Motion of myosin fragments during actin-activated ATPase: fluorescence correlation spectroscopy study.

The rates of the translational motion of myosin fragments, heavy meromyosin (HMM), and heavy meromyosin subfragment-1 (HMM S-1) were measured during actin-activated ATPase reaction by the method of fluorescence correlation spectroscopy. This technique monitors the random fluctuations in the concentration of fluorescent molecules in an open volume which result from the translational diffusion of the molecular species under observation. The statistical behavior of the fluctuations is represented in the form of the autocorrelation function, which is related to the translational diffusion coefficient of the fluorescent molecules. The translational motion of fluorescently labeled myosin fragments was progressively slowed down after additions of increasing amounts of actin in the presence of excess MgATP. When these results are interpreted according to a simple binding scheme, the extent of the retardation can be used to obtain the apparent association constant for binding of S-1 and HMM to actin in the presence of MgATP. In 0.1M KCl and at 23°C, the apparent association constants were determined as K_app^HMM = 2.2 × 10⁴M^?1 and K_app^S-1 = 8.8 × 10³ for HMM and S-1, respectively.     

Interaction of pollen F-actin with rabbit muscle myosin and its subfragments (HMM,S1)

Summary Actin purified from maize pollen grains can be polymerized into F-actin which increased the ATPase activities of proteolytic fragments (HMM, S₁) of rabbit muscle myosin. The values of K_app is 232 M for HMM and 290 M for S₁, which are six- and seven-fold higher than those of rabbit muscle F-actin under the same conditions. Pollen actin and rabbit muscle myosin form hybrid actomyosin showing increase in viscosity and turbidity of solution. Viscosity and turbidity of the actomyosin dropped and then increased again with addition of ATP. Polymerized pollen actin can be decorated in vitro with both rabbit muscle HMM and S₁ to form an arrowhead-shaped structure like that observed in living plant cells. The results show that pollen actin is similar to muscle actin at a qualitative level. But there are differences between them at a quantitative level.Abbreviations HMM heavy meromyosin - S₁ myosin subfragment 1 - ATP adenosine-5-triphosphate     

Steady-state kinetic studies on the actin activation of skeletal muscle heavy meromyosin subfragments: Effects of skeletal,smooth and non-muscle tropomyosins

The addition of either smooth muscle or brain tropomyosin to skeletal muscle actoheavy meromyosin (HMM) or acto-myosin subfragment-1 (SF1) produces an activation of the actin-activated ATPase activity up to 100%. This contrasts with the opposite, inhibitory effect produced by skeletal muscle tropomyosin. The degree of activation or inhibition depends on the ionic conditions, which influence the affinities of tropomyosin and HMM or SF1 for actin as well as on the molar ratio of actin to myosin.Enzyme kinetic analysis indicates that the inhibitory effect of skeletal muscle tropomyosin results from an approximately six- to tenfold increase in the apparent affinity (K_app) of the myosin head for the F-actin-tropomyosin complex with a concomitant six- to tenfold reduction in the maximal turnover rate (V_max). Thus, there is no direct competition of skeletal muscle tropomyosin and myosin for the same site on actin. Brain tropomyosin has an opposite effect, decreasing the apparent affinity with concomitant increase in the V_max.The effect of smooth muscle tropomyosin is more complex. At high ratios of myosin to actin this tropomyosin produces the same change in the K_app as skeletal muscle tropomyosin but yields a value of V_max that is about twofold higher. At lower molar ratios (below about 1 to 5 myosin subfragments to actin) the activating effect of this tropomyosin remains unchanged while the apparent affinity decreases to that observed for pure F-actin.On the basis of these data as well as from experiments carried out at fixed actin and varying SF1 concentrations, it is concluded that tropomyosins act in general as allosteric un-competitive inhibitors or activators of actomyosin by increasing or reducing the co-operative activation of myosin by actin at the level of product release.     

