Ibogaine, a noncompetitive inhibitor of serotonin transport, acts by stabilizing the cytoplasm-facing state of the transporter

Ibogaine, a hallucinogenic alkaloid with purported anti-addiction properties, inhibited serotonin transporter (SERT) noncompetitively by decreasing V(max) with little change in the K(m) for serotonin (5-HT). Ibogaine also inhibited binding to SERT of the cocaine analog 2beta-2-carbomethoxy-3-(4-[(125)I]iodophenyl)tropane. However, inhibition of binding was competitive, increasing the apparent K(D) without much change in B(max). Ibogaine increased the reactivity of cysteine residues positioned in the proposed cytoplasmic permeation pathway of SERT but not at nearby positions out of that pathway. In contrast, cysteines placed at positions in the extracellular permeation pathway reacted at slower rates in the presence of ibogaine. These results are consistent with the proposal that ibogaine binds to and stabilizes the state of SERT from which 5-HT dissociates to the cytoplasm, in contrast with cocaine, which stabilizes the state that binds extracellular 5-HT.     

Conformational changes of tarantula (Eurypelma californicum) haemocyanin detected with a fluorescent probe, 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole

Different fluorescent labels were tested in order to monitor conformational transitions of the four-hexamer haemocyanin from the tarantula Eurypelma californicum during the oxygenation process. When the four-hexamer was labelled with 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole, the maximum wavelength lambda max of the fluorescence emission spectrum was significantly shifted up to 5 nm, depending on pH and the degree of oxygenation. The values for lambda max of the fully oxygenated haemocyanin were 531.5 nm (pH less than 7.8) and 530.0 nm (pH greater than 7.8). For deoxygenated haemocyanin the values were 533.5 nm (pH less than 7.2) and 535.2 nm (pH greater than 7.2). The occurrence of four distinct emission maxima supports the hypothesis of four conformational species for the tarantula haemocyanin, which have been predicted by the nesting model [Robert, C. H., Decker, H., Richey, B., Gill, S. J. & Wyman, J. (1987) Proc. Natl Acad. Sci. USA 84, 1891-1895]. Only four amino acids of the four-hexamer were labelled with 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole. They were identified as lysine 484 on the purified peptide Leu-Arg-Lys-Phe-His-Arg. This amino acid is located on the surface of the four copies of subunit d. The sharp shift of the maxima of the emission wavelengths during oxygenation indicates that the four copies of subunits d synchronously take part in the conformational switch. This points to a concerted mechanism for the conformational transitions of the tarantula haemocyanin.     

Fluorescence energy transfer between ligand binding sites on chloroplast coupling factor 1.

The method of fluorescence energy transfer is used to measure the distance from the tight nucleotide binding sites to the 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole reactive sites on solubilized spinach chloroplast coupling factor 1 (CF1). The fluorescent adenine nucleotide analogs 1,N-6-ethenoadenosine diphosphate and 1,N-6-ethenoadenylyl imidodiphosphate were used as donors and 4-nitrobenzo-2-oxa-1,3-diazole bound to a tyrosine group and to an amino group were used as acceptors of energy transfer. Using three different donor-acceptor pairs, the distance measured varied from 38 to 43 A assuming both donor sites are equidistant from the acceptor site. A model is proposed for the location of the tight nucleotide binding sites and the active site on the alpha and beta subunits of CF1.     

