The binding of cytochalasin D to monomeric actin

The binding of cytochalasin D to monomeric actin has been measured directly. In the presence of 200 microM Ca2+, actin binds cytochalasin D in a 1:1 molar ratio with a KD of 18 microM. After incubation with 250 microM Mg2+ for 10 minutes, actin binds cytochalasin D with a KD of 2.6 microM but with one mole of cytochalasin D per 2 moles of actin. This suggests that cytochalasin D induces dimerization of Mg2+-induced actin monomers.     

Divalent cation binding to the high- and low-affinity sites on G-actin

Metal binding to skeletal muscle G-actin has been assessed by equilibrium dialysis using 45Ca2+ and by kinetic measurements of the increase in the fluorescence of N-acetyl-N'-(5-sulfo-1-naphthyl)-ethylenediamine-labeled actin. Two classes of cation binding sites were found on G-actin which could be separated on the basis of their Ca2+ affinity: a single high-affinity site with a Kd considerably less than 1 microM and three identical moderate-affinity binding sites with a Kd of 18 microM. The data for the Mg2+-induced fluorescence enhancement of actin labeled with N-acetyl-N'-(5-sulfo-1-naphthyl)ethylenediamine support a previously suggested mechanism [Frieden, C. (1982) J. Biol. Chem. 257, 2882-2886] in which Ca2+ is replaced by Mg2+ at the moderate affinity site(s), followed by a slow actin isomerization. This isomerization occurs independently of Ca2+ release from the high-affinity site. The fluorescence data do not support a mechanism in which this isomerization is directly related to Ca2+ release from the high-affinity site. Fluorescence changes of labeled actin associated with adding metal chelators are complex and do not reflect the same change induced by Mg2+ addition. Fluorescence changes in the labeled actin have also been observed for the addition of Cd2+ or Mn2+ instead of Mg2+. It is proposed actin may undergo a host of subtle conformational changes dependent on the divalent cation bound. We have also developed a method by which progress curves of a given reaction can be analyzed by nonlinear regression fitting of kinetic simulations to experimental reaction time courses.(ABSTRACT TRUNCATED AT 250 WORDS)     

Formation of actin dimers as studied by small angle neutron scattering

Small angle neutron scattering has been used to study the dimensions of G-actin and the formation of low molecular weight actin oligomers under conditions where rapid polymerization does not take place. In the presence of 200 microM Ca2+, actin in solution consists of a single component with a radius of gyration (Rg) of 19.9 +/- 0.4 A, consistent with the known molecular dimensions of the G-actin molecule. In the presence of 50 microM Mg2+, however, formation of an actin species with a larger Rg occurs over a 4-h period. Multicomponent fits were tried and the data were best fit assuming two components, the monomer and a species with an Rg of 29 +/- 1 A. This latter value is consistent with the dimensions expected for certain actin dimers. The apparent dissociation constant for dimer formation is approximately 150 microM with forward and reverse rate constants of 6.0 X 10(-7) microM-1 s-1 and 8.8 X 10(-5) s-1, respectively. Kinetic fluorescence experiments show that the dimer formed in the presence of low levels of Mg2+ is a nonproductive complex which does not participate in the polymerization process. However, the addition of cytochalasin D to actin in the presence of 50 microM Mg2+ rapidly induces the formation of dimers, presumably related to cytochalasin's ability to nucleate actin polymerization.     

The effects of Mg2+ at the high-affinity and low-affinity sites on the polymerization of actin and associated ATP hydrolysis

