Interaction of fluorescent adenine nucleotide derivatives with the ADP/ATP carrier in mitochondria. 2. [5-(Dimethylamino)-1-naphthoyl]adenine nucleotides as probes for the transition between c and m states of the ADP/ATP carrier

The binding to the ADP/ATP carrier in mitochondrial membranes of the 3'-O-(dimethylamino)naphthoyl (DAN) derivatives of AMP, ADP, and ATP was quantitatively analyzed. The sidedness of the fluorescent type binding to the "m" side only was shown comparing the mitochondrial membranes in various stages of integrity and surface orientation. In particles displacement by bongkrekate (BKA) is direct, whereas in the case of carboxyatractylate (CAT) the requirement for ADP and ATP demonstrates the transition from the "m" to the "c" state. Quantitatively the "physical" binding of [3H]DAN-AMP and fluorescence are well correlated, allowing for a little nonfluorescent binding to the c side. For DAN-AMP KD is 1.6 microM, for DAN-ADP KD is 0.8 microM, and in the Hill plot a straight line with n = 1.25 is obtained. The maximum number of binding sites for [3H]DAN-AMP (1.5 mumol/g of protein) is about equal to the sites found for [3H]BKA if the unspecific binding of both ligands is differentiated by blocking carrier sites with CAT. [3H]CAT binding is somewhat lower in accordance with the limited access of CAT to inverted vesicles. ADP is able to decrease fluorescence only by about 35% at high concentrations (10 mM) whereas GDP has virtually no effect. With ADP, DAN-AMP binding decreases by 30% of the total binding sensitive to BKA. Binding to ATPase is low because of the absence of Mg2+.(ABSTRACT TRUNCATED AT 250 WORDS)     

Probing the nucleotide binding domain of the osmoregulator EnvZ using fluorescent nucleotide derivatives

EnvZ is a histidine protein kinase important for osmoregulation in bacteria. While structural data are available for this enzyme, the nucleotide binding pocket is not well characterized. The ATP binding domain (EnvZB) was expressed, and its ability to bind nucleotide derivatives was assessed using equilbrium and stopped-flow fluorescence spectroscopy. The fluorescence emission of the trinitrophenyl derivatives, TNP-ATP and TNP-ADP, increase upon binding to EnvZB. The fluorescence enhancements were quantitatively abolished in the presence of excess ADP, indicating that the fluorescent probes occupy the nucleotide binding pocket. Both TNP-ATP and TNP-ADP bind to EnvZB with high affinity (K(d) = 2-3 microM). The TNP moiety attached to the ribose ring does not impede access of the fluorescent nucleotide into the binding pocket. The association rate constant for TNP-ADP is 7 microM(-1) s(-1), a value consistent with those for natural nucleotides and the eucaryotic protein kinases. Using competition experiments, it was found that ATP and ADP bind 30- and 150-fold more poorly, respectively, than the corresponding TNP-derivatized forms. Surprisingly, the physiological metal Mg(2+) is not required for ADP binding and only enhances ATP affinity by 3-fold. Although portions of the nucleotide pocket are disordered, the recombinant enzyme is highly stable, unfolding only at temperatures in excess of 70 degrees C. The unusually high affinity of the TNP derivatives compared to the natural nucleotides suggests that hydrophobic substitutions on the ribose ring enforce an altered binding mode that may be exploited for drug design strategies.     

Interaction of fluorescent 3'-[1,5-(dimethylamino)naphthoyl]adenine nucleotides with the solubilized ADP/ATP carrier