Visualizing Dopamine and Serotonin Transporters in the Human Brain with the Potent Cocaine Analogue [125I]RTI-55: In Vitro Binding and Autoradiographic Characterization

Abstract: The cocaine analogue RTI-55 was evaluated as a probe for in vitro labeling and localization of dopamine and serotonin transporters after death in the human brain. Kinetic, saturation, and competition binding experiments indicated complex interactions of the radioligand with the identification of multiple recognition sites. In membrane binding assays, the association of [¹²⁵I]RTI-55 at 25°C to putamen membranes was monophasic. In contrast, dissociation of [¹²⁵I]RTI-55 occurred in two phases with t_1/2 values of 9.4 and 36.5 min, respectively. Saturation analysis of [¹²⁵I]RTI-55 binding demonstrated two binding sites in the human putamen with K_D values of 0.10 ± 0.02 and 1.81 ± 0.46 nM. The binding of [¹²⁵I]RTI-55 was displaced by a wide range of cocaine analogues and monoamine uptake inhibitors. The rank order of potency demonstrated in competition assays with human putamen membranes indicates that the radioligand labels cocaine recognition sites on the dopamine transporter (mazindol > GBR 12909 > GBR 12935 > paroxetine > nisoxetine > desipramine ≥ fluoxetine > citalopram). In the human occipital cortex, [¹²⁵I]RTI-55 recognized multiple binding sites with K_D values of 0.02 ± 0.01 and 4.18 ± 0.46 nM. The rank order of potency for inhibition of [¹²⁵I]RTI-55 binding to cerebral cortex membranes (paroxetine > citalopram > GBR 12909 ≥ mazindol ≥ nisoxetine > benztropine) suggests that [¹²⁵I]RTI-55 labels the serotonin transporter in the human occipital cortex. Autoradiographic mapping of [¹²⁵I]RTI-55 revealed very high densities of cocaine recognition sites over areas known to be rich in dopaminergic innervation, including the caudate, putamen, and nucleus accumbens. Moderately elevated densities of [¹²⁵I]RTI-55 binding sites were also seen throughout the thalamus, hypothalamus, and substantia nigra. [¹²⁵I]RTI-55 binding sites were prevalent throughout the cerebral cortex and amygdala. In autoradiographic studies, the addition of the selective serotonin transport blocker citalopram completely prevented [¹²⁵I]RTI-55 labeling in the thalamus, hypothalamus, and throughout most of the cerebral cortex. In the presence of citalopram, [¹²⁵I]RTI-55 binding site densities remained elevated over the striatum and substantia nigra, with selective residual labeling also seen in the external segment of the globus pallidus and the lateral nucleus of the amygdala. These results demonstrate that in the human brain, [¹²⁵I]RTI-55 labels multiple recognition sites on dopamine and serotonin transporters.     

The role of conserved tryptophan and acidic residues in the human dopamine transporter as characterized by site-directed mutagenesis

The human dopamine (DA) transporter (hDAT) contains multiple tryptophans and acidic residues that are completely or highly conserved among Na(+)/Cl(-)-dependent transporters. We have explored the roles of these residues using non-conservative substitution. Four of 17 mutants (E117Q, W132L, W177L and W184L) lacked plasma membrane immunostaining and were not functional. Both DA uptake and cocaine analog (i.e. 2beta-carbomethoxy-3beta-(4-fluorophenyl)tropane, CFT) binding were abolished in W63L and severely damaged in W311L. Four of five aspartate mutations (D68N, D313N, D345N and D436N) shifted the relative selectivity of the hDAT for cocaine analogs and DA by 10-24-fold. In particular, mutation of D345 in the third intracellular loop still allowed considerable [(3)H]DA uptake, but caused undetectable [(3)H]CFT binding. Upon anti-C-terminal-hDAT immunoblotting, D345N appeared as broad bands of 66-97 kDa, but this band could not be photoaffinity labeled with cocaine analog [(125)I]-3beta-(p-chlorophenyl)tropane-2beta-carboxylic acid ([(125)I]RTI-82). Unexpectedly, in this mutant, cocaine-like drugs remained potent inhibitors of [(3)H]DA uptake. CFT solely raised the K(m) of [(3)H]DA uptake in wild-type hDAT, but increased K(m) and decreased V(max) in D345N, suggesting different mechanisms of inhibition. The data taken together indicate that mutation of conserved tryptophans or acidic residues in the hDAT greatly impacts ligand recognition and substrate transport. Additionally, binding of cocaine to the transporter may not be the only way by which cocaine analogs inhibit DA uptake.     