Actin contains a single high-affinity cation-binding site, for which Ca2+ and Mg2+ can compete, and multiple low-affinity cation-binding sites, which can bind Ca2+, Mg2+, or K+. Binding of cations to the low-affinity sites causes polymerization of monomeric actin with either Ca2+ or Mg2+ at the high-affinity site. A rapid conformational change occurs upon binding of cations to the low-affinity sites (G----G) which is apparently associated with the initiation of polymerization. A much slower conformational change (G----G', or G----G' if the low-affinity sites are also occupied) follows the replacement of Ca2+ by Mg2+ at the high-affinity site. This slow conformational change is reflected in a 13% increase in the fluorescence of G-actin labeled with the fluorophore 7-chloro-4-nitrobenzene-2-oxadiazole (NBD-labeled actin). The rate of the ATP hydrolysis that accompanies elongation is slower with Ca-G-actin than with Mg-G'-actin (i.e. with Ca2+ rather than Mg2+ at the high-affinity site) although their rates of elongation are similar. The slow ATP hydrolysis on Ca-F-actin causes a lag in the increase in fluorescence associated with the elongation of actin labeled with the fluorophore N-pyrene iodoacetamide (pyrenyl-labeled actin), even though there is no lag in the elongation rate, because pyrenyl-labeled ATP-F-actin subunits have a lower fluorescence intensity than pyrenyl-labeled ADP-F-actin subunits. The effects of the cation bound to the high-affinity binding site must, therefore, be considered in quantitatively analyzing the kinetics of polymerization of NBD-labeled actin and pyrenyl-labeled actin. Although their elongation rates are not very different, the rate of nucleation is much slower for Ca-G-actin than for Mg-G'-actin, probably because of the slower rate of ATP hydrolysis when Ca2+ is bound to the high-affinity site.     

Differences in G-actin containing bound ATP or ADP: the Mg2+-induced conformational change requires ATP

The role of adenosine 5'-triphosphate (ATP) in the Mg2+-induced conformational change of rabbit skeletal muscle G-actin has been investigated by comparing actin containing bound ADP with actin containing bound ATP. As previously described [Frieden, C. (1982) J. Biol. Chem. 257, 2882-2886], N-acetyl-N'-(5-sulfo-1-naphthyl)ethylenediamine-labeled G-actin containing ATP undergoes a time-dependent Mg2+-induced fluorescence change that reflects a conformational change in the actin. Addition of Mg2+ to labeled G-actin containing ADP gives no fluorescence change, suggesting that the conformational change does not occur. The fluorescence change can be restored on the addition of ATP. Examination of the time courses of these experiments suggests that ATP must replace ADP prior to the Mg2+-induced change. The Mg2+-induced polymerization of actin containing ADP is extraordinarily slow compared to that of actin containing ATP. The lack of the Mg2+-induced conformational change, which is an essential step in the Mg2+-induced polymerization, is probably the cause for the very slow polymerization of actin containing ADP. On the other hand, at 20 degrees C, at pH 8, and in 2 mM Mg2+, the elongation rate from the slow growing end of an actin filament, measured by using the protein brevin to block growth at the fast growing end, is only 4 times slower for actin containing ADP than for actin containing ATP.     

Conformational changes in subdomain 2 of G-actin: fluorescence probing by dansyl ethylenediamine attached to Gln-41.

Gln-41 on G-actin was specifically labeled with a fluorescent probe, dansyl ethylenediamine (DED), via transglutaminase reaction to explore the conformational changes in subdomain 2 of actin. Replacement of Ca2+ with Mg2+ and ATP with ADP on G-actin produced large changes in the emission properties of DED. These substitutions resulted in blue shifts in the wavelength of maximum emission and increases in DED fluorescence. Excitation of labeled actin at 295 nm revealed energy transfer from tryptophans to DED. Structure considerations and Cu2+ quenching experiments suggested that Trp-79 and/or Trp-86 serves as energy donors to DED. Energy transfer from these residues to DED on Gln-41 increased with the replacement of Ca2+ with Mg2+ and ATP with ADP. Polymerization of Mg-G-actin with MgCl2 resulted in much smaller changes in DED fluorescence than divalent cation substitution. This suggests that the conformation of loop 38-52 on actin is primed for the polymerization reaction by the substitution of Ca2+ with Mg2+ on G-actin.     

