4-acetamido-4--isothiocyanatostilbene-2-2--disulfonic-acid and 4-benzamido-4--isothiocyanostilbene-2-2--disulfonate

A range of fac-[Re(CO)(3)(L,L'-Bid)(H(2)O)](n) (L,L'-Bid = neutral or monoanionic bidentate ligands with varied L,L' donor atoms, N,N', N,O, or O,O': 1,10-phenanthroline, 2,2'-bipydine, 2-picolinate, 2-quinolinate, 2,4-dipicolinate, 2,4-diquinolinate, tribromotropolonate, and hydroxyflavonate; n = 0, +1) has been synthesized and the aqua/methanol substitution has been investigated. The complexes were characterized by UV-vis, IR and NMR spectroscopy and X-ray crystallographic studies of the compounds fac-[Re(CO)(3)(Phen)(H(2)O)]NO(3)·0.5Phen, fac-[Re(CO)(3)(2,4-dQuinH)(H(2)O)]·H(2)O, fac-[Re(CO)(3)(2,4-dQuinH)Py]Py, and fac-[Re(CO)(3)(Flav)(CH(3)OH)]·CH(3)OH are reported. A four order-of-magnitude of activation for the methanol substitution is induced as manifested by the second order rate constants with (N,N'-Bid) < (N,O-Bid) < (O,O'-Bid). Forward and reverse rate and stability constants from slow and stopped-flow UV/vis measurements (k(1), M(-1) s(-1); k(-1), s(-1); K(1), M(-1)) for bromide anions as entering nucleophile are as follows: fac-[Re(CO)(3)(Phen)(MeOH)](+) (50 ± 3) × 10(-3), (5.9 ± 0.3) × 10(-4), 84 ± 7; fac-[Re(CO)(3)(2,4-dPicoH)(MeOH)] (15.7 ± 0.2) × 10(-3), (6.3 ± 0.8) × 10(-4), 25 ± 3; fac-[Re(CO)(3)(TropBr(3))(MeOH)] (7.06 ± 0.04) × 10(-2), (4 ± 1) × 10(-3), 18 ± 4; fac-[Re(CO)(3)(Flav)(MeOH)] 7.2 ± 0.3, 3.17 ± 0.09, 2.5 ± 2. Activation parameters (ΔH(k1)(++), kJmol(-1); ΔS(k1)(), J K(-1) mol(-1)) from Eyring plots for entering nucleophiles as indicated are as follows: fac-[Re(CO)(3)(Phen)(MeOH)](+) iodide 70 ± 1, -35 ± 3; fac-[Re(CO)(3)(2,4-dPico)(MeOH)] bromide 80.8 ± 6, -8 ± 2; fac-[Re(CO)(3)(Flav)(MeOH)] bromide 52 ± 5, -52 ± 15. A dissociative interchange mechanism is proposed.

Topics: 2,2'-Dipyridyl; 4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic Acid; Crystallography, X-Ray; Kinetics; Ligands; Magnetic Resonance Spectroscopy; Methanol; Models, Molecular; Molecular Structure; Organometallic Compounds; Phenanthrolines; Picolinic Acids; Radiopharmaceuticals; Rhenium; Scattering, Small Angle; Thermodynamics; Water; X-Ray Diffraction

We have reported that chymotrypsin B (CtrB) is not just a digestive enzyme but is also stored in lysosomes. Herein, we demonstrated a broad distribution of CtrB and explored the involvement of CtrB in apoptosis. Exposure of RH-35 cells to H(2)O(2) or palmitate induced the redistribution of lysosomal CtrB into the cytoplasm as a result of lysosomal membrane permeabilization (LMP). Suppression of CtrB significantly blocked apoptosis, while overexpression of CtrB sensitized apoptosis markedly. CtrB could cleave Bid under neutral conditions. In RH-35 cells with Bid silenced, apoptosis induced by CtrB protein was attenuated, suggesting that CtrB mediates apoptosis of RH-35 cells mainly through processing Bid. Our data also suggest that LMP occurs earlier than mitochondrial outer membrane permeabilization; Bid activation initiated by caspase-8 might be reinforced by CtrB in consequence of LMP, which causes a positive feedback loop leading to the accumulation of tBid, and results in lysosome- and mitochondrion-dependent apoptosis.