Study of the binding environment of alpha-factor in its G protein-coupled receptor using fluorescence spectroscopy.

Mating in Saccharomyces cerevisiae is induced by the interaction of alpha-factor (W1H2W3L4Q5L6K7P8G9Q10P11M12Y13) with its cognate G protein-coupled receptor (Ste2p). Fifteen fluorescently labeled analogs of alpha-factor in which the 7-nitrobenz-2-oxa-1,3-diazol-4-yl (NBD) group was placed at the alphaN-terminus and in side-chains at positions 1, 3, 4, 6, 7, 12 and 13 were synthesized and assayed for biological activity and receptor affinity. Eleven of the analogs retained 6-60% of the biological activity of the alpha-factor, as judged using a growth arrest assay. The binding affinities depended on the position of NBD attachment in the peptide and the distance of the tag from the backbone. Derivatization of the positions 3 and 7 side-chains with the NBD group resulted in analogs with affinities of 17-35% compared with that of alpha-factor. None of the other NBD-containing agonists had sufficient receptor affinity or strong enough emission for fluorescence analysis. The position 3 and 7 analogs were investigated using fluorescence spectroscopy and collisional quenching by KI in the presence of Ste2p in yeast membranes. The results showed that the lambda max of NBD in the position 7 side-chain shifted markedly to the blue (510 nm) when separated by 4 or 6 bonds from the peptide backbone and that this probe was shielded from quenching by KI. In contrast, separation by 3, 5, 10 or more bonds resulted in lambda max ( approximately 540 nm) and collisional quenching constants consistent with increasing degrees of exposure. The NBD group in the position 3 side-chain was also found to be blue shifted (lambda max=520 nm) and shielded from solvent. These results indicate that the position 7 side-chain is likely interacting with a pocket formed by extracellular domains of Ste2p, whereas the side-chain of Trp3 is in a hydrophobic pocket possibly within the transmembrane region of the receptor.     

Biochemical characterization and structural analysis of a highly proficient cocaine esterase

The bacterial cocaine esterase, cocE, hydrolyzes cocaine faster than any other reported cocaine esterase. Hydrolysis of the cocaine benzoyl ester follows Michaelis-Menten kinetics with k(cat) = 7.8 s(-1) and K(M) = 640 nM. A similar rate is observed for hydrolysis of cocaethylene, a more potent cocaine metabolite that has been observed in patients who concurrently abuse cocaine and alcohol. The high catalytic proficiency, lack of observable product inhibition, and ability to hydrolyze both cocaine and cocaethylene make cocE an attractive candidate for rapid cocaine detoxification in an emergency setting. Recently, we determined the crystal structure of this enzyme, and showed that it is a serine carboxylesterase, with a catalytic triad formed by S117, H287, and D259 within a hydrophobic active site, and an oxyanion hole formed by the backbone amide of Y118 and the Y44 hydroxyl. The only enzyme previously known to use a Tyr side chain to form the oxyanion hole is prolyl oligopeptidase, but the Y44F mutation of cocE has a more deleterious effect on the specificity rate constant (k(cat)/K(M)) than the analogous Y473F mutation of prolyl oligopeptidase. Kinetic studies on a series of cocE mutants both validate the proposed mechanism, and reveal the relative contributions of active site residues toward substrate recognition and catalysis. Inspired by the anionic binding pocket of the cocaine binding antibody GNC92H2, we found that a Q55E mutation within the active site of cocE results in a modest (2-fold) improvement in K(M), but a 14-fold loss of k(cat). The pH rate profile of cocE was fit to the ionization of two groups (pK(a1) = 7.7; pK(a2) = 10.4) that likely represent titration of H287 and Y44, respectively. We also describe the crystal structures of both S117A and Y44F mutants of cocE. Finally, urea denaturation studies of cocE by fluorescence and circular dichroism show two unfolding transitions (0.5-0.6 M and 3.2-3.7 M urea), with the first transition likely representing pertubation of the active site.     

