Fluorescence energy transfer between epsilon-ATP at the nucleotide binding site and N-(4-dimethylamino-3,5-dinitrophenyl)-maleimide at Cys-373 of G-actin.

The method of fluorescence energy transfer is used to measure the distance from the nucleotide binding site to Cys-373 of G-actin. The fluorescent ATP analogue 1-N6-ethenoadenosine 5'-triphosphate was used as donor and N-(4-dimethylamino-3,5-dinitrophenyl)-maleimide was used as acceptor. From the measurements of the efficiency of fluorescence energy transfer by both static and time resolved fluorometries, the distance between nucleotide binding site and Cys-373 residue of G-actin was calculated to be about 30 A.

Compressibility and specific volume of actin decrease upon G to F transformation.

We measured the densities as well as the sound velocities in solutions of G-actin, F-actin and the reconstituted thin filament. Using the data obtained, we determined their partial specific volumes and partial specific adiabatic compressibilities. The objectives were to investigate the volume change of actin upon polymerization and to detect the conformational change associated with the ca2+-binding to the reconstituted thin filament. The partial specific volume and the partial specific adiabatic compressibility of G-actin were 0.749 cm3/g and 9.3 x 10(-12) cm2/dyne, respectively. The results suggest that G-actin is a rather soft protein compared with other globular proteins. The partial specific volumes of F-actin were in a range of 0.63 -0.66 cm3/g depending on the solvent conditions. The partial specific adiabatic compressibilities of F-actin were negative (-(7-13) x 10(-12) cm3/dyne). These data indicate that the amount of hydration may increase by several times upon polymerization assuming that the size of the cavity remains constant. We detected little difference between the partial specific adiabatic compressibility of the reconstituted thin filament in a Ca2+-bound state and that in a Ca2+-unbound state. This suggests that the Ca2+ binding affected not the subunit itself but the inter-subunit junction.

Fluorescence study of N-(3-pyrene)maleimide conjugated to rabbit skeletal F-actin and plasmodium actin polymers.

A fluorescent probe N-(3-pyrene)maleimide was conjugated to rabbit skeletal F-actin at the site of most reactive sulfhydryl group (Cys-373). Its fluorescence anisotropy decay showed a single correlation time of 560 ns at 25 degrees C, which is in a very good agreement with the correlation time of the dansyl-L-cysteine group conjugated to the same site of F-actin reported very recently [Wahl, Ph., Mihashi, K, and Auchet, J-C. (1975) FEBS Lett. 8, 164-167]. Actin from plasmodia of myxomycates, Physarum polycepharum, was also conjugated with N-(3-pyrene) maleimide and the fluorescence anisotropy was compared with rabbit skeletal F-actin using the classical steady excitation method. It was found that the internal mobility of the magnesium polymer of plasmodium actin is remarkably larger than both plasmodium F-actin and rabbit skeletal F-actin.

Torsional motion of eosin-labeled F-actin as detected in the time-resolved anisotropy decay of the probe in the sub-millisecond time range.

The internal motion of F-actin in the time range from 10(-6) to 10(-3) second has been explored by measuring the transient absorption anisotropy of eosin-labeled F-actin using laser flash photolysis. The transient absorption anisotropy of eosin-F-actin at 20 degrees C has a component that decays in the submicrosecond time scale to an anisotropy of about 0.3. This anisotropy then decays with a relaxation time of about 450 microseconds to a residual anisotropy of about 0.1 after 2 ms. When the concentration of eosin-F-actin was varied in the range from 7 to 28 microM, the transient absorption anisotropy curves obtained were almost indistinguishable from each other. These results show that the anisotropy decay arises from internal motion of eosin-F-actin. Analysis of the transient absorption anisotropy curves indicates that the internal motion detected by the decay in anisotropy is primarily a twisting of actin protomers in the F-actin helix; bending of the actin filament makes a minor contribution only to the measured decay. The torsional rigidity calculated from the transient absorption anisotropy is 0.2 X 10(-17) dyn cm2 at 20 degrees C, which is about an order of magnitude smaller than the flexural rigidity determined from previous studies. Thus, we conclude that F-actin is more flexible in twisting than in bending. The calculated root-mean-square fluctuation of the torsional angle between adjacent actin protomers in the actin helix is about 4 degrees at 20 degrees C. We also found that the torsional rigidity is approximately constant in the temperature range from 5 to approximately 35 degrees C, and that the binding of phalloidin does not appreciably affect the torsional motion of F-actin.