Topics: 4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic Acid; Apoptosis; Caspase 8; Chymotrypsin; Humans; Hydrogen Peroxide; Intracellular Membranes; Lysosomes; Mitochondria; Mitochondrial Membranes; Proteins

Previous fluorescence resonance energy transfer (FRET) measurements, using BIDS (4-benzamido-4'-isothiocyanostilbene-2,2'-disulfonate) as a label for the disulfonic stilbene site and FM (fluorescein-5-maleimide) as a label for the cytoplasmic SH groups on band 3 (AE1), combined with data showing that the cytoplasmic SH groups lie about 40 A from the cytoplasmic surface of the lipid bilayer, would place the BIDS sites very near the membrane's inner surface, a location that seems to be inconsistent with current models of AE1 structure and mechanism. We reinvestigated the BIDS-FM distance, using laser single photon counting techniques as well as steady-state fluorescence of AE1, in its native membrane environment. Both techniques agree that there is very little energy transfer from BIDS to FM. The mean energy transfer (E), based on three-exponential fits to the fluorescence decay data, is 2.5 +/- 0.7% (SEM, N = 12). Steady-state fluorescence measurements also indicate <3% energy transfer from BIDS to FM. These data indicate that the BIDS sites are probably over 63 A from the cytoplasmic SH groups, placing them near the middle or the external half of the lipid bilayer. This relocation of the BIDS sites fits with other evidence that the disulfonic stilbene sites are located farther toward the external membrane surface than Glu-681, a residue near the inner membrane surface whose modification affects the pH dependence and anion selectivity of band 3. The involvement of two relatively distant parts of the AE1 protein in transport function suggests that the transport mechanism requires coordinated large-scale conformational changes in the band 3 protein.

Topics: 4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic Acid; 4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid; Anion Exchange Protein 1, Erythrocyte; Cell Membrane; Chlorides; Dimerization; Erythrocytes; Fluoresceins; Fluorescence Resonance Energy Transfer; Humans; Kinetics; Models, Molecular; Protein Structure, Tertiary

The transport inhibitor, eosin 5-maleimide, reacts specifically at an external site on the membrane-bound domain of the anion exchange protein, Band 3, in the human erythrocyte membrane. The fluorescence of eosin-labeled resealed ghosts or intact cells was found to be resistant to quenching by CsCl, whereas the fluorescence of labeled inside-out vesicles was quenched by about 27% at saturating CsCl concentrations. Since both Cs+ and eosin maleimide were found to be impermeable to the red cell membrane and the vesicles were sealed, these results indicate that after binding of the eosin maleimide at the external transport site of Band 3, the inhibitor becomes exposed to ions on the cytoplasmic surface. The lifetime of the bound eosin maleimide was determined to be 3 ns both in the absence and presence of CsCl, suggesting that quenching is by a static rather than a dynamic (collisional) mechanism. Intrinsic tryptophan fluorescence of erythrocyte membranes was also investigated using anion transport inhibitors which do not appreciably absorb light at 335 nm. Eosin maleimide caused a 25% quenching and 4,4'-dibenzamidodihydrostilbene-2,2'-disulfonate) caused a 7% quenching of tryptophan fluorescence. Covalent labeling of red cells by either eosin maleimide or BIDS (4-benzamido-4'-isothiocyanostilbene-2,2'-disulfonate) caused an increase in the susceptibility of membrane tryptophan fluorescence to quenching by CsCl. The quenching constant was similar to that for the quenching of eosin fluorescence and was unperturbed by the presence of 0.5 M KCl. Neither NaCl nor Na citrate produced a large change in the relative magnitude of the tryptophan emission. The tryptophan residues that can be quenched by CsCl appear to be different from those quenched by eosin or BIDS and are possibly located on the cytoplasmic domain of Band 3. The results suggest that a conformational change in the Band 3 protein accompanies the binding of certain anion transport inhibitors to the external transport site of Band 3 and that the inhibitors become exposed on the cytoplasmic side of the red cell membrane.