pH-induced changes in G-actin conformation and metal affinity

Metal-induced conformational changes in actin at 20 degrees C have been investigated as a function of pH using actin labeled at Cys-374 with N-(iodoacetyl)-N'-(5-sulfo-1-naphthyl)ethylenediamine. At pH 8, the addition of a high Ca2+ concentration (2 mM) to G-actin gives an instantaneous fluorescence increase while the addition of a high Mg2+ concentration gives both an instantaneous and a slow fluorescence increase. The instantaneous increase is interpreted as divalent cation binding to low-affinity, relatively nonspecific sites, while the slow response is attributed to Mg2+ binding to specific sites of moderate affinity [Zimmerle, C.T., Patane, K., & Frieden, C. (1987) Biochemistry 26, 6545-6552]. The magnitudes of both the instantaneous and slow fluorescence increases associated with Mg2+ addition to G-actin are shown here to decrease as the pH is lowered while the fluorescence of labeled G-actin in the presence of low or moderate Ca2+ concentrations (less than 200 microM) increases. The pH-dependent data suggest that protonation of a single class of residues with an approximate pK of 6.8 alters the immediate environment of the label differently depending upon the cation bound at the moderate-affinity site. The pH-dependent changes in the magnitude of the slow fluorescence response upon Mg2+ addition to Ca2+-actin are not associated with changes in the Mg2+ affinity at the moderate-affinity site but result from protonation altering the fluorescence response to Mg2+ binding. Protonation of this same class of residues is proposed to induce an actin conformation similar to that induced by cation binding at the low-affinity sites.(ABSTRACT TRUNCATED AT 250 WORDS)     

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.     

Tight binding of divalent cations to monomeric actin. Binding kinetics support a simplified model

Using the fluorescent Ca2+ selective chelator Quin2 to induce and measure the dissociation of Ca2+ from actin, we have recently found that actin binds Ca2+ and Mg2+ much more tightly than previously thought (Gershman, L.C., Selden, L.A., and Estes, J.E. (1986) Biochem. Biophys. Res. Commun. 135, 607-614). In this report, we show that the kinetics of dissociation of Ca2+ from Ca-actin and Mg2+ from Mg-actin closely parallel the fluorescence changes in 1,5-I-N-iodoacetyl-N'-(5-sulfo-1-naphthyl)ethylenediamine (AEDANS)-actin, suggesting that the 1,5-I-AEDANS-actin fluorescence directly reflects slow first-order cation exchange rather than a slow Mg2+-induced isomerization as originally proposed by Frieden (Frieden, C. (1982) J. Biol. Chem. 257, 2882-2886). Measuring divalent cation exchange directly, we have determined the dissociation rate constants for Ca2+ (k-Ca) and Mg2+ (k-Mg), the equilibrium dissociation constants for Ca2+ (KCa), and the ratio of cation binding affinities, KMg/Kca, to actin over the pH range 7-8. We have found that k-Ca is 5-10 times greater than k-Mg and KMg is about 4 times greater than KCa. From the data we calculate the association rate constants for Ca2+ (kCa) and Mg2+ (kMg) to be about 7 X 10(6) M-1 s-1 and 2 X 10(5) M-1 s-1, respectively. kCa appears to be diffusion-limited, but kMg is significantly smaller due to the characteristics of the Mg2+ aquo ion. These findings are consistent with a simple first-order binding model for the tight binding of divalent cations to actin.     

The accessibility of etheno-nucleotides to collisional quenchers and the nucleotide cleft in G- and F-actin.