Regulation of Rat Dopamine Transporter mRNA and Protein by Chronic Cocaine Administration

This study describes a direct comparison of dopamine transporter (DAT) mRNA and protein, as well as its binding sites, in tissue from the same animals after chronic cocaine administration. Rats were treated twice daily with 25 mg/kg cocaine or with saline. After 8 days of cocaine administration, changes in DAT mRNA levels in the substantia nigra pars compacta and ventral tegmental area were measured by in situ hybridization, and DAT protein in the striatum was quantified by immunoblotting. Whereas chronic cocaine treatment significantly reduced levels of DAT mRNA in the substantia nigra pars compacta and ventral tegmental area as compared with vehicle-treated controls, cocaine treatment did not alter DAT protein levels in the striatum. Furthermore, the density of DAT binding sites was also measured in the striatum by quantitative autoradiography using two DAT radioligands, 33-(4-[125I]iodophenyl)tropane-2-carboxylic acid methyl ester ([125I]RTI-55) and [3H]propanoyl-3beta-(4-tolyl)tropane ([3H]PTT). Similar to the results of immunoblotting of DAT protein, [1251]RTI-55 and [3H]PTT binding site levels also remained unaltered. These results indicate a dissociation in the regulation of DAT mRNA and its protein levels as a result of cocaine administration in rats. This study also indicates that the DAT ligands [3H]PTT and [125I]RTI-55 provide an accurate assessment of DAT protein levels.     

Single- and multi-photon excited fluorescence from serotonin complexed with beta-cyclodextrin.

The fluorescence of serotonin on binding with beta-cyclodextrin has been studied using both steady state and time-resolved methods. Steady state fluorescence intensity of serotonin at 340 nm showed approximately 30% increase in intensity on binding with K(A) approximately 60 dm(3) mol(-1) and the fluorescence lifetimes showed a corresponding increase. In contrast, the characteristic green fluorescence ('hyperluminescence') of serotonin observed upon multiphoton near-infrared excitation with sub-picosecond pulses was resolved into two lifetime components assigned to free and bound serotonin. The results are of interest in relation to selective imaging and detection of serotonin using the unusual hyperluminescence emission and in respect to recent determinations of serotonin by capillary electrophoresis in the presence of cyclodextrin. The results also suggest that hyperluminescence occurs from multiphoton excitation of a single isolated serotonin molecule.     

Fluorescence energy transfer measurements between the nucleotide binding site and Cys-373 in actin and their application to the kinetics of actin polymerization

Intramonomer fluorescence energy transfer between the donor epsilon-ATP bound to the nucleotide-binding site and the acceptor 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole bound to Cys-373 in G-actin was measured by steady-state fluorimetry. Assuming for the orientation factor its dynamic limit K2 = 2/3, the donor and acceptor distance in a G-actin molecule was calculated to be about 3 nm. The intermonomer energy transfer in F-actin occurring between the donor bound to an actin monomer and the acceptor bound to the nearest-neighbour actin monomer was also measured and the distance was calculated to be about 4 nm. The kinetics of the actin polymerization process was studied by following the decrease in fluorescence intensity upon addition of salts to G-actin solution. The initial velocity of the fluorescence intensity change was proportional to the square of the initial G-actin concentration. The temperature dependence of the velocity was proportional to the square of the initial G-actin concentration. The temperature dependence of the velocity was proportional to exp(-10/RT). These results indicated that the initial fluorescence intensity change corresponds to monomer-dimer transformation and its activation enthalpy was 10 kcal/mol.     

