Anion transport inhibitor binding to band 3 in red blood cell membranes

The inhibitor of anion exchange 4,4''-dibenzoamido-2,2''-disulfonic stilbene (DBDS) binds to band 3, the anion transport protein in human red cell ghost membranes, and undergoes a large increase in fluorescence intensity when bound to band 3. Equilibrium binding studies performed in the absence of transportable anions show that DBDS binds to both a class of high-affinity (65 nM) and low-affinity (820 nM) sites with stoichiometry equivalent to 1.6 nmol/mg ghost protein for each site, which is consistent with one DBDS site on each band 3 monomer. The kinetics of DBDS binding were studied both by stopped-flow and temperature-jump experiments. The stopped-flow data indicate that DBDS binding to the apparent high-affinity site involves association with a low-affinity site (3 microM) followed by a slow (4 s-1) conformational change that locks the DBDS molecule in place. A detailed, quantitative fit of the temperature-jump data to several binding mechanisms supports a sequential-binding model, in which a first DBDS molecule binds to one monomer and induces a conformational change. A second DBDS molecule then binds to the second monomer. If the two monomers are assumed to be initially identical, thermodynamic characterization of the binding sites shows that the conformational change induces an interaction between the two monomers that modifies the characteristics of the second DBDS binding site.     

A two-site kinetic mechanism for ATP binding and hydrolysis by E. coli Rep helicase dimer bound to a single-stranded oligodeoxynucleotide.

Escherichia coli Rep helicase catalyzes the unwinding of duplex DNA in reactions that are coupled to ATP binding and hydrolysis. We have investigated the kinetic mechanism of ATP binding and hydrolysis by a proposed intermediate in Rep-catalyzed DNA unwinding, the Rep "P2S" dimer (formed with the single-stranded (ss) oligodeoxynucleotide, (dT)16), in which only one subunit of a Rep homo-dimer is bound to ssDNA. Pre-steady-state quenched-flow studies under both single turnover and multiple turnover conditions as well as fluorescence stopped-flow studies were used (4 degrees C, pH 7.5, 6 mM NaCl, 5 mM MgCl2, 10 % (v/v) glycerol). Although steady-state studies indicate that a single ATPase site dominates the kinetics (kcat=17(+/-2) s-1; KM=3 microM), pre-steady-state studies provide evidence for a two-ATP site mechanism in which both sites of the dimer are catalytically active and communicate allosterically. Single turnover ATPase studies indicate that ATP hydrolysis does not require the simultaneous binding of two ATP molecules, and under these conditions release of product (ADP-Pi) is preceded by a slow rate-limiting isomerization ( approximately 0.2 s-1). However, product (ADP or Pi) release is not rate-limiting under multiple turnover conditions, indicating the involvement of a second ATP site under conditions of excess ATP. Stopped-flow fluorescence studies monitoring ATP-induced changes in Rep's tryptophan fluorescence displayed biphasic time courses. The binding of the first ATP occurs by a two-step mechanism in which binding (k+1=1.5(+/-0.2)x10(7) M-1 s-1, k-1=29(+/-2) s-1) is followed by a protein conformational change (k+2=23(+/-3) s-1), monitored by an enhancement of Trp fluorescence. The second Trp fluorescence quenching phase is associated with binding of a second ATP. The first ATP appears to bind to the DNA-free subunit and hydrolysis induces a global conformational change to form a high energy intermediate state with tightly bound (ADP-Pi). Binding of the second ATP then leads to the steady-state ATP cycle. As proposed previously, the role of steady-state ATP hydrolysis by the DNA-bound Rep subunit may be to maintain the DNA-free subunit in an activated state in preparation for binding a second fragment of DNA as needed for translocation and/or DNA unwinding. We propose that the roles of the two ATP sites may alternate upon binding DNA to the second subunit of the Rep dimer during unwinding and translocation using a subunit switching mechanism.     