The binding of the 3'-[1,5-(dimethylamino)naphthoyl] (DAN) derivatives of AMP, ADP, and ATP to the solubilized ADP/ATP carrier is studied, evaluating primarily the fluorescence enhancement and 3H-labeled compound binding. DAN nucleotides also fluoresce when adsorbed to Triton X-100 micelles that are used for solubilization of the carrier. The partition of DAN-AMP between water and Triton X-100 micelles is measured, and it is shown to be shifted toward a higher content in Triton micelles with increasing salt concentration. In order to maintain a low level of fluorescence, the Triton content is decreased. The fraction of DAN nucleotide fluorescence due to carrier binding is determined by the suppression with bongkrekate (BKA). In contrast to the membrane-bound carrier, the solubilized preparation shows an increase of total BKA-sensitive fluorescence by 30-60% upon addition of ATP or ADP. In the solubilized atractylate-protein complex, the ADP-stimulated fluorescence amounts even to 80%. The suppression of fluorescence by BKA is independent of the presence of ADP or ATP, while that by carboxyatractylate (CAT) depends on ADP or ATP. The quantitation with [3H]BKA and [3H]CAT of these ligand interactions with DAN-AMP fluorescence shows that DAN-AMP fluorescence reflects the "m"-state carrier population and its redistribution under the influence of ADP or ATP. Thus, besides the "c"/"m" distribution, the kinetics of the c to m transition in the solubilized carrier also can be determined. The m share is increased to 80% when SO4, Pi, or pyrophosphate is present during solubilization. The rate of the ADP- or ATP-stimulated transition to the m state is markedly dependent on pH and on the presence of various anions, whereas the extent is little varied. The affinity decreases 4-fold going from DAN-AMP to DAN-ADP and to DAN-ATP (KD = 0.9, 1.6, and 3.2 microM). Comparison with physical binding of [3H]DAN nucleotides shows that the fluorescence yield of bound DAN-AMP is about 1.4 times higher than that of bound DAN-ATP. DAN substitution causes more than a 100-fold affinity increase for AMP and a 50-fold increase for ADP or ATP, probably because of interaction of the DAN group with a hydrophobic niche. A less specific, low-affinity displacement of DAN nucleotides by GDP, ADP, GTP and ATP (Ki = 1-2 mM) probably reflects primarily the ionic interactions at the binding center.     

The extent of mitochondrial F1-ATPase and adenine nucleotide carrier activity with epsilon-ATP.

1. The use of 1,N6-ethenoadenosine 5'-triphosphate (epsilon-ATP), a synthetic, fluorescent analog of ATP, by whole rat liver mitochondria and by submitochondrial particles produced via sonication has been studied. 2. Direct [3H]adenine nucleotide uptake studies with isolated mitochondria, indicate the epsilon-[3H]ATP is not transported through the inner membrane by the adenine nucleotide carrier and is therefore not utilized by the 2,4-dinitrophenol-sensitive F1-ATPase (EC 3.6.1.3) that functions in oxidative phosphorylation. However, epsilon-ATP is hydrolyzed by a Mg2+-dependent, 2,4-dinitrophenol-insensitive ATPase that is characteristic of damaged mitochondria. 3. epsilon-ATP can be utilized quite well by the exposed F1-ATPase of sonic submitochondrial particles. This epsilon-ATP hydrolysis activity is inhibited by oligomycin and stimulated by 2,4-dinitrophenol. The particle F1-ATPase displays similar Km values for both ATP and epsilon-ATP; however, the V with ATP is approximately six times greater than with epsilon-ATP. 4. Since epsilon-ATP is a capable substrate for the submitochondrial particle F1-ATPase, it is proposed that the fluorescent properties of this ATP analog might be employed to study the submitochondrial particle F1-ATPase complex, and its response to various modifiers of oxidative phosphorylation.     

Cubebin and derivatives as inhibitors of mitochondrial complex I. Proposed interaction with subunit B8

The effects on mitochondrial respiration and complex I NADH oxidase activity of cubebin and derivatives were evaluated. The compounds inhibited the state 3 glutamate/malate-supported respiration of hamster liver mitochondria with IC(50) values ranging from 12.16 to 83.96 microM. NADH oxidase reaction was evaluated in submitochondrial particles. The compounds also inhibited this activity, showing the same order of potency observed for effects on state 3 respiration, as well as a tendency towards a non-competitive type of inhibition (K(I) values ranging from 0.62 to 16.1 microM). A potential binding mode of these compounds with complex I subunit B8, assessed by docking calculations, is proposed.     

