Synthesis and biological activity of isopentenyl diphosphate analogues

A series of analogues of isopentenyl diphosphate (IPP) having a dicarboxylate moiety in place of the diphosphate were synthesized and investigated as inhibitors of undecaprenyl diphosphate (UPP) synthase and protein farnesyltransferase (PFTase). PFTase is involved in control of cell proliferation and is known to be inhibited by certain maleic acid derivatives bearing long alkyl substituents (> or =12 carbons, e.g., chaetomellic acid). UPP synthase is a potential target for antimicrobial agents and utilizes isopentenyl diphosphate (IPP) as a substrate. A number of dicarboxylate-containing IPP analogues were prepared in 2-5 steps from commercially available starting materials with good overall yield (20-78%). These syntheses involved the conjugate addition of an organocuprate to dimethyl acetylenedicarboxylate (DMAD) followed by basic ester hydrolysis. The E-pentenylbutanedioic acid 32 showed inhibition of UPP synthase with an IC(50) of 135 microM. Compound 30 displays competitive inhibition of PFTase with a K(i) of 287 microM.     

Photolytic release of MgADP reduces rigor force in smooth muscle

Photolytic release of MgADP (25-300 microM) from caged ADP in permeabilized tonic (rabbit femoral artery-Rfa) and phasic (rabbit bladder-Rbl) smooth muscle in high-tension rigor state, in the absence of Ca(2+), caused an exponential decline (approximately 1.5% in Rfa and approximately 6% in Rbl) of rigor force, with the rate proportional to the liberated [MgADP]. The apparent second-order rate constant of MgADP binding was estimated as approximately 1.0 x 10(6) M(-1) s(-1) for both smooth muscles. In control experiments, designed to test the specificity of MgADP, photolysis of caged ADP in the absence of Mg(2+) did not decrease rigor force in either smooth muscle, but rigor force decreased after photolytic release of Mg(2+) in the presence of ADP. The effects of photolysis of caged ADP were similar in smooth muscles containing thiophosphorylated or non-phosphorylated regulatory myosin light chains. Stretching or releasing (within range of 0.1-1.2% of initial Ca(2+)-activated force) did not affect the rate or relative amplitude of the force decrease. The effect of additions of MgADP to rigor cross-bridges could result from rotation of the lever arm of smooth muscle myosin, but this need not imply that ADP-release is a significant force-producing step of the physiological cross-bridge cycle.     

Inhibition of actin-activated myosin Mg(2+)-ATPase in smooth muscle by ruthenium red.

Ruthenium red was found to inhibit actin-activated myosin Mg(2+)-ATPase in smooth muscle and to bind to myosin heavy chain, but not to F-actin. The inhibition by Ruthenium red of actin-activated Mg(2+)-ATPase was of the competitive type with respect to actin (Ki 4.4 microM) and of the non-competitive type with respect to ATP (Ki 6.6 microM). However, Ruthenium red scarcely dissociated the acto-heavy meromyosin complex during the ATPase reaction. These results suggest that Ruthenium red interacts directly with the binding site for F-actin on the myosin heavy chain. This site is considered to be necessary not for maintaining the binding affinity of myosin for F-actin, but for activation of the Mg(2+)-ATPase.     

Phosphorylation regulates the ADP-induced rotation of the light chain domain of smooth muscle myosin.

We have observed the effects of MgADP and thiophosphorylation on the conformational state of the light chain domain of myosin in skinned smooth muscle. Electron paramagnetic resonance (EPR) spectroscopy was used to monitor the orientation of spin probes attached to the myosin regulatory light chain (RLC). Two spectral states were seen, termed here "intermediate" and "final", that are distinguished by a approximately 24 degrees axial rotation of spin probes attached to the RLC. The two observed conformations are similar to those found previously for smooth muscle myosin S1; the final state corresponds to the major conformation of S1 in the absence of ADP, while the intermediate state corresponds to the conformation of S1 with ADP bound. Light chain domain orientation was observed as a function of the MgADP concentration and the extent of RLC thiophosphorylation. In rigor (no MgADP), LC domains were distributed equally between the intermediate state and the final state; upon addition of saturating (3.5 mM) MgADP, about one-third of the LC domains in the final state rotated approximately 20 degrees axially to the intermediate state. The progression of the change in populations was fit to a simple binding equation, yielding an apparent dissociation constant of approximately 110 microM for skinned smooth muscle fibers and approximately 730 microM for thiophosphorylated, skinned smooth muscle fibers. These observations suggest a model that explains the behavior of "latch bridges" in smooth muscle.     

Didehydrofarnesyl diphosphate: an intrinsically fluorescent inhibitor of protein farnesyltransferase

Didehydrofarnesyl diphosphate (delta delta FPP), a fluorescent pentaene analogue of farnesyl diphosphate (FPP), was synthesized using stereoselective Wittig reactions. Although delta delta FPP was not an alternative substrate for yeast protein farnesyltransferase (FTase), the fluorescent analogue was a potent competitive inhibitor with a K(i) value of 8.8 microM (K (m) (FPP) = 27 microM).     