Equilibrium and kinetic studies on the binding of gluconolactone to almond beta-glucosidase in the absence and presence of glucose

The binding of glucono-1,5-lactone (gluconolactone) with almond beta-glucosidase was studied at pH 5.0 and 25 degrees C, in the absence and presence of glucose, by monitoring the enzyme fluorescence as a probe. From the results of fluorometric titration, the dissociation constant Kd and the maximum fluorescence intensity increase (percent) of the enzyme-gluconolactone complex relative to the enzyme alone, delta Fmax, were determined to be 12.7 microM and 14.7%, respectively. From the study of the temperature dependence of Kd, delta G degrees, delta H degrees and delta S degrees for the binding were evaluated to be -6.7 kcal mol-1, -3.5 kcal mol-1, and 10.8 e.u. (cal mol-1 deg-1), respectively, at 25 degrees C. The analysis of the fluorometric titration data in the presence of glucose revealed that these ligands bind competitively to the enzyme, probably at the same site. The results of a stopped-flow kinetic study are consistent with the following two-step mechanism: (formula; see text) which indicates that gluconolactone (L) and the enzyme (E) transiently form a loosely bound complex, ELtr (k-1/k+1 = 4.5 mM), in the first rapid bimolecular association step, and ELtr is converted into a more tightly bound complex EL (k+2 = 94 s-1, k-2 = 0.36 s-1) in the subsequent slow unimolecular process. The fluorescence intensity increase occurs solely in the latter step.     

Thermodynamic and kinetic analysis of carbohydrate binding to the basic lectin from winged bean (Psophocarpus tetragonolobus)

A basic lectin (pI approximately 10.0) was purified to homogeneity from the seeds of winged bean (Psophocarpus tetragonolobus) by affinity chromatography on Sepharose 6-aminocaproyl-D-galactosamine. The lectin agglutinated trypsinized rabbit erythrocytes and had a relative molecular mass of 58,000 consisting of two subunits of Mr 29,000. The lectin binds to N-dansylgalactosamine, leading to a 15-fold increase in dansyl fluorescence with a concomitant 25-nm blue shift in the emission maximum. The lectin has two binding sites/dimer for this sugar and an association constant of 4.17 X 10(5) M-1 at 25 degrees C. The strong binding to N-dansylgalactosamine is due to a relatively positive entropic contribution as revealed by the thermodynamic parameters: delta H = -33.62 kJ mol-1 and delta S0 = -5.24 J mol-1 K-1. Binding of this sugar to the lectin shows that it can accommodate a large hydrophobic substituent on the C-2 carbon of D-galactose. Studies with other sugars indicate that a hydrophobic substituent in alpha-conformation at the anomeric position increases the affinity of binding. The C-4 and C-6 hydroxyl groups are critical for sugar binding to this lectin. Lectin difference absorption spectra in the presence of N-acetylgalactosamine indicate perturbation of tryptophan residues on sugar binding. The results of stopped flow kinetics with N-dansylgalactosamine and the lectin are consistent with a simple one-step mechanism for which k+1 = 1.33 X 10(4) M-1 s-1 and k-1 = 3.2 X 10(-2) s-1 at 25 degrees C. This k-1 is slower than any reported for a lectin-monosaccharide complex so far. The activation parameters indicate an enthalpically controlled association process.     

Thermodynamic and kinetic studies on saccharide binding to soya-bean agglutinin.

The fluorescence of N-dansylgalactosamine [N-(5-dimethylaminonaphthalene-1-sulphonyl)galactosamine] was enhanced 11-fold with a 25 nm blue-shift in the emission maximum upon binding to soya-bean agglutinin (SBA). This change was used to determine the association constants and thermodynamic parameters for this interaction. The association constant of 1.51 X 10(6) M-1 at 20 degrees C indicated a very strong binding, which is mainly due to a relatively small entropy value, as revealed by the thermodynamic parameters: delta G = -34.7 kJ X mol-1, delta H = -37.9 kJ X mol-1 and delta S = -10.9 J X mol-1 X K-1. The specific binding of this sugar to SBA shows that the lectin can accommodate a large hydrophobic substituent on the C-2 of galactose. Binding of non-fluorescent ligands, studied by monitoring the fluorescence changes when they are added to a mixture of SBA and N-dansylgalactosamine, indicates that a hydrophobic substituent at the anomeric position increases the affinity of the interaction. The C-6 hydroxy group also stabilizes the binding considerably. Kinetics of binding of N-dansylgalactosamine to SBA studied by stopped-flow spectrofluorimetry are consistent with a single-step mechanism and yielded k+1 = 2.4 X 10(5) M-1 X s-1 and k-1 = 0.2 s-1 at 20 degrees C. The activation parameters indicate an enthalpicly controlled association process.     