Photoreceptor channel activation by nucleotide derivatives

Cyclic nucleotide activated sodium currents were recorded from photoreceptor outer segment membrane patches. The concentration of cGMP and structurally similar nucleotide derivatives was varied at the cytoplasmic membrane face; currents were generated at each concentration by the application of a voltage ramp. Nucleotide-activated currents were analyzed as a function of both concentration and membrane potential. For cGMP, the average K0.5 at 0 mV was 24 microM, and the activation was cooperative with an average Hill coefficient of 2.3. Of the nucleotide derivatives examined, only 8-[[(fluorescein-5-yl-carbamoyl)methyl]thio]-cGMP (8-Fl-cGMP) activated the channel at lower concentrations than cGMP with a K0.5 of 0.85 microM. The next most active derivative was 2-amino-6-mercaptopurine riboside 3',5'-monophosphate (6-SH-cGMP) which had a K0.5 of 81 microM. cIMP and cAMP had very high K0.5 values of approximately 1.2 mM and greater than 1.5 mM, respectively. All nucleotides displayed cooperativity in their response and were rapidly reversible. Maximal current for each derivative was compared to the current produced at 200 microM cGMP; only 8-Fl-cGMP produced an identical current. The partial agonists 6-SH-cGMP, cIMP, and cAMP activated currents which were approximately 90%, 80%, and 25% of the cGMP response, respectively. 5'-GMP, 2-aminopurine riboside 3',5'-monophosphate, and 2'-deoxy-cGMP produced no detectable current. The K0.5 values for cGMP activation, examined from -90 to +90 mV, displayed a weak voltage dependence of approximately 400 mV/e-fold; the index of cooperativity was independent of the applied field.(ABSTRACT TRUNCATED AT 250 WORDS)     

Synthesis and properties of fluorescent derivatives of atractyloside as potential probes of the mitochondrial ADP/ATP carrier protein

The chemical synthesis of fluorescent derivatives of atractyloside (ATR), an inhibitor of the mitochondrial ADP/ATP carrier protein, is described. These derivatives are the following: 6′-O-dansyl ATR, 6′-O-dansyl-aminobutyryl ATR, and 6′-O-naphthoyl ATR. The spectral properties of these analogs were analyzed, and their biological features were compared to those of ATR. The fluorescence emission of the dansyl ATR derivatives was increased in organic solvents and that of naphthoyl ATR was decreased; for both analogs, solubilization in organic solvents resulted in a blue shift of the emission peak. The fluorescent dansyl and naphthoyl ATR derivatives were specifically recognized by the mitochondrial ADP/ATP carrier protein. Because of their spectral properties and their biochemical reactivities, the fluorescent analogs of ATR can be considered as potential probes to investigate the topography of the ADP/ATP carrier in the mitochondrial membrane and to monitor conformational changes of the ADP/ATP carrier protein associated with transport.     

The recognition of two specific binding sites of the adenine nucleotide translocase by palmitoyl CoA in bovine heart mitochondria and submitochondrial particles.

Palmitoyl CoA which is an effective inhibitor of adenine nucleotide transport is able to remove bound [¹⁴C]ADP and [³H]atractylate from the translocator on the outer side of the inner mitochondrial membrane. Bongkrekic acid, when added to the incubation medium prior to palmitoyl CoA, can prevent the removal of bound [¹⁴C]ADP from the membrane by palmitoyl CoA, however, bongkrekic acid is ineffective if palmitoyl CoA is added first. Upon incubation with inverted submitochondrial particles, both palmitoyl CoA and bongkrekic acid prevent the uptake and transport of [¹⁴C]ADP by the particles. Moreover, when the submitochondrial particles are preincubated with [¹⁴C]ADP, palmitoyl CoA, like bongkrekic acid, is unable to remove the bound nucleotide from the inner face of the carrier. Thus, palmitoyl CoA which has a high affinity for the translocator on both sides of the inner mitochondrial membrane, nevertheless, interacts differently with the carrier on each side of the membrane. This suggests that the translocase contains binding sites in two specific states both of which accommodate palmitoyl CoA.     