Binding of chloride and a disulfonic stilbene transport inhibitor to red cell band 3

Summary The effect of chloride on 4,4-dibenzamido-2,2-disulfonic stilbene (DBDS) binding to band 3 in unsealed red cell ghost membranes was studied in buffer [NaCl (0 to 500mm) + Na citrate] at constant ionic strength (160 or 600mm). pH 7.4, 25°C. In the presence of chloride, DBDS binds to a single class of sites on band 3. At 160mm ionic strength, the dissociation constant of DBDS increases linearly with chloride concentration in the range [Cl]=450mm. The observed rate of DBDS binding to ghost membranes, as measured by fluorescence stopped-flow kinetic experiments, increases with chloride concentration at both 160 and 600mm ionic strength. The equilibrium and kinetic results have been incorporated into the following model of the DBDS-band 3 interaction: The equilibrium and rate constants of the model at 600mm ionic strength areK ₁=0.67±0.16 m,k ₂=1.6±0.7 sec^–1,k _–2=0.17±0.09 sec^–1,K ₁=6.3±1.7 m,k ₂=9±4 sec^–1 andk _–2=7±3 sec^–1. The apparent dissociation constants of chloride from band 3,K _Cl, are 40±4mm (160mm ionic strength) and 11±3mm (600mm ionic strength). Our results indicate that chloride and DBDS have distinct, interacting binding sites on band 3.     

Inorganic ion effects on the kinetic parameters of acetylcholinesterase

EARLY work on the effects of inorganic ions on the activity of acetylcholine acetyl-hydrolase (EC 3.1.1.7; AChE) from various sources has been summarized by COHEN and OOSTERBAAN (1963) and many other reports have been published subsequently (CHANGEUX, 1966; CRONE, 1973; HELLER and HANAHAN, 1972; IVANOVA, 1967; KITZ, BRASWELL and GINSBURG, 1970; ROUF-CALIS and QUIST, 1972; ROUFOGALIS and THOMAS, 1968; WINS, SCHOFFENIELS and FOIDART, 1970). Despite this work, no comprehensive study has yet been made to determine whether the observed effects are specific to particular ions or dependent only on the ionic strength of the medium (CHANGEUX, 1966). In some cases, specific ion effects have been observed (CHANGEUX, 1966; HELLER and HANAHAN, 1972; ROUFOGALIS and QUIST, 1972; ROUFOGALIS and THOMAS, 1968) at salt concentrations from 600 mM to below 1 mM, but the studies were not detailed enough and in some cases the total ionic strength was not rigidly controlled, so that no general deductions can be drawn. We have studied the hydrolysis of acetylcholine (ACh) by bovine erythrocyte AChE at subinhibitory substrate concentrations, and now present our results on the effect of inorganic salts at varying ionic strength on the kinetic parameters K_m, and V_max. The present work shows that this hydrolysis follows simple Michaelis kinetics very closely, and therefore these two constants suffice to define the complete pattern of initial reaction velocity as a function of substrate concentration (ATKINSON, 1966).     

Characteristics of immobilized invertase

Five kinds of immobilized invertases (IMI)—covalently of porous glass and ion-exchange resins and ionically on ion-exchange resins—have been prepared and their kinetic characteristics for sucrose hydrolysis, such as K_m, K, pH profile, and thermal stability were studied. Comparing the values of K_m and activation energy and the entropy of IMI with those of native invertase, it was concluded that the immobilization influences not binding but kinetic specificity. The effects of the immobilization method on thermal stability were also discussed.     