Pressure-jump study of the kinetics of ethidium bromide binding to DNA

Pressure-jump chemical relaxation has been used to investigate the kinetics of ethidium bromide binding to the synthetic double-stranded polymers poly[d(G-C)] and poly[d(A-T)] in 0.1 M NaCl, 10 mM tris(hydroxymethyl)aminomethane hydrochloride, and 1 mM ethylenediaminetetraacetic acid, pH 7.2, at 24 degrees C. The progress of the reaction was followed by monitoring the fluorescence of the intercalated ethidium at wavelengths greater than 610 nm upon excitation at 545 nm. The concentration of DNA was varied from 1 to 45 microM and the ethidium bromide concentration from 0.5 to 25 microM. The data for both polymers were consistent with a single-step bimolecular association of ethidium bromide with a DNA binding site. The necessity of a proper definition of the ethidium bromide binding site is discussed: it is shown that an account of the statistically excluded binding phenomenon must be included in any adequate representation of the kinetic data. For poly[d(A-T)], the bimolecular association rate constant is k1 = 17 X 10(6) M-1 s-1, and the dissociation rate constant is k-1 = 10 s-1; in the case of poly[d(G-C)], k1 = 13 X 10(6) M-1 s-1, and k-1 = 30 s-1. From the analysis of the kinetic amplitudes, the molar volume change, delta V0, of the intercalation was calculated. In the case of poly[d(A-T)], delta V0 = -15 mL/mol, and for poly[d(G-C)], delta V0 = -9 mL/mol; that is, for both polymers, intercalation is favored as the pressure is increased.(ABSTRACT TRUNCATED AT 250 WORDS)     

Tryptophan fluorescence in electron-transfer flavoprotein:ubiquinone oxidoreductase: fluorescence quenching by a brominated pseudosubstrate

We have studied the intrinsic fluorescence of the 12 tryptophan residues of electron-transfer flavoprotein:ubiquinone oxidoreductase (ETF:QO). The fluorescence emission spectrum (lambda ex 295 nm) showed that the fluorescence is due to the tryptophan residues and that the contribution of the 22 tyrosine residues is minor. The emission maximum (lambda m 334 nm) and the bandwidth (delta lambda 1/2 56 nm) suggest that the tryptophans lie in hydrophobic environments in the oxidized protein. Further, these tryptophans are inaccessible to a range of ionic and nonionic collisional quenching agents, indicating that they are buried in the protein. Enzymatic or chemical reduction of ETF:QO results in a 5% increase in fluorescence with no change of lambda m or delta lambda 1/2. This change is reversible upon reoxidation and is likely to reflect a conformational change in the protein. The ubiquinone analogue Q0(CH2)10Br, a pseudosubstrate of ETF:QO (Km = 2.6 microM; kcat = 210 s-1), specifically quenches the fluorescence of one tryptophan residue (Kd = 1.6-3.2 microM) in equilibrium fluorescence titrations. The ubiquinone homologue UQ-2 (Km = 2 microM; kcat = 162 s-1) and the analogue Q0(CH2)10OH (Km = 2 microM; kcat = 132 s-1) do not quench tryptophan fluorescence; thus the brominated analogue acts as a static heavy atom quencher. We also describe a rapid purification for ETF:QO based on extraction of liver submitochondrial particles with Triton X-100 and three chromatographic steps, which results in yields 3 times higher than previously published methods.     

Kinetics of conformational changes in Nereis sarcoplasmic calcium-binding protein upon binding of divalent ions