Glucosylceramidase from calf spleen. Characterization of its active site with 4-n-alkylumbelliferyl beta-glucosides and N-alkyl derivatives of 1-deoxynojirimycin

The beta-glucosides of 4-heptyl-, -nonyl-, and -undecylumbelliferone were synthesized and their substrate properties studied with calf spleen glucosylceramidase. Self-association of the free long chain alkylumbelliferones in aqueous buffer was inferred from their low fluorescence in the absence and strongly enhanced fluorescence in the presence of detergents. Association of the higher alkylumbelliferyl glucosides with detergent micelles was indicated by the influence of detergent on solubility and on enzyme activity which differed markedly between the methyl and the higher alkyl substrates. Compared to 4-methylumbelliferyl beta-glucoside their Km was 14 to 23 times smaller and Vmax/Km 20 to 30 times larger with no significant difference between the nonyl and undecyl derivatives. The enzyme was inhibited by 1-deoxynojirimycin (1,5-dideoxy-1,5-imino-D-glucitol, dNM) and a series of its N-alkyl derivatives with Ki-values that ranged from 390 microM for the parent compound to 330 microM for the butyl derivative and 0.08 microM for the tetradecyl derivative. The biphasic linear plot of - RT X 1n [Ki/Ki (dNM)] vs. chain length is interpreted in terms of an aglycon binding site that has an extended hydrophobic region starting at about 5 carbon atoms from the catalytic site. dNM inhibited greater than or equal to 10(3) times better than D-glucose, and N-decanoyl-dNM was a very weak inhibitor compared to N-decyl-dNM. It is concluded that the formation of an ion pair consisting of the protonated dNM derivative and an essential carboxylate at the catalytic site makes a large contribution to the binding energy. Strong shielding of this site from the aqueous environment is indicated by identical effects of ionic strength on Km and Ki.     

Use of modified adenine nucleotides in mechanistic studies on oxidative phosphorylation: Structure and space at the catalytic site

This report summarizes structure/activity investigations on 3-O-substituted adenine nucleotides derived from 3-O-naphthoyl-ADP. Among these are fluorescent nucleotides, which allow one to differentiate between two types of binding sites on the inner surface of the mitchondrial inner membrane. One type of site is highly fluorescent but is not located on F₁. It is attributed to the nucleotide carrier, because it stays on the membrane when F₁ is removed. The other type of sites, giving no or only very low fluorescence, is located on F₁ and shows high affinity to these analogs, which is modulated by the energy state of the membrane. On the basis of kinetic data, stability of magnesium complexes, and fluorescence properties, conclusions are drawn on the probable conformation of these nucleotides in the bound state. They allow one to explain why these nucleotide analogs are extremely strong inhibitors of oxidative phosphorylation and photophosphorylation, why the ADP derivatives cannot be phosphorylated, and why the ATP analogs are no substrates of ATPase. Furthermore, the results allow some insight into the mechanism of phosphorylation and the structural properties at the catalytic site.Abbreviations AP₅A 5,5-diadenosinepentaphosphate - SMP submitochondrial particles - F-AD(T,M)P fluorescent AD(T,M)P = 3-O-(5-dimethylaminonaphthoyl-1)-AD(T,M)P - FCCP carbonylcyanide 4-trifluoromethoxyphenylhydraxone     

Role of magnesium in nucleotide exchange on the small G protein rac investigated using novel fluorescent Guanine nucleotide analogues

Novel guanine nucleotide analogues have been used to investigate the role of Mg(2+) in nucleotide release and binding with the small G protein rac. The fluorescent analogues have 7-(ethylamino)-8-bromocoumarin-3-carboxylic acid attached to the 3'-position of the ribose via an ethylenediamine linker. This modification has only small effects on the interaction with rac. There are large fluorescence changes on binding of the triphosphate to rac, on hydrolysis, and then on release of the diphosphate. Furthermore, the fluorescence is sensitive to the presence of Mg(2+) in the active site. Using this signal, it was shown that, for a variety of conditions, the nucleotides dissociate by a two-step mechanism. Mg(2+) is released first followed by the nucleotide. With the diphosphate, Mg(2+) is fast and nucleotide release slow. For the fluorescent GMPPNP analogue, the rate of dissociation is limited by Mg(2+) release. In the latter case, Mg(2+) binds tightly with a K(d) of 61 nM, whereas for the diphosphate the K(d) is 11 microM (30 degrees C, pH 7.6).     