Recent publication of the atomic structure of G-actin (Kabsch, W., Mannherz, H. G., Suck, D., Pai, E. F., & Holmes, K. C., 1990, Nature 347, 37-44) raises questions about how the conformation of actin changes upon its polymerization. In this work, the effects of various quenchers of etheno-nucleotides bound to G- and F-actin were examined in order to assess polymerization-related changes in the nucleotide phosphate site. The Mg(2+)-induced polymerization of actin quenched the fluorescence of the etheno-nucleotides by approximately 20% simultaneously with the increase in light scattering by actin. A conformational change at the nucleotide binding site was also indicated by greater accessibility of F-actin than G-actin to positively, negatively, and neutrally charged collisional quenchers. The difference in accessibility between G- and F-actin was greatest for I-, indicating that the environment of the etheno group is more positively charged in the polymerized form of actin. Based on calculations of the change in electric potential of the environment of the etheno group, specific polymerization-related movements of charged residues in the atomic structure of G-actin are suggested. The binding of S-1 to epsilon-ATP-G-actin increased the accessibility of the etheno group to I- even over that in Mg(2+)-polymerized actin. The quenching of the etheno group by nitromethane was, however, unaffected by the binding of S-1 to actin. Thus, the binding of S-1 induces conformational changes in the cleft region of actin that are different from those caused by Mg2+ polymerization of actin.(ABSTRACT TRUNCATED AT 250 WORDS)     

Troponin-C-mediated calcium-sensitive changes in the conformation of troponin I detected by pyrene excimer fluorescence

Troponin I (TnI) from rabbit white skeletal muscle was labeled at cysteines 48 and 64 with the fluorescent reagent N-(1-pyrene)maleimide. The fluorescence spectra of pyrene-labeled TnI (pyr-TnI) exhibit peaks characteristic of pyrene in its monomeric form and an additional peak resulting from formation of excited dimers (excimers), indicating that the labeled cysteines are close together. Formation of a pyr-TnI-TnC complex in the absence of Ca2+ has little effect on the spectrum, but when Ca2+ is bound to the low-affinity sites of TnC there is a substantial decrease in excimer and a corresponding increase in monomer fluorescence. The involvement of the low-affinity sites in the Ca2+-induced effect is consistent with the fact that Mg2+ has no effect on pyrene fluorescence. On rapid mixing of the pyr-TnI-TnC complex with Ca2+ in a stopped-flow apparatus, most of the excimer decrease is complete within the instrumental dead time, indicating a rate constant k greater than 350 s-1, which is comparable to that of the conformational change in TnC resulting from Ca2+ binding to the low-affinity sites. Rapid mixing of the Mg2-TnC-pyr-TnI complex with Ca2+ yields similar results, suggesting that the type of metal ion present at the high-affinity sites has little, if any, effect on the probe. It has been suggested previously that Cys 48 and 64 are located in a TnT-binding region of TnI (Chong P.C.S. and Hodges, R.S. (1982) J. Biol. Chem. 255, 3757). Our results suggest that a Ca2+-induced structural change in the TnI-binding region of TnC could be transmitted to TnT by affecting the TnT-binding region of TnI as part of the chain of events in the regulation of muscle contraction.     

Kinetic determination of talin-actin binding

Smooth muscle talin prepared from chicken gizzard binds to skeletal muscle actin in vitro. The stoichiometry of 1:3 for talin:fluorescent labelled G-actin was confirmed by steady state titration and viscosity measurements under non-polymerizing conditions. The binding constant (Kd) of talin and G-actin was determined by continuous fluorescence titration and gave a value of approx 0.3 microM. The association rate constant of talin and fluorescent labelled G-actin of approx 7 x 10(6) M-1 x s-1 was ascertained by the stopped flow method; the dissociation rate constant was calculated at approx 2-3 s-1.     

Actin polymerization. The mechanism of action of cytochalasin D

Fluorescence changes using actin covalently labeled with N-(1-pyrenyl)iodoacetamide have been used to determine the effect of cytochalasin D on actin polymerization. A mechanism for the effect of cytochalasin D on actin polymerization is presented, which explains the experimental observation of a cytochalasin D-induced increase in the initial rate of polymerization and a decrease in the final extent of the reaction. Central to this mechanism is the Mg2+-dependent formation of cytochalasin D-induced dimers. The dimers serve as nuclei to enhance the polymerization rate. Binding of Mg2+ to a low affinity site on the dimer induces a conformational change which can be observed as a rapid fluorescence increase. A subsequent time-dependent fluorescence decrease observed prior to polymerization appears to represent ATP hydrolysis resulting in dissociation of the dimer and release of actin monomers containing ADP. We postulate that a slow rate of exchange of ATP for bound ADP relative to hydrolysis results in the accumulation of monomers containing ADP. As these monomers have a high critical concentration, the final extent of polymerization is reduced dramatically. The Mg2+ dependence of the final extent of polymerization in the presence of cytochalasin D is also explained in the context of this mechanism.     