The excimer fluorescence of N-(1-pyrenyl)iodoacetamide labeled to myosin and its subfragment 1.

Myosin and its subfragment 1 were labeled with the fluorescent probe N-(1-pyrenyl)iodoacetamide. Both of the labeled complexes exhibited the excimer band at 480 nm (pH 8.0, 25 degrees C). SH1 and SH2 are labeled with this probe as judged by Ca2+-ATPase of the labeled complex. Excimers arise both from the interaction of PIAAs in the two different heads within a single myosin molecule and also from the interaction of PIAAs in the same head. ATP affects these excimers depending on the concentration of Ca2+.

Nanosecond pulse fluorometry in polarized light of dansyl-L-cysteine linked to a unique SH group of F-actin; the influence of regulatory proteins and myosin moiety.

The order of magnitude of the correlation time, which characterizes the dansyl cysteine residue linked to F-actin is ten times greater than the correlation time of the G-actin monomer [1]. Still it is much smaller than the correlation times of the F-actin polymer as a whole. The dansyl chromophore reveals that the C terminal end of the actin peptide chain, is mobile. As Ebashi and his co-workers have shown (13), Ca2+ triggers muscular contraction by acting on F-actin through the mediation of the regulatory proteins troponin and tropomyosin. By using spin label technique, Tonomura et al. [14] found that Ca2+ induces a conformational change on the troponin, tropomyosin actin complex. The quasi elastic scattering of laser light measurement of Fujime and Ishiwata [15] showed that troponin-tropomyosin F-actin has a rotational correlation time in the millisecond range which characterizes the flexibility of this complex; Ca2+ induces an increase of this flexibility. The present pulse fluorometry study shows an increase of mobility of the fluorescent probe induced by Ca2+. It seems difficult to correlate the results of the two kinds of measurements as long as we do not know the exact nature of the fluorescent kinetics unit.

Anti-AIDS agents. 15. Synthesis and anti-HIV activity of dihydroseselins and related analogs.

Forty-two dihydroseselins based on the structure of suksdorfin (1) were synthesized in order to evaluate their anti-HIV activity. These synthetic derivatives include 3',4'-di-O-acyl- and 3'- or 4'-O-acyl-cis-dihydroseselins (8-21) and 3',4'-trans-dihydroseselins with O-acyl and/or O-alkyl groups at the 3' and 4' positions (6, 22-43). Two 4'-azido (44, 45) and three 4'-alkylamido (46, 48, 49) derivatives were also prepared. By using optically pure reagents, three pairs of diastereoisomers were synthesized and separated as optically pure compounds (14, 15; 16, 17; 38, 39). Together with the above synthetic derivatives, seselin (3) and (+/-)-cis-(4), (+)-cis- (5), and (+/-)-trans-dihydroseselin-3',4'-diol (7) were also tested for their in vitro anti-HIV activity. An optically pure compound, 3',4'-di-O-(-)-camphanoyl-(+)-cis-khellactone (16), showed potent inhibitory activity and remarkable selectivity against HIV replication. The EC50 value and in vitro therapeutic index (TI) of 16 are 4 x 10(-4) microM and 136,719, respectively, which are better than those shown by AZT in the same assay. In addition, compound 16 is also active against HIV replication in a monocytic cell line and in peripheral blood mononuclear cells (PBMCs). Our in vitro assay indicated that, like compound 1, compound 16 is not an inhibitor of HIV-1 reverse transcriptase. Moreover, the anti-HIV activity of 16 is stereoselective as its three diastereoisomers (17, 38, 39) are at least 10,000 times less active. Since other synthetic dihydroseselin derivatives with different substituents or without any substituents are inactive or are active only at much higher concentration, the antiviral potency of 16 could be associated with the camphanoyl moieties of its structure. Therefore, compound 16 represents a unique coumarin structure with promising anti-HIV activity.