The sarcoplasmic calcium-binding protein (SCP) of the sandworm Nereis possesses three Ca2(+)-Mg2+ sites but no Ca2(+)-specific site. Binding of Mg2+, but not of Ca2+, displays a marked positive cooperativity. The apparent cooperativity of Ca2+ binding in the presence of Mg2+ results from the allostery in Mg2+ dissociation. Binding of the first Ca2+ or Mg2+ induces all the conformational change, monitored by Trp fluorescence. In displacement reactions the conformational changes occur in the step SCP.Mg3----SCP.Ca1Mg2. Stopped-flow experiments indicate that Trp fluorescence changes upon Ca2(+)-binding are instantaneous whereas Mg2(+)-binding involves a fast pre-equilibrium (Keq = 28 M-1), followed by two slow consecutive conformational changes with k1 = 13.5 s-1 and k2 = 0.21 s-1. The fluorescence change after dissociation of Ca2+ from SCP is monophasic with k = 0.02 s-1; that after Mg2+ dissociation is biphasic with k1 = 0.8 s-1 and k2 = 0.1 s-1. Trp life time measurements also indicate that Ca2(+)- and Mg2(+)-induced conformational changes are completely different. Displacement of bound Ca2+ by Mg2+ can be described by two consecutive reactions in which the first (without fluorescence change) corresponds to the dissociation of the last Ca2+ (k1 = 2.4 s-1) and the second (k2 = 0.45 s-1) to the final conformational change observed upon direct Mg2+ binding. Displacement of bound Mg2+ by Ca2+ follows the kinetic scheme of simple competition; the conformational rate constant approaches asymptotically (up to the limit of 129 s-1) the dissociation rate of Mg2+ as the concentration of Ca2+ increases. In summary, after fast dissociation of Ca2+ or Mg2+, Nereis SCP slowly converts to the metal-free configuration, but in Ca2(+)-Mg2+ exchange reactions, the conformational changes are nearly as fast as the cation dissociation reactions.     

Stopped-flow fluorescence kinetic studies of Glu-plasminogen. Conformational changes triggered by AH-site ligand binding

Binding of 6-aminohexanoic acid to the AH-site, a weak lysine binding site in Glu-plasminogen, alters the conformation of the molecule. The kinetics of the binding and the accompanying conformational change are investigated at pH 7.8, 25 degrees C. Changes of intrinsic protein fluorescence were measured as a function of time after rapid mixing in a stopped-flow apparatus. The results reflect a two-step reaction mechanism: Rapid association of Glu-plasminogen and 6-aminohexanoic acid (K1 = 44 mM) followed by the conformational change (k2 = 69 s-1 and k-2 = 3 s-1) with an overall dissociation constant Kd = 2.0 mM. Thus the conformational change is rather fast, t12 = 0.01 s. Its importance for the rates of Glu-plasminogen activation reactions is discussed.     

Desferrioxamine protects human red blood cells from hemin-induced hemolysis

Hemin binding to red cell membranes, its effect on red cell hemolysis, and it interaction with desferrioxamine (DFO) in these processes were investigated. DFO interacted with hemin via the iron moiety. Blockage of the binding groups in DFO prevented interaction of DFO with hemin, implying the importance of the hydroxamic acid groups in DFO-hemin interactions. Since hemolysis is a result of hemin association with the membrane components, its binding in the presence and absence of DFO was studied. DFO strongly inhibited hemin-induced lysis in a concentration-dependent manner. With 50 microM hemin, 1 mM DFO completely inhibited lysis. Preincubation of ghost membranes with DFO (1 mM) inhibited binding of hemin (50 microM) to membranes by 42%. After ghost membranes were preincubated with hemin (50 microM), the addition of DFO (1 mM) removed 20% of the membrane-bound hemin. It is suggested that DFO may have an important role in alleviating the hemin-induced deleterious effects on the red cell membrane, especially in hemolytic anemias associated with unstable, autoxidized hemoglobins.     

Stop-flow analysis of cooperative interactions between GLUT1 sugar import and export sites.