Ca2+-dependent binding of severin to actin: a one-to-one complex is formed

Severin is a protein from Dictyostelium that severs actin filaments in a Ca2+-dependent manner and remains bound to the filament fragments (Brown, S. S., K. Yamamoto, and J. A. Spudich , 1982, J. Cell Biol., 93:205-210; Yamamoto, K., J. D. Pardee , J. Reidler , L. Stryer , and J. A. Spudich , 1982, J. Cell Biol. 95:711-719). Further characterization of the interaction of severin with actin suggests that it remains bound to the preferred assembly end of the fragmented actin filaments. Addition of severin in molar excess to actin causes total disassembly of the filaments and the formation of a high-affinity complex containing one severin and one actin. This severin -actin complex does not sever actin filaments. The binding of severin to actin, measured directly by fluorescence energy transfer, requires micromolar Ca2+, as does the severing and depolymerizing activity reported previously. Once bound to actin in the presence of greater than 1 microM Ca2+, severin is not released from the actin when the Ca2+ is lowered to less than 0.1 microM by addition of EGTA. Tropomyosin, DNase I, phalloidin, and cytochalasin B have no effect on the ability of severin to bind to or sever actin filaments. Subfragment 1 of myosin, however, significantly inhibits severin activity. Severin binds not only to actin filaments, but also directly to G-actin, as well as to other conformational species of actin.     

Structural analysis of Mg2+ and Ca2+ binding to CaBP1, a neuron-specific regulator of calcium channels

CaBP1 (calcium-binding protein 1) is a 19.4-kDa protein of the EF-hand superfamily that modulates the activity of Ca(2+) channels in the brain and retina. Here we present data from NMR, microcalorimetry, and other biophysical studies that characterize Ca(2+) binding, Mg(2+) binding, and structural properties of recombinant CaBP1 purified from Escherichia coli. Mg(2+) binds constitutively to CaBP1 at EF-1 with an apparent dissociation constant (K(d)) of 300 microm. Mg(2+) binding to CaBP1 is enthalpic (DeltaH = -3.725 kcal/mol) and promotes NMR spectral changes, indicative of a concerted Mg(2+)-induced conformational change. Ca(2+) binding to CaBP1 induces NMR spectral changes assigned to residues in EF-3 and EF-4, indicating localized Ca(2+)-induced conformational changes at these sites. Ca(2+) binds cooperatively to CaBP1 at EF-3 and EF-4 with an apparent K(d) of 2.5 microM and a Hill coefficient of 1.3. Ca(2+) binds to EF-1 with low affinity (K(d) >100 microM), and no Ca(2+) binding was detected at EF-2. In the absence of Mg(2+) and Ca(2+), CaBP1 forms a flexible molten globule-like structure. Mg(2+) and Ca(2+) induce distinct conformational changes resulting in protein dimerization and markedly increased folding stability. The unfolding temperatures are 53, 74, and 76 degrees C for apo-, Mg(2+)-bound, and Ca(2+)-bound CaBP1, respectively. Together, our results suggest that CaBP1 switches between structurally distinct Mg(2+)-bound and Ca(2+)-bound states in response to Ca(2+) signaling. Both conformational states may serve to modulate the activity of Ca(2+) channel targets.     