Topics: 4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic Acid; Anion Exchange Protein 1, Erythrocyte; Blood Proteins; Eosine Yellowish-(YS); Erythrocyte Membrane; Erythrocytes; Fluorescent Dyes; Humans; Kinetics; Protein Binding; Protein Conformation; Serum Albumin, Bovine; Spectrometry, Fluorescence; Tryptophan

Evidence is presented that the binding of aromatic disulfonates to the external transport sites of the red cell anion-exchange protein (band 3) can exhibit negative cooperativity. Fluorescence resonance energy transfer has been used to compare the affinities of an aromatic disulfonate 4,4'-bis-(4-nitro-2,1,3-benzoxadiazolyl)dihydrostilbene-2,2'-disulfonate[H2(NBD)2DS] for "empty" band 3 dimers (in which neither external transport site is occupied) and for "half-filled" dimers (in which one site per dimer is occupied by a covalently attached fluorescent stilbenedisulfonate). H2(NBD)2DS apparently binds to the external anion transport site since it is a potent inhibitor of [35S]sulfate influx into red cells (Ki = 20-50 nM), binds reversibly to approximately one site per band 3 monomer (1.6 X 10(6) sites/cell), and is displaced by covalent labeling with a disulfonic stilbene. The affinity of H2(NBD)2DS for membranes in which 80% of the transport sites are occupied by covalently attached 4-benzamido-4'-isothiocyanostilbene-2,2'-disulfonate (BIDS) was approximately 1 order of magnitude lower than that for unmodified membranes. However, when a similar proportion of the transport sites on red cells was blocked by reaction with BIDS, [35S]sulfate was taken up with a lower Vmax but with a Km identical with that observed for unmodified cells, suggesting that no subunit interactions are necessary for transport. Therefore, in order to test whether the observed negative cooperativity of aromatic disulfonate binding could be ascribed simply to steric hindrance, the distance between transport sites was measured by fluorescence resonance energy transfer. H2(NBD)2DS and eosin maleimide were used as acceptors, with BIDS as donor. Transfer efficiencies were determined by donor fluorescence quenching, by acceptor fluorescence enhancement, and from donor lifetime changes. Uncertainties in the distance were estimated from measured depolarization factors. The donor-acceptor distance was found to be only 28-52 A. Since the probes are large molecules, they could therefore be very close together, and the observed negative cooperativity might be explained by overlapping sites. The results suggest that the subunits of a band 3 dimer transport anions independently but that access to the transport sites may be provided by a cavity between the subunits.

Topics: 4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic Acid; Anion Exchange Protein 1, Erythrocyte; Anions; Binding Sites; Biological Transport; Blood Proteins; Energy Transfer; Erythrocyte Membrane; Erythrocytes; Humans; Membrane Proteins; Protein Binding; Spectrometry, Fluorescence; Sulfates

Anion exchange across the erythrocyte membrane can be inhibited competitively by stilbenedisulfonates, which bind to the external transport of the band 3 protein, and noncompetitively by external NAP-taurine [2-[N-(4-azido-2-nitrophenyl)amino]ethanesulfonate], which it has been suggested binds to a "modifier site" [Knauf, P. A., Ship. S., Breur, W., MCCulloch, L., & Rothstein, A. (1978) J. Gen. Physiol. 72, 604-630]. The binding of the two types of inhibitor of erythrocyte membranes is shown in the present study to be competitive, indicating that binding to the same subunit of band 3 is mutually exclusive. Covalent labeling of red cells with a stilbenedisulfonate [4-benzamido-4'-isothiocyano-stilbene-2,2'-disulfonate (BIDS)] to 80% saturation had no detectable effect upon the Ki for inhibition of [32P]phosphate influx by NAP-taurine, indicating that when bound to adjacent subunits in the band 3 dimer, the two types of inhibitor do not interact. In addition to the external NAP-taurine site, a second high-affinity NAP-taurine site (Kd = 15 microM) was detected on the cytoplasmic side of red cell membranes. This site is less than 51 A from the disulfonic stilbene binding site, as judged by fluorescence resonance energy transfer from BIDS to NAP-taurine. Binding at this site is not affected by covalent attachment of BIDS, and no clear role for this site in transport could be determined. On the basis of these studies we present a model indicating that disulfonic stilbenes bind to a site which overlaps both the anion transport site and the modifier site on a band 3 monomer and suggests that the modifier site may be part of a transporting gate.

Topics: 4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic Acid; Anion Exchange Protein 1, Erythrocyte; Binding Sites; Binding, Competitive; Biological Transport, Active; Blood Proteins; Erythrocyte Membrane; Erythrocytes; Humans; Membrane Proteins; Models, Chemical; Spectrometry, Fluorescence; Stilbenes; Taurine