Identification of a general anesthetic binding site in the diacylglycerol-binding domain of protein kinase Cdelta

Protein kinase C (PKC) is an important signal transduction protein that has been proposed to interact with general anesthetics at its cysteine-rich diacylglycerol/phorbol ester-binding domain C1, a tandem repeat of C1A and C1B subdomains. To test this hypothesis, we expressed, purified, and characterized the high affinity phorbol-binding subdomain, C1B, of mouse protein kinase Cdelta, and studied its interaction with general anesthetic alcohols. When the fluorescent phorbol ester, sapintoxin-D, bound to PKCdelta C1B in buffer at a molar ratio of 1:2, its fluorescence emission maximum, lambda(max), shifted from 437 to 425 nm. The general anesthetic alcohols, butanol and octanol, further shifted lambda(max) of the PKCdelta C1B-bound sapintoxin-D in a concentration-dependent, saturable manner to approximately 415 nm, suggesting that alcohols interact at a discrete allosteric binding site. To identify this site, PKCdelta C1B was photolabeled with three photo-activable diazirine alcohol analogs, 3-azioctanol, 7-azioctanol, and 3-azibutanol. Mass spectrometry showed photoincorporation of all three alcohols in PKCdelta C1B at a stoichiometry of 1:1 in the labeled fraction. The photolabeled PKCdelta C1B was subjected to tryptic digest, the fragments were separated by online chromatography and sequenced by mass spectrometry. Each azialcohol photoincorporated at Tyr-236. Inspection of the known structure of PKCdelta C1B shows that this residue is situated adjacent to the phorbol ester binding pocket, and within approximately 10 A of the bound phorbol ester. The present results provide direct evidence for an allosteric anesthetic site on protein kinase C.     

Oxidation of specific tryptophan residues inhibits high-affinity binding of cocaine and its metabolites to a humanized anticocaine mAb

Cocaine addiction remains a serious problem lacking an effective pharmacological treatment. Thus, we have developed a high-affinity anti-cocaine monoclonal antibody (mAb), h2E2, for the treatment of cocaine use disorders. We show that selective tryptophan (Trp) oxidation by 2,2′-azobis(2-amidinopropane) dihydrochloride (AAPH) resulted in a loss of high-affinity binding of cocaine to this mAb. The newly developed use of excess methionine (Met) to protect mAb met residues from AAPH oxidation did not substantially attenuate the effects of oxidation on cocaine binding but greatly decreased the modification of met residues in the mAb. Similar large decreases in ligand affinity (5000–10,000-fold) upon oxidation were observed using cocaine and two cocaine metabolites, cocaethylene and benzoylecgonine, which also bind with nanomolar affinity to this h2E2 mAb. The decrease in binding affinity was accompanied by a decrease of approximately 50% in Trp fluorescence, and increases in mAb 310 to 370 nm absorbance were consistent with the presence of oxidized forms of Trp. Finally, mass spectral analysis of peptides derived from control and AAPH-oxidized mAb indicated that excess free met did effectively protect mAb met residues from oxidation, and that AAPH-oxidized mAb heavy-chain Trp33 and light-chain Trp91 residues are important for cocaine binding, consistent with a recently derived h2E2 Fab fragment crystal structure containing bound benzoylecgonine. Thus, protection of the anti-cocaine h2E2 mAb from Trp oxidation prior to its clinical administration is critical for its proposed therapeutic use in the treatment of cocaine use disorders.     

Cooperativity and pseudo-cooperativity in the glutathione S-transferase from Plasmodium falciparum

Binding and catalytic properties of glutathione S-transferase from Plasmodium falciparum (PfGST) have been studied by means of fluorescence, steady state and pre-steady state kinetic experiments, and docking simulations. This enzyme displays a peculiar reversible low-high affinity transition, never observed in other GSTs, which involves the G-site and shifts the apparent K(D) for glutathione (GSH) from 200 to 0.18 mM. The transition toward the high affinity conformation is triggered by the simultaneous binding of two GSH molecules to the dimeric enzyme, and it is manifested as an uncorrected homotropic behavior, termed "pseudo-cooperativity." The high affinity enzyme is able to activate GSH, lowering its pK(a) value from 9.0 to 7.0, a behavior similar to that found in all known GSTs. Using 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole, this enzyme reveals a potential optimized mechanism for the GSH conjugation but a low catalytic efficiency mainly due to a very low affinity for this co-substrate. Conversely, PfGST efficiently binds one molecule of hemin/monomer. The binding is highly cooperative (n(H) = 1.8) and occurs only when GSH is bound to the enzyme. The thiolate of GSH plays a crucial role in the intersubunit communication because no cooperativity is observed when S-methylglutathione replaces GSH. Docking simulations suggest that hemin binds to a pocket leaning into both the G-site and the H-site. The iron is coordinated by the amidic nitrogen of Asn-115, and the two carboxylate groups are in electrostatic interaction with the epsilon-amino group of Lys-15. Kinetic and structural data suggest that PfGST evolved by optimizing its binding property with the parasitotoxic hemin rather than its catalytic efficiency toward toxic electrophilic compounds.     