The two ADF-H domains of twinfilin play functionally distinct roles in interactions with actin monomers

Twinfilin is a ubiquitous and abundant actin monomer-binding protein that is composed of two ADF-H domains. To elucidate the role of twinfilin in actin dynamics, we examined the interactions of mouse twinfilin and its isolated ADF-H domains with G-actin. Wild-type twinfilin binds ADP-G-actin with higher affinity (K(D) = 0.05 microM) than ATP-G-actin (K(D) = 0.47 microM) under physiological ionic conditions and forms a relatively stable (k(off) = 1.8 s(-1)) complex with ADP-G-actin. Data from native PAGE and size exclusion chromatography coupled with light scattering suggest that twinfilin competes with ADF/cofilin for the high-affinity binding site on actin monomers, although at higher concentrations, twinfilin, cofilin, and actin may also form a ternary complex. By systematic deletion analysis, we show that the actin-binding activity is located entirely in the two ADF-H domains of twinfilin. Individually, these domains compete for the same binding site on actin, but the C-terminal ADF-H domain, which has >10-fold higher affinity for ADP-G-actin, is almost entirely responsible for the ability of twinfilin to increase the amount of monomeric actin in cosedimentation assays. Isolated ADF-H domains associate with ADP-G-actin with rapid second-order kinetics, whereas the association of wild-type twinfilin with G-actin exhibits kinetics consistent with a two-step binding process. These data suggest that the association with an actin monomer induces a first-order conformational change within the twinfilin molecule. On the basis of these results, we propose a kinetic model for the role of twinfilin in actin dynamics and its possible function in cells.     

Effect of pH on the mechanism of actin polymerization

The effect of pH on the Mg2+-induced polymerization of rabbit skeletal muscle G-actin at 20 degrees C was examined. Polymerization data were obtained at various initial concentrations of Mg2+, Ca2+, and G-actin between pH 6 and 7.5. The data were found to fit a kinetic mechanism for actin polymerization previously proposed at pH 8 in which Mg2+ binding at a moderate-affinity site on actin induces an isomerization of the protein enabling more favorable nucleation [Frieden, C. (1982) J. Biol. Chem. 257, 2882-2886]. The data also suggest the formation of actin dimers induced by Mg2+ binding is over 2 orders of magnitude more favorable at pH 6 than at pH 8. Little effect on trimer formation is found over this pH range. In addition, the conformation induced by nonspecific binding of metal to low-affinity sites becomes more favorable as the pH is lowered. The critical concentration for filament formation is also decreased at lower pH. The kinetic data do not support fragmentation occurring under any of the conditions examined. Furthermore, as Mg2+ exchange for Ca2+ at a high-affinity site (Kd less than 10(-9) M) fails to alter significantly the polymerization kinetics, Ca2+ release from this site appears unnecessary for either the nucleation or the elongation of actin filaments.     

Effect of temperature on the mechanism of actin polymerization

The rate of the Mg2+-induced polymerization of rabbit skeletal muscle G-actin has been measured as as function of temperature at pH 8 by using various concentrations of Mg2+, Ca2+, and G-actin. A polymerization mechanism similar to that proposed at this pH [Frieden, C. (1983) Proc. Natl. Acad. Sci. U.S.A. 80, 6513-6517] was found to fit the data from 10 to 35 degrees C. From the kinetic data, no evidence for actin filament fragmentation was found at any temperature. Dimer formation is the most temperature-sensitive step, with the ratio of forward and reverse rate constants changing 4 orders of magnitude from 10 to 35 degrees C. Over this temperature change, all other ratios of forward and reverse rate constants change 7-fold or less, and the critical concentration remains nearly constant. The reversible Mg2+-induced isomerization of G-actin monomer occurs to a greater extent with increasing temperature, measured either by using N-(iodoacetyl)-N'-(5-sulfo-1-naphthyl)ethylenediamine-labeled actin or by simulation of the full-time course of the polymerization reaction. This is partially due to Mg2+ binding becoming tighter, and Ca2+ binding becoming weaker, with increasing temperature. Elongation rates from the filament-pointed end, determined by using actin nucleated by plasma gelsolin, show a temperature dependence slightly larger than that expected for a diffusion-limited reaction.     