Dynamic light-scattering study of synthetic myosin filaments

Synthetic myosin filaments of rabbit were prepared. Electron microscopy showed that the number-average length (L_n = 470 nm) and sharpness in length distribution (L_w/L_n = 1.03₆) were independent of ionic strengths of 134, 74, and 44 mM, whereas the number ratio of M-filaments (about 15 nm in diameter at the bare zone) to m-filaments (about 10 nm) strongly depended on ionic strength (IS); the major filaments were M-filaments at IS = 134 mM, m-filaments at IS = 74 mM, and almost exclusively m-filaments at IS = 44 mM. Dynamic light scattering showed that the change in diameter with the change in ionic strength by 2-h dialysis was reversible. Combination of dynamic light scattering and sedimentation studies suggested a dynamic equilibrium between M- and m-filaments. Dynamic light-scattering spectra at IS = 134 and 74 mM could be analyzed by a theory for rigid rods, whereas those at IS = 44 mM only by introducing semiflexibility of filaments; m-filaments are more flexible at IS = 44 than at 74 mM.     

Flow kinetics of L-asparaginase attached to nylon tubing

L -Asparaginase has been attached by chemical means to the inner surface of nylon tubing. An experimental study has been carried out of the flow kinetics for such a system, asparagine solutions at various concentrations being passed through two lengths of tubing at various flow rates. Measurements were made of the concentration of the product ammonia at the tube exit, and of the rate of formation of ammonia, under the various conditions. Apparent Michaelis constants, K_m(app), were some three orders of magnitude higher than the K_m for the enzyme in free solution (～13 × 10^?6JM). The results were analyzed with respect to the theoretical treatment described in the preceding paper (Kobayashi and Laidler), three different methods being employed. It is concluded that at lower substrate concentrations and flow rates the reactions are largely diffusion-controlled, the enhanced K_m(app) values being largely if not entirely due to the diffusion control; ionic strength studies showed electrostatic repulsion effects to be unimportant. At high concentrations and high flow rates (when the diffusion layer is of negligible thickness) the diffusional effects are minimized, and K_m(app) approaches the true K_m value for the immobilized enzyme.     

Coenzyme models. IV. Effect of polymer micelles on rate and equilibrium of addition of cyanide ion to N-substituted 3-carboxamidopyridinium ions.

The water-soluble poly(1-vinyl-2-ethylimidazole) quaternized with ethyl bromide and lauryl bromide was prepared; lauryl group content, 8.8 mol% (L-9), 28.9 mol% (L-29), and 40.9 mol% (L-41). The λ_max value of methyl orange near 460 nm shifted to shorter wavelengths (417–433 nm) in the aqueous solution of L-29 and L-41, and the intrinsic viscosity of L-29 was more than ten times smaller than that of L-9. The rate and equilibrium constants (k_? and K) for addition of cyanide ion to the N-substituted 3-carboxamidopyridinium ions were studied at 30°C, where N-substituents employed were n-propyl, n-hexyl, benzyl, 2,6-dichlorobenzyl, and n-lauryl. The kinetic parameters for n-lauryl-3-carboxamidopyridinium were markedly increased in the presence of L-29 and L-41 and with increasing polymer concentrations (84-fold for k_? and 7800-fold for K), especially at low ionic strength, whereas L-9 decelerated the addition reaction. These distinct behaviors mean that L-29 and L-41 are classified as micellelike polymers and L-9 as a polyelectrolytelike polymer. However, L-29 depressed the rate of the forward reaction for benzyl-3-carboxamidopyridinium, acting like a simple polyelectrolyte. Therefore, the polymer micelle can provide both the microenvironments characteristic of polyelectrolytes and micelles, depending on the hydrophobicity of substrates.     

Multispectroscopic DNA-binding studies of a terbium(III) complex containing 2,2′-bipyridine ligand

Agarose gel electrophoresis, absorption, fluorescence, viscosity, and circular dichroism (CD) have been used in exploring the interaction of terbium(III) complex, [Tb(bpy)₂Cl₃(OH₂)] where bipy is 2,2′-bipyridine, with Fish salmon DNA. Agarose gel electrophoresis assay, along with absorption and fluorescence studies, reveal interaction between the corresponding complex and FS-DNA. Also, the binding constants (K_b) and the Stern–Volmer quenching constants (K_sv) of Tb(III) complex with FS-DNA were determined. The calculated thermodynamic parameters suggested that the binding of mentioned complex to FS-DNA was driven mainly by hydrophobic interactions. A comparative study of this complex with respect to the effect of iodide-induced quenching, ionic strength effect, and ethidium bromide exclusion assay reflects binding of explicit to the FS-DNA primarily in a groove fashion. CD and viscosity data also support the groove binding mode. Furthermore, Tb(III) complex have been simultaneously screened for their antibacterial and antifungal activities.     