Ca-dependent regulation of the subunit exchange of F-actin under the influence of tropomyosin and troponin.

A quasi-steady state rate of actin subunit incorporation into a complex of F-actin, tropomyosin, and troponin was studied in a solution containing MgCl2 0.5 mM, ATP 0.2 mM, Tris-HCl 5 mM (pH 8.0), and either CaCl2 64 muM or EGTA 644 muM at 33 degrees C by adding a small amount (less than 1 muM) of fluorescence-labeled actin monomer. Incorporation of the labeled actin monomer was remarkably much slower in the presence of EGTA, but the rate recovered instantaneously upon addition of an excess amount of CaCl2. The rate was very much reduced by the presence of cytochalasin D (1 muM) and again showed Ca-dependence.

Effects of subtilisin cleavage of monomeric actin on its nucleotide binding.

The kinetics of ATP exchange on subtilisin-cleaved G-actin was investigated by measuring the fluorescence of 1,N6-ethenoadenosine 5'-triphosphate. The apparent dissociation rate of ATP (k-ATP) was 2.8-fold larger than that of intact G-actin in the presence of 300 microM free Ca2+. Analysis of the dependence of k-ATP on free Ca2+ showed that the dissociation rate constant of tightly bound Ca2+ was not significantly changed by subtilisin cleavage. On the other hand, an equilibrium binding study using 8-amino-2-[(2-amino-5-methylphenoxy)-methyl]-6-methoxyquinoline N,N,N',N'-tetraacetic acid (Quin 2) showed that the affinity of tightly bound Ca2+ for G-actin was reduced by about 13-fold after subtilisin treatment. Consequently, the stabilization by Ca2+ of ATP was weak in cleaved G-actin. Furthermore, the kinetic analysis of ATP exchange revealed that the binding equilibrium between ATP and divalent cation-free cleaved G-actin was much slower than that in the case of intact G-actin.

The binding of myosin subfragment-1 to F-actin in the absence of nucleotide. Evidence for dependence on F-actin concentration.

The binding of myosin subfragment-1 (S-1) to F-actin in the absence of nucleotide was examined by the sedimentation method using 1,5-IAEDANS-labeled S-1. We found that the binding affinity of F-actin to S-1 was dependent on the concentration of F-actin, and the binding was weaker at higher concentrations of F-actin. The apparent association constant determined from a linearly extrapolated Scatchard plot was 6.5 x 10(6) M-1 at 8.1 microns F-actin, and 1.7 x 10(7) M-1 at 2.0 microns F-actin in 120 mM KC1, 2 mM MgCl2, 0.1 mM CaCl2, and 20 mM Tris-acetate (pH 7.6) at 20 degrees C. Furthermore, the Scatchard plot revealed the existence of cooperativity in the binding of S-1 to F-actin. In order to obtain higher precision we developed a new method for the chromatographic determination of free S-1 in acto-S-1 solution. By this method we could determine free S-1 concentrations of the order of 10(-9) M easily and accurately. The above conclusion obtained by the sedimentation method was confirmed by this chromatographic method, and these effects can be well explained by considering the length distribution of F-actin. We propose an allosteric model in which both the length distribution and the polarity of F-actin are taken into consideration.

High-affinity binding of cytochalasin B to the B-end of F-actin loses its inhibitory effect on subunit exchange when the bound nucleotide is ADP.

We investigated the mode of binding of cytochalasin B (CB) to F-actin in an ADP-solution with and without inorganic phosphate (Pi). In the presence of Pi (20 mM), a filament of F-actin had a single high-affinity CB binding site (Kd = 1.4 nM), just like in the case of an ATP-solution [Kd = 5.0 nM: Suzuki, N. & Mihashi, K. (1991) J. Biochem. 109, 19-23]. But in the absence of Pi, there were two low-affinity (Kd = 200 nM) CB binding sites as well as one high-affinity site (Kd = 1.6 nM). We determined the concentration of CB necessary for half-maximal inhibition of growth or shortening of F-actin (Ki) using of pyrene-labeled actin. We obtained Ki = 80 nM for growth and Ki = 800 nM for shortening in the presence of ATP. The addition of Pi to the ATP-solution reduced Ki for growth to 9 nM. We propose a model explaining these results. In the model, high-affinity CB binding to the terminal subunit dimer can inhibit subunit exchange at the B-end only when the terminal subunits bind ATP or ADP.Pi. When the terminal subunits bind ADP, additional low-affinity CB bindings to the terminal subunits are needed to inhibit the subunit exchange.