The human erythrocyte sugar transporter is thought to function either as a simple carrier (sugar import and sugar export sites are presented sequentially) or as a fixed-site carrier (sugar import and sugar export sites are presented simultaneously). The present study examines each hypothesis by analysis of the rapid kinetics of reversible cytochalasin B binding to the sugar export site in the presence and absence of sugars that bind to the sugar import site. Cytochalasin B binding to the purified, human erythrocyte glucose transport protein (GLUT1) induces quenching of GLUT1 intrinsic tryptophan fluorescence. The time-course of GLUT1 fluorescence quenching reflects a second-order process characterized by simple exponential kinetics. The pseudo-first-order rate constant describing fluorescence decay (kobs) increases linearly with [cytochalasin B] while the extent of fluorescence quenching increases in a saturable manner with [cytochalasin B]. Rate constants for cytochalasin B binding to GLUT1 (k1) and dissociation from the GLUT1.cytochalasin B complex (k-1) are obtained from the relationship: kobs = k-1 + k1[cytochalasin B]. Low concentrations of maltose, D-glucose, 3-O-methylglucose, and other GLUT1 import-site reactive sugars increase k-1(app) and reduce k1(app) for cytochalasin B interaction with GLUT1. Higher sugar concentrations decrease k1(app) further. The simple carrier mechanism predicts that k1(app) alone is modulated by import- and export-site reactive sugars and is thus incompatible with these findings. These results are consistent with a fixed-site carrier mechanism in which GLUT1 simultaneously presents cooperative sugar import and export sites.     

Evidence that inhibitors of anion exchange induce a transmembrane conformational change in band 3

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.     

Kinetic study on chemical modification of taka-amylase A. I. Location and role of tryptophan residues

1. Five and four tryptophan residues in Taka-amylase A [EC 3.2.1.1] of A. oryzae (TAA) were modified with dimethyl(2-hydroxy-5-nitrobenzyl)-sulfonium bromide (K-IWS) in the absence and the presence of 15% maltose (substrate analog), respectively. Only one tryptophan residue was modified with dimethyl(2-methoxy-5-nitrobenzyl)-sulfonium bromide (K-IIWS) irrespective of the presence or absence of maltose. Kinetic parameters (molecular activity, k0, Michaelis constant, Km, and inhibitor constant, Ki) of the enzyme modified with K-IWS and K-IIWS were determined. The k0 value decreased with increase in the number of modified residues, but Km and Ki values and the type of inhibition were not altered by the modification. 2. The fluorescence quenching reaction of TAA with N-bromosuccinimide (NBS) proceeded in three phases. The second-order rate constants of the three phases were determined to be (4.3 +/- 0.5) x 10(5) M-1 . s-1, (2.1 +/- 0.3) x 10(3) M-1 . s-1 and (1.7 +/- 0.2) x 10(2) M-1 . s-1, respectively. In the presence of maltose, the first phase was further separated into two phases with rate constants of (4.6 +/- 0.6) x 10(6) M-1 . s-1 and (6.9 +/- 1.1) x 10(4) M-1 . s-1, respectively. On the basis of the results, it is estimated that five out of nine tryptophan residues are accessible to the solvent and among them, two tryptophan residues are substantially exposed: one is located in the maltose binding site near the catalytic site (its modification affects the catalytic function), and the other exists on the enzyme surface far from the active site.     

Study on the interaction between soybean beta-amylase and substrate by the stopped-flow method.

The hydrolysis of substrates (maltoheptaose, maltopentaose, and maltotetraose) catalyzed by soybean beta-amylase [EC 3.2.1.2] at pH 5.4 and 25 degrees C was followed by monitoring small changes in the quenching of fluorescence due to tryptophan residues by the stopped-flow method. By analysis of whole time course, the dissociation constants, KdS, of enzyme-substrate and enzyme-product complexes were reasonably evaluated; and the difference in fluorescence intensities per mol between the enzyme-complex (ES or EP) and the free enzyme, delta F, was determined. The molecular activity, k0, was also determined by a new method of half time analysis. The KdS and k0 values are in good agreement with our kinetic data reported previously. The delta Fs of substrates were of smaller magnitude than those of products (G2 and G3), which means that the higher the binding affinity of the ligand is, the smaller the delta F value is. This indicates that at least two tryptophan residues must be located in the active site if the enzyme is rigid, or that if there is only one, the active site must undergo a structural change caused by the binding of ligand.     