Effects of ethacrynic acid and furosemide on phosphorylation reactions of kidney mitochondria. Inhibition of the adenine nucleotide translocase.

Previous reports that ethacrynic acid and furosemide diminish mitochondrial P : O ratios and reduce (Na+ + K+)-ATPase activity suggested that these diuretics may inhibit mitochondrial phosphorylation reactions. This possibility was initially studied by determining the effects of ethacrynic acid and furosemide on [32P]ATP exchange activity of rat kidney mitochondria. Concentrations of both drugs at 10(-4) M or greater, significantly inhibited [32P]ATP exchange. To investigate the mechanism of this inhibition, the effects of ethacrynic acid and furosemide on the ATPase activity of intract mitochondria and sonicated submitochondrial particles were determined. Both diuretics inhibited ATPase activity of intact mitochondria at 10(-4) M. In contrast, ATPase of submitochondrial particles was significantly less susceptible to inhibition by the diuretics. These results suggested that ethacrynic acid anf furosemide inhibit adenine nucleotide transport across the mitochondrial membrane. This was directly tested by determining the effects of the diretics on the mitochondrial adenine nucleotide translocase. At 5-10(-4) M, both ethacrynic acid and furosemide significantly inhibited adenine nucleotide transport. These findings suggest that ethacrynic acid and furosemide may diminish renal tubular solute reabsorption by direct inhibition of adenine nucleotide transport across the mitochondrial inner membrane.     

Differences in nucleotide-binding site of isoapyrases deduced from tryptophan fluorescence

Comparative studies of intrinsic and extrinsic fluorescence of apyrases purified from two potato tuber varieties (Pimpernel and Desirée) were performed to determine differences in the microenvironment of the nucleotide binding site. The dissociation constants (K(d)) of Pimpernel apyrase for the binding of different fluorescent substrate analogs: methylanthranoyl (MANT-), trinitrophenyl (TNP-), and epsilon -derivatives of ATP and ADP were determined from the quenching of Trp fluorescence, and compared with K(d) values previously reported for Desirée enzyme. Binding of non-fluorescent substrate analogues decreased the Trp emission of both isoapyrases, indicating conformational changes in the vicinity of these residues. Similar effect was observed with fluorescent derivatives where, in the quenching effect, the transfer of energy from tryptophan residues to the fluorophore moiety could be additionally involved. The existence of energy transfer between Trp residues in the Pimpernel enzyme was demonstrated with epsilon -analogues, similar to our previous observations with the Desirée. From these results we deduced that tryptophan residues are close to or in the nucleotide binding site in both enzymes. Experiments with quenchers like acrylamide, Cs(+) and I(-), both in the presence and absence of nucleotide analogues, suggest the existence of differences in the nucleotide binding site of the two enzymes. From the results obtained in this work, we can conclude that the differences found in the microenvironment of the nucleotide binding site can explain, at least in part, the kinetic behaviour of both isoenzymes.     

Characteristics of 8-substituted adenine nucleotide derivatives utilized in affinity chromatography.

Improved methods for the preparation of several 8-substituted adenine nucleotide derivatives are described. Enzymatic properties of these 8-substituted derivatives were investigated by steady state kinetic and inhibition studies. It was found that 8-(6-aminohexyl)-amino DPN⁺ and TPN⁺ exhibit relatively high affinity for most DPN⁺ and TPN⁺ dependent dehydrogenases. Preliminary nmr studies indicate that the 8-substituted adenine nucleotide derivatives may exist in slightly different ribosyl as well as glycosyl conformations from those of the natural adenine nucleotides. The chemical shift difference between geminal C₄ protons of dihydropyridine moiety of DPN⁺ and TPN⁺ changes from 0.1 to 0.2 ppm upon the 8-hexyl substitution of the natural coenzymes, indicating a strong interaction between 8-hexyl side chain of adenine moiety and the dihydropyridine moiety of these coenzyme derivatives. However, the folding and fluorescence properties of 8-(6-aminohexyl)-amino DPN⁺ and TPN⁺ as well as their reduced analogs in aqueous solutions are not significantly altered as compared to those of natural DPN⁺ and TPN⁺. Purification of glucose-6-phosphate dehydrogenase from yeast extracts and human erythrocytes using 8-(6-aminohexyl)-amino-TPN⁺ -Sepharose column is reported. Preliminary studies on the purification of various kinases using 8-substituted ADP and ATP Sepharose columns are also presented.     