Disease-causing mutations in the cellular retinaldehyde binding protein tighten and abolish ligand interactions

Mutations in the human cellular retinaldehyde binding protein (CRALBP) gene cause retinal pathology. To understand the molecular basis of impaired CRALBP function, we have characterized human recombinant CRALBP containing the disease causing mutations R233W or M225K. Protein structures were verified by amino acid analysis and mass spectrometry, retinoid binding properties were evaluated by UV-visible and fluorescence spectroscopy and substrate carrier functions were assayed for recombinant 11-cis-retinol dehydrogenase (rRDH5). The M225K mutant was less soluble than the R233W mutant and lacked retinoid binding capability and substrate carrier function. In contrast, the R233W mutant exhibited solubility comparable to wild type rCRALBP and bound stoichiometric amounts of 11-cis- and 9-cis-retinal with at least 2-fold higher affinity than wild type rCRALBP. Holo-R233W significantly decreased the apparent affinity of rRDH5 for 11-cis-retinoid relative to wild type rCRALBP. Analyses by heteronuclear single quantum correlation NMR demonstrated that the R233W protein exhibits a different conformation than wild type rCRALBP, including a different retinoid-binding pocket conformation. The R233W mutant also undergoes less extensive structural changes upon photoisomerization of bound ligand, suggesting a more constrained structure than that of the wild type protein. Overall, the results show that the M225K mutation abolishes and the R233W mutation tightens retinoid binding and both impair CRALBP function in the visual cycle as an 11-cis-retinol acceptor and as a substrate carrier.     

Aladan3]TIPP: a fluorescent delta-opioid antagonist with high delta-receptor binding affinity and delta selectivity

Fluorescent analogues of the potent and highly selective delta-opioid antagonist TIPP (H-Tyr-Tic-Phe-Phe-OH) and TIP (H-Tyr-Tic-Phe-OH) containing the exceptionally environmentally sensitive fluorescent amino acid beta-(6'-dimethylamino-2'-naphthoyl)alanine (Aladan [Ald]) in place of Phe3 were synthesized. The Ald3- and D-Ald3 analogues of TIPP and TIP all retained delta-opioid antagonist properties. The most potent analogue, [Ald3]TIPP, showed a K(e) value of 2.03 nM in the mouse vas deferens assay and five times higher delta vs. mu selectivity (K(i)mu/K(i)delta = 7930) than the TIPP parent peptide in the opioid receptor binding assays. Theoretical conformational analyses of [Ald3]TIPP and [Ald3]TIP using molecular mechanics calculations resulted in a number of low-energy conformers, including some showing various patterns of aromatic ring stacking and others with the Ald side chain and a carbonyl group (fluorescence quencher) in close proximity. These ensembles of low-energy conformers are in agreement with the results of steady-state fluorescence experiments (fluorescence emission maxima and quantum yields) and fluorescence decay measurements (fluorescence lifetime components), which indicated that the fluorophore was either engaged in intramolecular hydrophobic interactions or in proximity of a fluorescence quencher (e.g., a carbonyl group). These fluorescent TIP(P) delta-opioid antagonists represent valuable pharmacological tools for various applications, including studies on membrane interactions, binding to receptors, cellular uptake and intracellular distribution, and tissue distribution.     