Depicting the binding of furazolidone/furacilin with DNA by multiple spectroscopies,voltammetric as well as molecular docking

The interaction between DNA and furazolidone/furacillin was investigated using various analytical techniques including spectroscopy and electroanalysis and molecular modelling. With the aid of acridine orange (AO), the fluorescence lifetimes of DNA–AO, DNA–furazolidone/furacillin–AO remained almost the same, which proved that the ground state complex was formed due to furazolidone/furacillin binding with DNA. Circular dichroism spectra and Fourier transform infrared spectroscopy showed that the second structure of DNA changed. Viscosity experiments presented that relative viscosity of DNA was increased with the increasing concentrations of furazolidone and almost unchanged for furacilin. In addition, the results of melting temperature (T_m), ionic strength, site competition experiments, cyclic voltammetry, and molecular docking all proved the intercalation binding mode for furazolidone and groove binding mode for furacilin. The binding constants (K_a) obtained from Wolfe–Shimmer equation were calculated as 3.66 × 10⁴ L mol^?1 and 3.95 × 10⁴ L mol^?1 for furazolidone–DNA and furacilin–DNA, respectively.     

Comparison of the reaction of N-ethylmaleimide with myosin and heavy meromyosin subfragment 1

Myosin reacted at low ionic strength with NEM forms an actomyosin which is Ca⁺⁺ insensitive. With HMM S-1 the reaction with NEM causes a marked loss of the actin activated ATPase activity and the Ca⁺⁺ sensitivity is reduced but not eliminated. The presence of actin during the sulfhydryl reaction does not significantly alter this result. HMM S-1 prepared from myosin previously desensitized by NEM regains Ca⁺⁺ sensitivity. These results indicate that the conformations of myosin and HMM S-1 are different and could reflect a difference between insoluble (filamentous) myosin and myosin, or its fragments, in solution.     

Effects of phosphorylation of light chain residues threonine 18 and serine 19 on the properties and conformation of smooth muscle myosin

Smooth muscle myosin can be phosphorylated by myosin light chain kinase at the serine 19 and threonine 18 residues of the two 20,000-dalton light chains (Ikebe, M., Hartshorne, D. J., and Elizinga, M. (1986) J. Biol. Chem. 261, 36-39). These studies with myosin and heavy meromyosin (HMM) compare the effects induced by phosphorylation of serine 19 (M2P and HMM2P) and serine 19 plus threonine 18 (M4P and HMM4P). Formation of M4P altered the KCl dependence of viscosity and Mg2+-ATPase and higher values were maintained at lower ionic strengths, compared to M2P or dephosphorylated myosin (Mo). This is consistent with the stabilization of the 6 S conformation. The tendency for aggregation, as judged by light scattering, followed the sequence M4P greater than M2P greater than Mo. Filaments formed with M4P were more resistant to dissociation by ATP compared to filaments of M2P. Phosphorylation of HMM2P doubled Vmax of actin-activated ATPase with little effect on the apparent affinity for actin. The Mg2+-ATPase of HMM4P exhibited a higher activity at low ionic strength compared to HMM2P and HMMo. Hydrodynamic differences were detected at low ionic strength in the presence of ATP by sedimentation velocity measurements with HMM4P, HMM2P, and HMMo. Proteolysis by papain indicated an increased susceptibility of the head-neck junction of HMM4P compared to HMM2P. These data suggest that the phosphorylation of threonine 18 in addition to serine 19 change the conformation of myosin and HMM and this is associated with altered biological properties.     