Exploration of the nucleotide binding sites of the isolated ADP/ATP carrier protein from beef heart mitochondria. 1. Probing of the nucleotide sites by Naphthoyl-ATP, a fluorescent nontransportable analogue of ATP

The ADP/ATP carrier protein was extracted and purified from beef heart mitochondria, and its binding parameters with respect to 3'-O-naphthoyladenosine 5'-triphosphate (N-ATP), a fluorescent nontransportable analogue of ATP, were studied. The binding of N-ATP to the isolated carrier protein was accompanied by a decrease in fluorescence. Conversely, the release of bound N-ATP upon addition of carboxyatractyloside (CATR) or ATP resulted in a fluorescence increase. The bound N-ATP that was released upon addition of an excess of CATR or ATP was referred to as specifically bound N-ATP, i.e., N-ATP bound to the nucleotide sites of the carrier protein. Two classes of binding sites for N-ATP could be identified; the number of high-affinity sites (Kd less than 10 nM) was equal to the number of low-affinity sites (Kd = 0.45 microM). CATR behaved apparently as a noncompetitive inhibitor of the binding of N-ATP. The amount of N-ATP released increased linearly with the amount of CATR added, indicating an extremely high affinity of the carrier protein for CATR. The number of CATR binding sites was equal to half the total number of N-ATP binding sites (high- and low-affinity sites); at saturating concentrations of N-ATP, the binding of 1 mol of CATR resulted in the release of 2 mol of bound N-ATP, one from the high-affinity site and the other from the low-affinity site, showing unambiguously that each CATR site is interacting with a pair of probably interdependent N-ATP sites. A clear competition between N-ATP and ATP for binding to the carrier protein was demonstrated. The Kd values of the high- and low-affinity sites for ATP were less than 50 nM and 5 microM, respectively. In the presence of high concentrations of ATP, the two classes of N-ATP binding sites became indistinguishable, suggesting interconversion. It is proposed that the asymmetry in affinity for N-ATP binding is induced by the binding step itself, the carrier protein exhibiting a negative cooperativity for N-ATP binding.     

Equilibrium and kinetic analysis of nucleotide binding to the DEAD-box RNA helicase DbpA

The Escherichia coli DEAD-box protein A (DbpA) is an RNA helicase that utilizes the energy from ATP binding and hydrolysis to facilitate structural rearrangements of rRNA. We have used the fluorescent nucleotide analogues, mantADP and mantATP, to measure the equilibrium binding affinity and kinetic mechanism of nucleotide binding to DbpA in the absence of RNA. Binding generates an enhancement in mant-nucleotide fluorescence and a corresponding reduction in intrinsic DbpA fluorescence, consistent with fluorescence resonance energy transfer (FRET) from DbpA tryptophan(s) to bound nucleotides. Fluorescent modification does not significantly interfere with the affinities and kinetics of nucleotide binding. Different energy transfer efficiencies between DbpA-mantATP and DbpA-mantADP complexes suggest that DbpA adopts nucleotide-dependent conformations. ADP binds (K(d) approximately 50 microM at 22 degrees C) 4-7 times more tightly than ATP (K(d) approximately 400 microM at 22 degrees C). Both nucleotides bind with relatively temperature-independent association rate constants (approximately 1-3 microM(-1) s(-1)) that are much lower than predicted for a diffusion-limited reaction. Differences in the binding affinities are dictated primarily by the dissociation rate constants. ADP binding occurs with a positive change in the heat capacity, presumably reflecting a nucleotide-induced conformational rearrangement of DbpA. At low temperatures (<22 degrees C), the binding free energies are dominated by favorable enthalpic and unfavorable entropic contributions. At physiological temperatures (>22 degrees C), ADP binding occurs with positive entropy changes. We favor a mechanism in which ADP binding increases the conformational flexibility and dynamics of DbpA.     