A continuous fluorescence assay for lecithin cholesterol acyltransferase

A continuous fluorescence assay was adapted to the measurement of the phospholipase reaction of lecithin cholesterol acyltransferase (LCAT). The fluorescent phospholipid 1-acyl-2-(N-4-nitrobenzo-2-oxa-1,3-diazole)aminocaproyl phosphatidylcholine (C6-NBD-PC) in micelle form reacted with LCAT to yield NBD-caproic acid, resulting in up to 5-fold increases in fluorescence in 30 min. The reaction rates were optimal in mixtures containing 0.1 M NaCl and 4 mM beta-mercaptoethanol at 37 degrees C. Apolipoprotein A-I did not activate the enzyme and bovine serum albumin bound monomeric substrate and interfered with the fluorescence assay. Under similar reaction conditions, bee venom phospholipase A2 was almost 100-fold more reactive than LCAT.     

Synthesis and properties of a fluorescent derivative of phosphatidylcholine

1-acyl-2-(N-4-nitrobenzo-2-oxa-1,3-diazole)-aminocaproyl phosphatidyl choline, (NBD-PC) was prepared by alkylation of ?-amino caproic acid with 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole (NBD-C1), followed by esterification of lysophosphatidylcholine. The compound was purified by silicic acid chromatography, and exhibited a single spot on thin layer chromatography using acidic, basic and neutral solvent systems, when visualized by UV, molybdate spray, primuline, or charring. The UV-visible absorption spectrum, and the uncorrected fluorescence excitation spectrum of NBD-PC in absolute ethanol showed maxima at approximately 340 and 460 nm, while the fluorescence emission spectrum showed a single peak at 525 nm. Fluorescence intensity and emission maximum wavelength of NBD-PC are strongly dependent on solvent dielectric constant, and the relative fluorescent intensity of NBD-PC in absolute ethanol is directly proportional to its concentration from 1 ng/ml to approximately 3 μg/ml. Incorporation of NBD-PC into membranes of human lymphocytes cultures in the presence or absence of phytohemagglutinin (PHA) resulted in a marked increase in the relative fluorescent intensity of the bound fluorochrome, and a 15 nm blue shift in its emission maximum wavelength. Fluorescence titration data indicate that the unstimulated lymphocytes bound 912 pmoles NBD-PC/mg protein with an association constant of 3.45 × 10⁷ M, while the PHA stimulated cells bound 1200 pmoles NBD-PC/mg protein with an association constant of 2.82 × 10⁷ M. The temperature dependence of the fluorescent intensity of NBD-PC incorporated in control, and PHA stimulated lymphocytes showed discontinuities at 15 and 24 °C respectively. Fluorescence polarization of NBD-PC incorporated in the membranes of stimulated lymphocytes was greater than the polarization of the fluorochrome in non-stimulated cells, suggesting that the plasma membranes of PHA stimulated lymphocytes contain regions of higher microviscosity.     

The Effects of ADF/Cofilin and Profilin on the Conformation of the ATP-Binding Cleft of Monomeric Actin

Actin depolymerizing factor (ADF)/cofilin and profilin are small actin-binding proteins, which have central roles in cytoskeletal dynamics in all eukaryotes. When bound to an actin monomer, ADF/cofilins inhibit the nucleotide exchange, whereas most profilins accelerate the nucleotide exchange on actin monomers. In this study the effects of ADF/cofilin and profilin on the accessibility of the actin monomer''s ATP-binding pocket was investigated by a fluorescence spectroscopic method. The fluorescence of the actin bound ɛ-ATP was quenched with a neutral quencher (acrylamide) in steady-state and time dependent experiments, and the data were analyzed with a complex form of the Stern-Volmer equation. The experiments revealed that in the presence of ADF/cofilin the accessibility of the bound ɛ-ATP decreased, indicating a closed and more compact ATP-binding pocket induced by the binding of ADF/cofilin. In the presence of profilin the accessibility of the bound ɛ-ATP increased, indicating a more open and approachable protein matrix around the ATP-binding pocket. The results of the fluorescence quenching experiments support a structural mechanism regarding the regulation of the nucleotide exchange on actin monomers by ADF/cofilin and profilin.     