Analysis of ATP binding to CheA containing tryptophan substitutions near the active site

Signal transduction in the chemotaxis system of Escherichia coli involves an autophosphorylating protein histidine kinase, CheA. At the active site of CheA, phenylalanine residues 455 and 459 occupy positions near the ATP-binding pocket, immediately adjacent to one of the hinge regions of a loop that undergoes an ATP-induced conformational change ("lid closure") that has been characterized previously in X-ray crystal structures [Bilwes et al. (2001) Nat. Struct. Biol. 8, 353-360]. We generated versions of CheA carrying F455W and F459W replacements and investigated whether the fluorescence properties of the introduced tryptophan side chains were affected by nucleotide binding in a manner that would provide a signal for investigating the dynamics of active site events, such as ATP binding and lid closure. Our results indicate that CheA(F455W) is useful in this regard, but CheA(F459W) is not. CheA(F455W) retained full catalytic activity and exhibited easily monitored fluorescence changes upon binding nucleotide: we observed a 25-30% decrease in CheA(F455W) fluorescence emission intensity at 330 nm upon binding ATP in the absence of Mg(2+); in the presence of Mg(2+), the emission spectrum of the CheA(F455W):ATP complex was red-shifted by 5 nm and exhibited an increased intensity (approximately 20% higher at 345 nm relative to that of uncomplexed CheA(F455W)). Different fluorescence changes were observed when two ATP analogues (ADPNP and ADPCP) were used instead of ATP and when Mn(2+) or Ca(2+) was used in place of Mg(2+). We exploited the fluorescence changes induced by Mg(2+)-ATP to explore the kinetics and mechanism of nucleotide binding by CheA(F455W). In the absence of Mg(2+), binding appears to involve a simple one-step equilibrium (k(assn) = 0.7 microM(-1) s(-1) and k(dissn) = 270 s(-1) at 4 degrees C). In the presence of Mg(2+), the binding mechanism involves at least two steps: (i) rapid, relatively weak binding followed by (ii) a rapid, reversible step (k(forward) = 300 s(-1) and k(reverse) =15 s(-1) at 4 degrees C) that enhanced the overall affinity of the complex and generated an increase in W455 fluorescence. This second step could reflect a conformational change at the CheA active site, such as lid closure. These results provide the first insight into the dynamics of nucleotide binding and substrate-induced conformational changes at the active site of a protein histidine kinase.     

Kinetics of the low pH-induced conformational changes and fusogenic activity of influenza hemagglutinin.

The decrease of the intrinsic tryptophan fluorescence intensity of purified influenza (X31 strain) hemagglutinin (HA) was used to monitor the low pH-induced conformational change of this protein. The kinetics of the fluorescence decrease depended strongly on the pH. At pH optimal for fusion, the change in tryptophan fluorescence was fast and could be fitted to a monoexponential function. We measured a rate constant of 5.78 s-1 (t1/2 = 120 ms) at pH 4.9 using rapid stopped-flow mixing. Under suboptimal conditions (higher pH), the rate constant was decreased by an order of magnitude. In addition, a slow component appeared and the fluorescence decrease followed a sum of two exponentials. The kinetics of conformational changes were compared with those of the fusion of influenza virus with red blood cell membranes as assessed by the R18-dequenching assay. At optimal pH the HA conformational change was not rate-limiting for the fusion process. However, at sub-optimal pH, the slow transition to the fusogenic conformational of HA resulted in slower kinetics and decreased extent of fusion.     

Tryptophan fluorescence as a probe of placental ribonuclease inhibitor binding to angiogenin

The binding of human placental ribonuclease inhibitor (PRI) to angiogenin, a human protein that induces neovascularization, occurs with a 1:1 stoichiometry and is accompanied by a 50% increase in tryptophan fluorescence. In contrast, the binding of PRI to bovine pancreatic RNase A or to angiogenin oxidized at its single tryptophan residue results in a quenching of fluorescence. These observations suggest that there is a change in the local environment of Trp-89 of angiogenin. Quenching experiments with acrylamide are consistent with the view that Trp-89 is exposed in the native protein and becomes less accessible upon formation of the complex with PRI. Stopped-flow kinetic measurements monitoring the fluorescence enhancement indicate a two-step mechanism for the binding of PRI to angiogenin. The first step involves rapid formation of an enzyme-inhibitor complex, EI, followed by a slower isomerization of EI to a tight enzyme-inhibitor complex, EI*: (Formula: see text). In 0.1 M NaCl at pH 6 and 25 degrees C, the values of K1 and K2 are 0.53 microM and 97 s-1, respectively. The apparent second-order rate constant of association at protein concentrations much less than K1 is approximated by K2/K1 and equals 1.8 X 10(8) M-1 s-1. The corresponding value for the association of PRI with RNase A is only slightly higher, 3.4 X 10(8) M-1 s-1. The effects of pH and sodium chloride concentration on the association rate of PRI with angiogenin suggest the importance of ionizable groups and ionic interactions, respectively, in the association process.(ABSTRACT TRUNCATED AT 250 WORDS)     