Fluorescent derivatives of bile salts. II. Suitability of NBD-amino derivatives of bile salts for the study of biological transport.

Interaction of unconjugated and taurine-conjugated NBD-amino-dihydroxy-5 beta-cholan-24-oic acids bearing the fluorophor in the 3 alpha, 3 beta, 7 alpha, 7 beta, 12 alpha, or 12 beta position with albumin results in a small hypsochromic shift of the emission maximum and an increase in quantum yield, suggesting binding by hydrophobic interactions. The different unconjugated fluorescent bile salt derivatives are metabolized by intact rat liver in different ways. The unconjugated 3 beta-NBD-amino derivative is completely transformed to its taurine conjugate and secreted as such, whereas the 3 alpha-NBD-amino derivative is completely transformed to a polar fluorescent compound not identical with its taurine conjugate. The unconjugated 7 alpha- and 7 beta-NBD-amino derivatives are only partially conjugated with taurine and mainly secreted in unmetabolized form. The unconjugated 12 alpha- and 12 beta-NBD-amino derivatives are not at all transformed to their taurine conjugates, but are partially metabolized to unidentified compounds. They are predominantly secreted as the unmetabolized compounds. In contrast to the unconjugated derivatives, all taurine-conjugated fluorescent bile salt derivatives are secreted into bile unmetabolized. With the exception of the 3 alpha-compound, all synthesized taurine-conjugated fluorescent derivatives interfere with the secretion of cholyltaurine. Differential photoaffinity labeling studies using (7,7-azo-3 alpha,12 alpha- dihydroxy-5 beta-cholan-24-oyl)-2'-[2'-3H(N)]aminoethanesulfonate as a photolabile derivative revealed that in liver cells all fluorescent bile salt derivatives interact with the same polypeptides as the physiological bile salts. The hepatobiliary transport of taurine-conjugated NBD-amino bile salt derivatives is, due to hydrophobic interactions, accompanied by an increase in fluorescence intensity which is favorable for the study of biological bile salt transport by fluorescence microscopy.     

Insights into nucleotide binding in protein kinase A using fluorescent adenosine derivatives

The binding of the methylanthraniloyl derivatives of ATP (mant-ATP), ADP (mant-ADP), 2'deoxyATP (mant-2'deoxyATP), and 3'deoxyATP (mant-3'deoxyATP) to the catalytic subunit of protein kinase A was studied to gain insights into the mechanism of nucleotide binding. The binding of the mant nucleotides leads to a large increase in fluorescence energy transfer at 440 nm, allowing direct measurements of nucleotide affinity. The dissociation constant of mant-ADP is identical to that for ADP, while that for mant-ATP is approximately threefold higher than that for ATP. The dissociation constant for mant-3'deoxyATP is approximately fivefold higher than that for 3'deoxyATP while derivatization of 2'deoxyATP does not affect affinity. The time-dependent binding of mant-ATP, mant-2'deoxyATP, and mant-ADP, measured using stopped-flow fluorescence spectroscopy, is best fit to three exponentials. The fast phase is ligand dependent, while the two slower phases are ligand independent. The slower phases are similar but not identical in rate, and have opposite fluorescence amplitudes. Both isomers of mant-ATP are equivalent substrates, as judged by reversed-phase chromatography, although the rate of phosphorylation is approximately 20-fold lower than the natural nucleotide. The kinetic data are consistent with a three-step binding mechanism in which initial association of the nucleotide derivatives produces a highly fluorescent complex. Either one or two conformational changes can occur after the formation of this binary species, but one of the isomerized forms must have low fluorescence compared to the initial binary complex. These data soundly attest to the structural plasticity within the kinase core that may be essential for catalysis. Overall, the mant nucleotides present a useful reporter system for gauging these conformational changes in light of the prevailing three-dimensional models for the enzyme.     