Binding mode of cytochalasin B to F-actin is altered by lateral binding of regulatory proteins.

The binding of cytochalasin B (CB) to F-actin was studied using a trace amount of [3H]-cytochalasin B. F-Actin-bound CB was separated from free CB by ultracentrifugation and the amount of F-actin-bound CB was determined by comparing the radioactivity both in the supernatant and in the precipitate. A filament of pure F-actin possessed one high-affinity binding site for CB (Kd = 5.0 nM) at the B-end. When the filament was bound to native tropomyosin (complex of tropomyosin and troponin), two low-affinity binding sites for CB (Kd = 230 nM) were created, while the high-affinity binding site was reserved (Kd = 3.4 nM). It was concluded that the creation of low-affinity binding sites was primarily due to binding of tropomyosin to F-actin, as judged from the following two observations: (1) a filament of F-actin/tropomyosin complex possessed one high-affinity binding site (Kd = 3.9 nM) plus two low-affinity binding sites (Kd = 550 nM); (2) the Ca2(+)-receptive state of troponin C in F-actin/native tropomyosin complex did not affect CB binding.

Kinetics of conformational change of troponin-C induced by binding or removal of calcium ion.

The kinetics of conformational change of troponin-C (TN-C) induced by binding or removal of calcium ion were studied in the presence or absence of magnesium ion by measuring the fluorescence of tyrosyl residues by stopped-flow spectrofluorometry. The result was analyzed in terms of first-order kinetics. Two phases were observed both in pCa-up and in pCa-down experiments. The dependence of the rate constants on pCa was explained by a simple mechanism as follows; (see article). The dissociation constants of calcium bound to TN-C, K and K', calculated from the experimentally determined rate constants were K = 3.16 X 10(-7) M, K' = 1.58 X 10(-6) M in the absence of magnesium ion, and K = K' = 1 X 10(-6) M in the presence of 2 mM MgCl2.

Kinetics of conformational change of troponin C induced by calcium.

The kinetics of conformational change of troponin C (TN-C) induced by binding and removal of calcium ions were studied by measuring the fluorescence of tyrosine by stopped-flow spectrofluorometry. When the concentration of free calcium ions in the solution [Ca2+] was increased rapidly from 4X10(-9)M to 1X10(-3)M at neutral pH, a first-order reaction with a rate constant of 13.7 sec-1, which was preceded by a much faster reaction, was observed. In contrast, when [Ca2+] was reduced from 2X10(-3)M to 4.4X10(-8)M, two first-order reactions with rate constants of 7.4 and 0.78 sec-1, preceded by a much faster reaction, were observed.

Evidence for the existence of two equilibrium conformations of the ternary complex of myosin subfragment-1, ADP, and orthovanadate.

In order to investigate the flexibility of the ternary complex consisting of myosin subfragment-1 (S1), ADP, and orthovanadate (Vi), i.e., S1.ADP.Vi, the exchangeability of the bound ADP was examined. After isolation of the ternary complex of S1.ADP.Vi by gel filtration, 3'-O-(N-methylanthraniloyl)-ADP (Mant-ADP), a fluorescent analogue of ADP, was added at 0.5 degrees C. The added Mant-ADP was incorporated into the ternary complex very slowly by replacing the bound ADP. The nucleotide exchange occurred without regeneration of the ATPase activity of S1. Similarly, the ternary complex of S1.Mant-ADP.Vi prepared and isolated by gel filtration according to Hiratsuka (3, 4), was incubated with ADP (2.4 mM) at 4.5 degrees C. The nucleotide exchange of S1.Mant-ADP.Vi with ADP occurred in two phases with the apparent rates of 4.5 x 10(-4) s-1 (the fast phase) and 6.7 x 10(-6) s-1 (the slow phase). Biphasic exchange of the bound nucleotide was also observed with S1(A1) isozyme, indicating that the biphasic exchange did not correspond to two S1 isozymes. The apparent rates of the fast and the slow phases increased with the concentration of the added ADP, but they became saturated at an ADP concentration of the order of 2 mM, indicating that the nucleotide exchange reaction involves a step (or steps) which is insensitive to the concentration of free ADP in the solution. This step might be a reversible isomerization.

