensitive fluorescent product was detected on thin layer chromatography profiles of the reaction mixtures. P450 was also observed to be modified by a fluorescent derivative of ANS, when the fluorescence was enhanced by butanol. These results also show that an organic compound which can not be metabolized by the reconstituted system of cytochrome P450 and NADPH-P450 reductase is metabolized by the reconstituted system of P450 and hydroperoxide, suggesting the activities of these two systems may not be completely comparable. (Mol Cell Biochem 167: 159-168, 1997)     

Early Aggregated States in the Folding of Interleukin-1β

Kinetic data measured from folding of the protein interleukin-1β fits best to three exponential phases when studied with tryptophan fluorescence but only two exponential phases when measured using other methods. The technique of ANS fluorescence was used to determine whether the additional phase observed in tryptophan fluorescence was also detected with ANS dye binding. Unlike trytophan fluorescence, the ANS fluorescence was highly dependent on the concentration of protein present during the folding experiment. Experimental controls provide evidence that ANS binds to protein aggregates, present at higher concentrations and absent at lower concentrations. Protein concentration-dependent folding studies demonstrate that, at lower interleukin-1β concentrations, tryptophan fluorescence kinetics can be fit adequately with a two exponential fit. This study indicates that (1) measured interleukin-1β folding kinetics fit to a 2 phase model and (2) at higher protein concentrations, transient association of IL-1β may result in a kinetic fit of 3 phases.     

Anion-binding centers of human serum albumin during an N-F conformation transition and in isolated albumin and blood serum

Fluorescent probe N-phenyl-1-amino-8-sulfonaphthalene (ANS) was used for studying pH-dependent structural N-F-transition in human serum albumin of two kinds: in commercial albumin and in natural blood serum. The kinetics of ANS fluorescence decay in albumin solutions was measured. There were found two types of the sites occupied by ANS in albumin under physiological conditions (pH 7.4). In the first binding site ANS fluorescence decay time was 16.6 +/- 0.3 nsec and it was not significantly changed at N-F transition (pH 4.0). In the second binding site the decay time was dependent on pH in commercial albumin and was not significantly changed in serum. In the second binding site there were individual differences of ANS decay time (4.3 +/- 0.6 nsec). The observed ANS fluorescence intensity enhancing (about 40-50%) in N-F transition may be explained by an increase of albumin binding sites capacity for ANS.     

能量化时线粒体内膜表面电荷的变化

本文报告用荧光探剂1,8—ANS和电泳激光光散射技术,研究鼠肝线粒体内膜在加入ATP的能量化过程中其膜表面电荷的变化。实验结果表明在加入ATP后线粒体内膜的能量化使其膜表面的负电荷减少。作者论讨了用上述二种方法研究线粒体内膜在能量化时表面电荷变化的有关问题。     

The interaction of the anionic fluorescence probe, 1-anilinonaphthalene-8-sulfonate, with hepatocytes and hepatoma tissue culture cells

The fluorescence probe 1-anilinonaphthalene-8-sulfonate (ANS) has been used to characterize the anion transport properties of normal hepatocytes and hepatoma tissue culture cells. Incubation of hepatocytes in the presence of ANS (20 micron) resulted in a 35-fold enhancement of fluorescence and a 50 nm blue shift. The time course of this process is biphasic. A rapid initial fluorescence enhancement suggests ANS binding to the plasma membrane, and a slower component reflects the uptake of ANS into intracellular compartments. Analysis of ANS uptake showed this latter process to be saturable, with a Km of 10 micron, to be temperature dependent and to occur only in viable cells. The above observations suggest a carrier-mediated anion transport mechanism. Incubation of hepatoma tissue culture cells with ANS (20 micron) gave a fluorescence emission spectrum similar to that obtained from purified plasma membranes. The kinetics of this interaction only exhibited a rapid initial binding of ANS. The second slow component was now absent, suggesting that ANS transport by the malignant cell system was greatly reduced. Transport of ANS could, however, be stimulated in the presence of the local anesthetic tetracaine. The observed transport was now saturable, temperature dependent, and as in normal hepatocytes, required viable cells, again indicating a carrier-mediated transport system. These studies suggest a significant alteration in membrane function in hepatoma tissue culture cells resulting in a major defect in anion transport.     

ANS fluorescence. I. Effect of self-association on the spectral properties of the probe]

The dependence of spectral properties of Mg2+ and NH4+ salt of 8-anilino-1-naphthalenesulfonic acid (Mg-(ANS)2 and NH4-ANS, respectively) on the dye concentration and solvent composition was investigated by means of steady-state and time-resolved fluorescence spectroscopy. We have shown that the increase in ANS concentrations leads to changes in the shape of absorption and fluorescence spectra of the dye, accompanied by the decrease in its fluorescence decay time values. Such changes, observed in aqueous and organic solvents for both salts of ANS, reflect the existence of self-association of the dye molecules. The decrease in fluorescence intensity induced by self-association of the probe molecules is too small to explain a weak fluorescence of ANS in water. At the same time, it expounds the difference between the decay times of protein-embedded ANS molecules upon interaction of this probe with native and molten globule proteins.     

Native states of adenylate kinase are two active sub-ensembles

There are two kinds of conformational forms of adenylate kinase (AK) in equilibrium in solution with different ANS-binding properties. Furthermore, the nature of AP(5)A inhibition suggests also that the native forms of AK for binding with different substrates pre-exist in the absence of substrates. In the present study, a kinetics approach was used to explore the native forms distinguished by ANS-binding properties and by the nature of AP(5)A inhibition. The results revealed that the native forms distinguished by ANS probe are two conformational sub-ensembles. Both sub-ensembles are active and consist of a series of forms, which pre-exist in solution and can bind with different substrates. The K(m) values of N(1) for AMP, ADP and MgATP are larger than that of N(2), indicating that the N(2) sub-ensemble is more specific for binding substrates. This is consistent with the previous observation that the activity of N(2) is about 1.8-fold of that of N(1).     

Rapid methods for differentiating Gram-positive from Gram-negative aerobic and facultative anaerobic bacteria

M anafi , M. & K neifel , W. 1990. Rapid methods for differentiating Gram-positive from Gram-negative aerobic and facultative anaerobic bacteria. Journal of Applied Bacteriology 69 , 822–827.
Different tests based on lysis by KOH and on reaction with fluorogenic and chromogenic substrates, L-alanine-4-nitroanilide (LANA); L-alanine-4-methoxy-β-naphthylamide (MNA); 4-alanine-2-amidoacridone (AAA); L-alanine-7-amido-4-methylcoumarin (AAMC); 8-anilino-l-naphthalene-sulphonic acid (ANS) were compared for their suitability to distinguish Gram-positive from Gram-negative bacteria. A concentration of 100 μg/ml was chosen for incorporating LANA, AAA, AAMC and ANS into the growth medium, based on sensitivity tests. MNA did not show any detectable reaction over a concentration range from 50 to 200 μg/ml, and led to inhibition of all bacteria at 200 μ/ml. In the examination of a total of 146 bacterial strains, including Yersinia enterocoiitica, Bacillus cereus , and B. subtilis the KOH test was not comparable with the Gram staining. A good correlation with Gram staining was found between LANA, AAA and AAMC added to plate count agar on one hand, and LANA and AAMC impregnated paper strips on the other hand, thereby utilizing the aminopeptidase activity. Agar containing ANS showed detectable fluorescence with all Gram-negative strains, but with Staphylococcus aureus and Staph. epidermidis a weak reaction was also observed. AAMC was selected for a rapid paper strip test With this substrate a pronounced blue fluorescence was obtained with Gram-negative colonies.