3' Esters of ADP as energy-transfer inhibitors and probes of the catalytic site of oxidative phosphorylation.

1. A large series of 3' esters of ADP has been synthesized. Several of these can serve as photoaffinity labels; others exhibit fluorescent properties. The corresponding AMP and ATP derivatives have also been synthesized in some cases. 2. The influence of the 3'-O-acyl nucleotides on energy-linked functions of beef-heart submitochondrial particles has been investigated. The following results were obtained. a) 3'Esters of ADP are powerful and highly specific inhibitors of oxidative phosphorylation. The inhibition is competitive to ADP and Ki values as low as 0.05 microM, for the 3'-O-(1)naphthoyl ester of ADP, could be observed. b) The inhibition of oxidative phosphorylation by 3' esters of ADP appears to be non-competitive versus inorganic phosphate. c) The nucleotide analogs are not phosphorylated themselves. The corresponding ATP analogs can not drive energy-linked process. d) The 3' esters of AMP are ineffective as inhibitors, whereas the ATP derivatives are only comparatively weak inhibitors. e) Uncoupled or solubilized ATPase is almost two orders of magnitude less sensitive against inhibition by 3' esters than coupled systems. The analogs exert maximal inhibition specifically in systems involving an 'energized' state of the coupling device. f) Azido-group-bearing analogs can be used for irreversible photoinactivation of the coupling ATPase. Photoinactivation also is most efficient when carried out with 'energized' particles. g) The inhibitory properties are similar also in ATP-driven NAD+ reduction by succinate, and in the uncoupler-sensitive ATP in equilibrium with Pi exchange. The required concentrations for half-maximal inhibition are somewhat higher than in oxidative phosphorylation, but lower than with uncoupled ATPase. 3. From molecular models, from substituent properties, and from the conditions required for inhibition it is concluded that these highly effective analogs of ADP may act as conformation-specific probes at the catalytic site of oxidative phosphorylation. The results are interpreted in terms of a model suggesting that, in the process of ATP synthesis, a hydrophobic cavity on the enzyme is exposed only in the energized state, accepting the large 3' substituent. The substituent is assumed to inhibit phosphoryl transfer and/or conformational transitions inherent in the process of ADP phosphorylation by steric hinderance.     

Substrate-induced modifications of the intrinsic fluorescence of the isolated adenine nucleotide carrier protein: demonstration of distinct conformational states

The effects of ATP or ADP and the specific inhibitors carboxyatractyloside (CATR) and bongkrekic acid (BA) on the conformation of the isolated adenine nucleotide (AdN) carrier protein were studied by fluorescence spectroscopy. The addition of ATP to the AdN carrier resulted in a rapid fluorescence increase of the tryptophanyl residue(s) at 355 nm, which leveled up in less than 1 s at 22 degrees C. Among the natural nucleotides, only ATP and ADP were effective. At 10 degrees C or below, the kinetics of the fluorescence increase induced by ATP were biphasic, consisting of a rapid phase of less than 1 s, followed by a slower phase that lasted for a few seconds and had virtually the same amplitude as the rapid one. Both phases were abolished when CATR was added prior to ATP or fully reversed when CATR was added after the fluorescence response to ATP had been elicited. The number of CATR binding sites present on the carrier protein was determined by CATR specific inhibition of the ATP-induced increase in intrinsic fluorescence. The calculated number of CATR sites was equal to that found by another method based on the use of the same preparation of AdN carrier loaded with fluorescent nucleotide naphthoyl-ATP and on the CATR-induced release of the bound naphthoyl-ATP, demonstrating the reliability of the intrinsic fluorescence assay. Addition of BA prior to or together with ATP nearly doubled the amplitude of the ATP-induced fluorescence signal. At 10 degrees C or below, the fluorescence response to ATP in the presence of BA could also be decomposed into rapid and slow phases.(ABSTRACT TRUNCATED AT 250 WORDS)