Phosphorylation of light chains of myosin from rabbit skeletal muscles affects the type of conformation changes of F-actin induced by heavy meromyosin

The changes in F-actin conformation in myosin-free single ghost fiber induced by the binding of heavy meromyosin (HMM) with dephosphorylated or phosphorylated light chains-2 (LC2) have been studied by measuring intrinsic tryptophan polarized fluorescence of F-actin. It has been found that at low concentrations of Ca2+ (pCa greater than or equal to 8), the binding of HMM with dephosphorylated LC2 to F-actin in ghost fibres increases, whereas the binding of HMM with phosphorylated LC2 decreases the anisotropy of polarized tryptophan fluorescence. The effect is reversed at high concentrations of Ca2+ (pCa = 5). It has been assumed that this effect of myosin light chains phosphorylation may be due to its influence on the type of myosin head binding to F-actin.     

Deoxygenation affects fluorescence photobleaching recovery measurements of red cell membrane protein lateral mobility.

We have used the fluorescence photobleaching recovery technique to study the dependence on oxygen tension of the lateral mobility of fluorescently labeled band 3, the phospholipid analogue fluorescein phosphatidylethanolamine, and glycophorins in normal red blood cell membranes. Band 3 protein and sialic acid moieties on glycophorins were labeled specifically with eosin maleimide and fluorescein thiosemicarbazide, respectively. The band 3 diffusion rate increased from 1.7 x 10(-11) cm2 s-1 to 6.0 x 10(-11) cm2 s-1 as oxygen tension was decreased from 156 to 2 torr, and a further increase to 17 x 10(-11) cm2 s-1 occurred as oxygen tension was decreased from 2 to 0 torr. The fractional mobility of band 3 decreased from 58 to 32% as oxygen tension was decreased from 156 to 0 torr. The phospholipid diffusion coefficient remained constant as oxygen tension was decreased from 156 to 20 torr, but increased from 2.3 x 10(-9) cm2 s-1 to 7.1 x 10(-9) cm2 s-1 as oxygen tension was decreased from 20 to 0 torr. Neither the diffusion coefficient nor the fractional mobility of glycophorins changed significantly at low oxygen tension. Under non-bleaching excitation conditions, intensities of fluorescence emission were identical for oxygenated and deoxygenated eosin-labeled RBCs. Deoxygenated eosin-labeled RBCs required 160-fold greater laser intensities than did oxygenated RBCs to achieve comparable extents of photobleaching, however. Oxygen seems to act as a facilitator of fluorophore photobleaching and may thereby protect the fluorescently labeled red cell membrane from photodamage.(ABSTRACT TRUNCATED AT 250 WORDS)     

Transient-kinetic studies of the adenosine triphosphatase activity of scallop heavy meromyosin.

Fluorescence stopped-flow experiments were performed to elucidate the elementary steps of the ATPase mechanism of scallop heavy meromyosin in the presence and in the absence of Ca2+. ATP binding and hydrolysis, as monitored by the change in tryptophan fluorescence, appear to be Ca2+-insensitive, whereas both Pi release and ADP release are markedly suppressed in the absence of Ca2+. Rate constants for Pi release are 0.2 s-1 and 0.002 s-1 and for ADP release are 6 s-1 and 0.01 s-1 in the presence and in the absence of Ca2+ respectively. Ca2+ binding to the specific site of the regulatory domain is rapid and its release occurs at 25 s-1, consistent with the time scale of a twitch of the striated adductor muscle. Nucleotide binding is a multi-step process requiring a minimum of three states. In such a model Ca2+ controls the rate of conformational changes at the active site in both the forward and the reverse direction, leading to a large dependence of the rate of nucleotide release, but a lesser effect on the overall equilibrium position. The kinetic trapping of nucleotides and Pi at the active site, in the absence of Ca2+, appears to be a fundamental step in suppressing the interaction of the myosin head with the thin filaments in relaxed molluscan muscle.