Reactions of the sarcoplasmic reticulum calcium adenosinetriphosphatase with adenosine 5'-triphosphate and Ca2+ that are not satisfactorily described by an E1-E2 model

Phosphorylation of the sarcoplasmic reticulum calcium ATPase, E, is first order with kb = 70 +/- 7 s-1 after free enzyme was mixed with saturating ATP and 50 microM Ca2+; this is one-third the rate constant of 220 s-1 for phosphorylation of enzyme preincubated with calcium, cE.Ca2, after being mixed with ATP under the same conditions (pH 7.0, Ca2+-loaded vesicles, 100 mM KCl, 5 mM Mg2+, 25 degrees C). Phosphorylation of E with ATP and Ca2+ in the presence of 0.25 mM ADP gives approximately 50% E approximately P.Ca2 with kobsd = 77 s-1, not the sum of the forward and reverse rate constants, kobsd = kf + kr = 140 s-1, that is expected for approach to equilibrium if phosphorylation were rate limiting. These results show that (1) kb represents a slow conformational change, rather than phosphoryl transfer, and (2) different pathways are followed for the phosphorylation of E and of cE.Ca2. The absence of a lag for phosphorylation of E with saturating ATP and Ca2+ indicates that all other steps, including the binding of Ca2+ ions and phosphoryl transfer, have rate constants of greater than 500 s-1. Chase experiments with unlabeled ATP or with ethylene glycol bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA) show that the rate constants for dissociation of [gamma-32P]ATP and Ca2+ are comparable to kb. Dissociation of ATP occurs at 47 s-1 from E.ATP.Ca2+ and at 24 s-1 from E.ATP. Approximately 20% phosphorylation occurs following an EGTA chase 4.5 ms after the addition of 300 microM ATP and 50 microM Ca2+ to enzyme. This shows that Ca2+ binds rapidly to the free enzyme, from outside the vesicle, before the conformational change (kb). The fraction of Ca2+-free E.[gamma-32P]ATP that is trapped to give labeled phosphoenzyme after the addition of Ca2+ and a chase of unlabeled ATP is half-maximal at 6.8 microM Ca2+, with a Hill slope of n = 1.8. The calculated dissociation constant for Ca2+ from E.ATP.Ca2 is approximately 2.2 X 10(-10) M2 (K0.5 = 15 microM). The rate constant for the slow phase of the biphasic reaction of E approximately P.Ca2 with 1.1 mM ADP increases 2.5-fold when [Ca2+] is decreased from 50 microM to 10 nM, with half-maximal increase at 1.7 microM Ca2+. This shows that Ca2+ is dissociating from a different species, aE.ATP.Ca2, that is active for catalysis of phosphoryl transfer, has a high affinity for Ca2+, and dissociates Ca2+ with k less than or equal to 45 s-1.(ABSTRACT TRUNCATED AT 400 WORDS)     

High affinity divalent cation binding to actin. Effect of low affinity salt binding

Monomeric actin labeled with the fluorescent probe N-iodoacetyl-N'-(5-sulfo-1-naphthyl)ethylenediamine (1,5-I-AEDANS-actin) displays a fast fluorescence intensity increase immediately upon addition of salt and then a slow fluorescence intensity change concurrent with Ca2+/Mg2+ exchange at the high affinity divalent cation binding site on actin. The fast change appears to reflect competitive binding of K+ at low affinity (nonspecific) sites and of Mg2+ or Ca2+ at low and intermediate affinity sites. Binding of cation at the low affinity sites (but apparently not at the intermediate affinity sites) results in an increase in k-Ca and k-Mg and thus a decrease in affinity for divalent cations at the high affinity site. The effect of Mg2+ on k-Ca is twice that of K+ for equal fractional saturations of the low affinity binding, and the effect of K+ and Mg2+ together on k-Ca reflects competitive binding at the low affinity sites. Thus the affinity and kinetics of divalent cation binding at the high affinity site of actin are significantly affected by concurrent cation binding at low affinity sites.