Characterization of wall-bound invertase isoforms of Picea abies cells and regulation by ectomycorrhizal fungi

In culture, the ectomycorrhiza-forming fungi Amanita muscaria (Pers. ex Fries) Hock. and Hebeloma crustuliniforme (Bull. ex Fries) Quel. only grow on media with glucose or fructose but not with sucrose as sole carbohydrate source. This is due to their lack of wall-bound invertase activity. Therefore, utilization of sucrose by the fungi within a mycorrhizal association is believed to depend on the wall-bound invertase activity of the host. This enzyme activity was studied in the apoplast of suspension cultured cells of Picea abies (L.) Karst. An ionically and a tightly wall-bound isoform of acid invertase were found that function as β-d -fructofuranoside-fructohydrolases (EC 3.2.1.26). The ionically bound enzyme could be easily released from walls of intact cells with buffer of high ionic strength. In its native form, the ionically bound invertase isoform is a monomeric protein with a molecular mass of 61 kDa, as determined by gel filtration and SDS-PAGE. Glycoprotein nature of the enzyme was demonstrated with antibodies directed against the digoxigenin-labeled protein. The K_m values of both enzymes for sucrose, their natural substrate, are relatively high (ionically bound invertase K_m= 16 mM, tightly bound invertase K_m= 8.6 mM). Activity of both wall-bound invertase isoforms strongly depends on the apoplastic pH. They have a narrow pH-optimum and exhibit highest activity at pH 4.5. with elevated activity between pH 4.5 and 6.0. Furthermore, fructose acts as competitive inhibitor of both isoforms, whereas glucose is not inhibitory. Unloading of sucrose from host cells to the apoplastic interface of the Hartig net in ectomycorrhizae appears to depend on the rate of hydrolysis by the wall-bound invertase of the host. Since the activity of the plant invertase depends on the actual pH value and the fructose concentration in the mycorrhizal interface, we suggest that the fungus can actively influence the activity of the plant invertase by acidification of the cell wall and by fructose uptake. Thus, the fungus itself can regulate its own supply of glucose and fructose.     

Analysis of cooperativity and ion effects in the interaction of quinacrine with DNA

The interaction of quinacrine with calf thymus DNA was monitored at several different ionic strengths using spectrophotometric and equilibrium dialysis techniques. The binding results can be explained, assuming each base pair is a potential binding site, using a model containing two negative cooperative effects: (1) ligand exclusion at binding sites adjacent to a filled binding site and (2) ligand–ligand negative cooperativity at adjacent filled binding sites. The logarithm of the observed equilibrium constant (K_obs) determined by this model varies linearily with log[Na⁺], as predicted by the ion condensation theory for polyelectrolytes. When the log K_obs plot is correlated for sodium release by DNA in the intercalation conformational change, the predicted number of ion pairs between the ligand and DNA is approximately two, as expected for the quinacrine dication. Even though K_obs depends strongly on ionic strength, the ligand negative cooperativity parameter ω was found to be indpendent of ionic strength within experimental error. This finding is also in agreement with the ion condensation theory, which predicts a relatively constant amount of condensed counterion on the DNA double helix over this ionic strength range. Drugs would, therefore, experience a relatively constant ionic environment when complexed to DNA even though the ionic conditions of the solvent could change considerably.     

Insights into the structural stability of nuclear matrix ribonucleoprotein,LMG160: thermodynamic and spectroscopic analysis