Kinetics of the conformational change of troponin-C induced by magnesium-binding or removal.

The kinetics of the conformation change of troponin-C (TN-C) induced by magnesium-binding or removal were studied in the absence of calcium ion by measuring the fluorescence intensity change of BIPM bound to TN-C by stopped-flow spectrofluorometry. The kinetic process of the conformational change was biphasic. The rate constants of the two phases were determined as a function of free magnesium ion concentration ([Mg]) of the solution. The [Mg]-dependence of the rate constants was explained by a simple molecular kinetic mechanism: (formula: see text) The dissociation constant of magnesium bound to TN-C was also determined to be 1 x 10(-3) M in the kinetic study.

Kinetics of conformational change of troponin-C induced by proton binding or removal in the absence of calcium ions.

The kinetics of the conformational change of troponin-C induced by binding or removal of protons was studied by a stopped-flow pH-jump spectrofluorometric method. In the pH-down experiment (to investigate the kinetics of conformational change from the deprotonated state to the protonated state), a single first-order reaction with a rate constant and amplitude of 1.75-2.4 sec-1 and around 10% respectively, was observed. On the other hand, two first-order reactions with rate constants of 0.84-1.6 sec-1 and 0.08-0.4 sec-1 were observed in the pH-up experiment, the total amplitudes of these reactions being around 10-20%. The pH dependences of the rate constants of these reactions were analyzed in terms of a three-species mechanism.

Absorption, fluorescence, and linear dichroism spectra of fluorescein mercuric acetate (FMA) bound to F-actin. Two kinds of effects of divalent cations.

Two kinds of F-actin were prepared; one binds magnesium at the unique divalent cation binding site of actin protomer and the other binds calcium at this site. They were designated as F(Mg)-actin and F(Ca)-actin, respectively. The binding of fluorescein mercuric acetate (FMA) to F(Mg)-actin and F(Ca)-actin was studied spectroscopically. The absorption and fluorescence spectra of bound FMA differed slightly but distinctly between F(Mg)-actin and F(Ca)-actin. Moreover, FMA bound to F(Mg)-actin showed linear dichroism in the presence of 2 mM MgCl2 (or 2mM CaCl2) in the solvent, while the dichroism was abolished by the removal of divalent cations from the solvent. In contrast, FMA bound to F(Ca)-actin did not show any appreciable linear dichroism irrespective of the presence (or absence) of divalent cations in the solvent. These results suggest that the structure of F-actin is characteristically regulated by divalent cations in a dual mode.

Internal motion of F-actin in 10(-6)-10(-3) s time range studied by transient absorption anisotropy: detection of torsional motion.

By means of laser flash photolysis, the transient absorption anisotropy (TAA) of the triplet probe, 5-iodoacetamide-Eosin, labeling rabbit skeletal F-actin was measured in the 10(-6)-10(-3) s time range. The TAA curve at 20 degrees C showed a relatively slow decay phase covering several hundred microseconds and a large residual anisotropy (approximately 0.1 at 2 ms). After analysis with Barkley & Zimm's formula, it was concluded that the TAA of Eosin-F-actin can be approximated by the anisotropy decay due to torsional motion of F-actin.

An ellagic compound and iridoids from Cornus capitata root cultures.

An ellagic acid derivative, 3,3'-di-O-methylellagic acid 4-(5"-acetyl)-alpha-L-arabinofuranoside, and two iridoid glucosides, 6alpha-dihydrocornic acid and 6beta-dihydrocornic acid, were isolated from Cornus capitata adventitious roots cultured in Murashige-Skoog (Murashige, T., Skoog, F., 1962. A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiol. Plant. 15, 473-487) liquid medium containing 10 microM CuSO(4). Three known related metabolites, i.e. stenophyllin H1, dihydrocornin and cornin were also produced in the root cultures. The chemical structures were characterized by analysis of spectroscopic data.