The photobleaching sequence of a short-wavelength visual pigment.

The photobleaching pathway of a short-wavelength cone opsin purified in delipidated form (lambda(max) = 425 nm) is reported. The batho intermediate of the violet cone opsin generated at 45 K has an absorption maximum at 450 nm. The batho intermediate thermally decays to the lumi intermediate (lambda(max) = 435 nm) at 200 K. The lumi intermediate decays to the meta I (lambda(max) = 420 nm) and meta II (lambda(max) = 388 nm) intermediates at 258 and 263 K, respectively. The meta II intermediate decays to free retinal and opsin at >270 K. At 45, 75, and 140 K, the photochemical excitation of the violet cone opsin at 425 nm generates the batho intermediate at high concentrations under moderate illumination. The batho intermediate spectra, generated via decomposing the photostationary state spectra at 45 and 140 K, are identical and have properties typical of batho intermediates of other visual pigments. Extended illumination of the violet cone opsin at 75 K, however, generates a red-shifted photostationary state (relative to both the dark and the batho intermediates) that has as absorption maximum at approximately 470 nm, and thermally reverts to form the normal batho intermediate when warmed to 140 K. We conclude that this red-shifted photostationary state is a metastable state, characterized by a higher-energy protein conformation that allows relaxation of the all-trans chromophore into a more planar conformation. FTIR spectroscopy of violet cone opsin indicates conclusively that the chromophore is protonated. A similar transformation of the rhodopsin binding site generates a model for the VCOP binding site that predicts roughly 75% of the observed blue shift of the violet cone pigment relative to rhodopsin. MNDO-PSDCI calculations indicate that secondary interactions involving the binding site residues are as important as the first-order chromophore protein interactions in mediating the wavelength maximum.     

Mechanism for activation of the 4-nitrobenzo-2-oxa-1,3-diazole-labeled sarcoplasmic reticulum ATPase by Ca2+ and its modulation by nucleotides

The mechanism for activation of sarcoplasmic reticulum ATPase by Ca2+ was investigated in 2 mM MgCl2 and 0.1 M KCl at pH 6.5 and 11 degrees C by using enzyme preparations in which a specific amino acid residue (Cys-344) was labeled with 4-nitrobenzo-2-oxa-1,3-diazole (NBD) [Wakabayashi, S., Imagawa, T., & Shigekawa, M. (1990) J. Biochem. (Tokyo) 107, 563-571]. We compared the kinetics of binding and dissociation of Ca2+ from the enzyme with those of the accompanying NBD fluorescence changes. The fluorescence rise following addition of Ca2+ proceeded monoexponentially. At 2-100 microM Ca2+ and in the absence of nucleotides, the Ca2(+)-induced fluorescence rise and Ca2+ binding to the enzyme proceeded at similar rates, which were almost independent of the Ca2+ concentration. In contrast, the fluorescence decrease induced by Ca2+ removal was slower than the Ca2+ dissociation, and both of these processes were inhibited markedly by increasing medium Ca2+. ATP by binding at 1 mol/mol of the phosphorylation site markedly accelerated both the Ca2(+)-induced fluorescence rise and Ca2+ binding, ADP and AMPPNP but not GTP also being effective. In contrast, ADP minimally affected the NBD fluorescence decrease and the Ca2+ dissociation. These data are consistent with a reaction model in which binding of Ca2+ occurs after the conformational transition of the free enzyme from a state (E2) having low affinity for Ca2+ to one (E1) having high affinity for Ca2+ and in which ATP bound at the catalytic site of E2, whose affinity for ATP is about 30-fold less than that of E1, accelerates this conformational transition.