Low-mobility group nonhistone chromatin protein, LMG_160, is a nuclear matrix ribonucleoprotein particle (RNP) which has a RNA molecule with approximately 300 bases. In this study, structural stability of the intact LMG₁₆₀ (I-LMG₁₆₀) was investigated at different ionic strength and in the absence of its RNA moiety (T-LMG₁₆₀) employing spectroscopic and thermodynamic techniques. The UV absorption spectra showed hypochromicity and red shift under increasing ionic strength for both forms of LMG₁₆₀ but in different extents. The fluorescence emission intensity was decreased as ionic strength was increased and the Stern–Volmer quenching constant (K_sv) for T-LMG₁₆₀ was 3.7 times less than for I-LMG₁₆₀. In the absence of sodium chloride, I-LMG₁₆₀ exhibited a very stable structure against the temperature change compared to T-LMG₁₆₀. The thermodynamic parameters showed that the positive values of ΔH_m and ΔS_m increased by increasing ionic strength in both forms of LMG₁₆₀. Removal of the RNA moiety altered secondary structure: as T-LMG₁₆₀ showed more helical content than I-LMG₁₆₀. From the results, it is concluded that I-LMG₁₆₀ is more sensitive to alteration of environment and the RNA has an important role in this RNP conformation. Also, interaction of both I- and T-LMG₁₆₀ with sodium chloride is entropy driven and is usually accompanied by surface hydrophobicity.     

Some properties of a ficin-papain inhibitor from avian egg white

A procedure has been established for the isolation, from sheep liver, of 6-phosphogluconate dehydrogenase which is homogeneous according to the criteria of the analytical ultracentrifuge, and isoelectric focusing. A systematic determination of the effects of pH, ionic strength, metal ions, and temperature, on the kinetic parameters of the isolated 6-phosphogluconate dehydrogenase has been carried out. Double-reciprocal plots of enzyme rate measurements as a function of substrate concentration indicate K_m values of 15 μm for 6-phosphogluconate, and 7 μm for NADP⁺, under optimum assay conditions, and show no effect of the concentration of one substrate on the K_m of the other substrate under the assay conditions employed. Ionic strength, monovalent and divalent metals, are apparently interchangeable in their ability to activate the enzyme, and act by decreasing the K_m values of the enzyme, not by increasing the reaction rate. Concentrations of metals above the optimum are strongly inhibitory. Plots of ?log K_m vs pH show inflection points at 8.3 for 6-phosphogluconate, and 6.5 for NADP⁺. At low substrate concentrations the pH optimum of the enzyme is at pH 7.7, but plots of V vs pH increase up to pH 9.1 (the enzyme is unstable at higher pH values). An Arrhenius plot shows a straight line between temperatures of 8.6 and 39.4 °C, and an energy of activation of 15,450 cal mole^?1.     

Na+-Coupled Alanine Transport in LLC-PK1 Cells: The Relationship Between the K m for Na+ at Low [Alanine] and Potential Dependence for the System

Analysis of the mechanistic basis by which sodium-coupled transport systems respond to changes in membrane potential is inherently complex. Algebraic expressions for the primary kinetic parameters (K _m and V _max ) consist of multiple terms that encompass most rate constants in the transport cycle. Even for a relatively simple cotransport system such as the Na⁺/alanine cotransporter in LLC-PK₁ cells (1:1 Na⁺ to substrate coupling, and an ordered binding sequence), the algebraic expressions for K _m for either substrate includes ten of the twelve rate constants necessary for modeling the full transport cycle. We show here that the expression of K _m of the first-bound substrate (Na⁺) simplifies markedly if the second-bound substrate (alanine) is held at a low concentration so that its' binding becomes the rate limiting step. Under these conditions, the expression for the K ^Na _m includes rate constants for only two steps in the full cycle: (i) binding/dissociation of Na⁺, and (ii) conformational `translocation' of the substrate-free protein. The influence of imposed changes in membrane potential on the apparent K ^Na _m for the LLC-PK₁ alanine cotransporter at low alanine thus provides insight to potential dependence at these sites. The data show no potential dependence for K ^Na _m at 5 μm alanine, despite marked potential dependence at 2 mm alanine when the full algebraic expression applies. The results suggest that neither translocation of the substrate-free form of the transporter nor binding/dissociation of extracellular sodium are potential dependent events for this transport system. Received: 10 April 1998/Revised: 6 July 1998