Calmodulin-linked equilibria in smooth muscle myosin light chain kinase

Competition experiments using 9-anthroylcholine, a fluorescent dye that undergoes calmodulin-dependent binding by smooth muscle myosin light chain kinase [Malencik, D. A., Anderson, S. R., Bohnert, J. L., & Shalitin, Y. S. (1982) Biochemistry 21, 4031], demonstrate a strongly stabilizing interaction between the adenosine 5'-triphosphate and myosin light chain binding sites operating within the enzyme-calmodulin complex but probably not in the free enzyme. The interactions in the latter case may be even slightly destabilizing. The fluorescence enhancement in solutions containing 5.0 microM each of the enzyme and calmodulin is directly proportional to the maximum possible concentration of bound calcium on the basis of four calcium binding sites. Evidently, all four calcium binding sites of calmodulin contribute about equally to the enhanced binding of 9-anthroylcholine by the enzyme. Fluorescence titrations on solutions containing 1.0 microM enzyme plus calmodulin yield a Hill coefficient of 1.2 and K = 0.35 +/- 0.08 microM calcium. Three proteolytic fragments of smooth muscle myosin light chain kinase, apparent products of endogenous proteolysis, were isolated and characterized. All three possess calmodulin-dependent catalytic activity. Their interactions with 9-anthroylcholine, in both the presence and absence of calmodulin, are similar to those of the native enzyme. However, the stabilities of their complexes with calmodulin vary. The corresponding dissociation constants range from 2.8 nM for the native enzyme and 8.5 nM for the 96K fragment to approximately 15 nM for the 68K and 90K fragments [0.20 N KCl, 50 mM 3-(N-morpholino)propanesulfonic acid, and 1 mM CaCl2, pH 7.3, 25 degrees C]. A coupled fluorometric assay, modified from a spectrophotometric assay for adenosine cyclic 3',5'-phosphate dependent protein kinase [Cook, P. F., Neville, M. E., Vrana, K. E., Hartl, F. T., & Roskoski, R. (1982) Biochemistry 21, 5794], has provided the first continuous recordings of myosin light chain kinase phosphotransferase activity. The results show that smooth muscle myosin light chain kinase is a responsive enzyme, whose activity adjusts rapidly to changes in solution conditions.     

Hormonal interactions with benzodiazepine binding sites in vitro

Prostaglandin A1 and hormones like corticosteroids and DL-Thyroxin (T4) inhibit binding of [3H]RO 5-4864 and [3H] Clonazepam to their respective binding sites with inhibition constants in the low micromolar range. The corticosteroid Cortisone inhibits [3H] RO 5-4864, but not [3H] Clonazepam binding in a competitive manner with an inhibition constant of 4.3 +/- 0.7 microM, Prostaglandin A1 inhibits [3H] Clonazepam, but not [3H] RO 5-4864 binding in a competitive manner with an inhibition constant of 6 +/- 1.2 microM and DL-Thyroxin (T4) inhibits both [3H] RO 5-4864 and [3H] Clonazepam binding with inhibition constants of 12.1 +/- 2.2 and 1.6 +/- 0.4 microM respectively. While the inhibition of [3H] RO 5-4864 binding by DL-Thyroxin (T4) is competitive, the inhibition of [3H] Clonazepam binding is of the mixed type as indicated by Scatchard Plot.     

Electrostatic contributions to the binding of myosin and myosin-MgADP to F-actin in solution

The ionic strength dependence of skeletal myosin subfragment 1 (S1) binding to unregulated F-actin was measured in solutions containing from 0 to 0.50 M added lithium acetate (LiOAc) in the absence and presence of MgADP. The data were analyzed by using a theory based on an ion interaction model that is rigorous for high ionic strength solutions [Pitzer, K. S. (1973) J. Phys. Chem. 77, 268-277] in order to obtain values for K, the equilibrium association constant when the ionic strength is zero, and for [zMzA[, the absolute value of the product of the net electric charges of the actin binding site on myosin (zM) and the myosin binding site on actin (zA). The presence of MgADP reduced K by a factor of 10, as expected, and reduced [zMzA[ by about 1 esu2. Because the presence of MgADP is not likely to change the net charge of the myosin binding site on actin, these data are consistent with a model in which MgADP binding to S1 reduces its affinity for actin by a mechanism that reduces the net electric charge of the acting binding site on S1. The value of [zMzA[ in the absence of ADP was 8.1 +/- 0.9 esu2, which, if one uses integer values, suggests that zM and zA are in the 8+ to 1+ esu and 1- to 8- esu ranges, respectively. ADP binding then reduces zM to the 7+ to 0.88+ esu range.     

Some motile properties of fast characean myosin

We improved a motility assay system by using an affinity-purified antibody against the C-terminal globular domain of characean myosin. This improvement allowed us to study the sensitivity to ionic strength or the processivity of characean myosin. The sliding velocity of actin filaments on a characean myosin-coated surface was unaffected by ionic strength. This property is unlike that of skeletal or smooth muscle myosin and suggests that the binding manner of characean myosin to actin is different from that in other muscle myosins. The sliding velocity decreased when the MgADP concentration was raised. The extent of inhibition by MgADP on the motile activity of characean myosin was almost the same as in skeletal muscle or cardiac myosin. The number of sliding filaments on the characean myosin-coated surface decreased drastically with a decrease in the motor density. The motor density required to produce a successful movement of actin filament was about 200 molecules/microm(2). These results suggest that the characean myosin is not a processive motor protein.     

The calmodulin binding domain of chicken smooth muscle myosin light chain kinase contains a pseudosubstrate sequence

Smooth muscle myosin light chain kinase contains a 64 residue sequence that binds calmodulin in a Ca2+-dependent manner (Guerriero, V., Jr., Russo, M. A., and Means, A. R. (1987) Biochemistry, in press). Within this region is a sequence with homology to the corresponding sequence reported for the calmodulin binding region of skeletal muscle myosin light chain kinase (Blumenthal, D. K., Takio, K., Edelman, A. M., Charbonneau, H., Titani, L., Walsh, K. A., and Krebs, E. G. (1985) Proc. Natl. Acad. Sci. U.S.A. 82, 3187-3191). Inspection of these sequences reveals that they both share a similar number and spatial arrangement of basic residues with those present in the myosin light chain substrate. We have synthesized a 22-residue peptide corresponding to residues 480-501 (determined from the cDNA) of the smooth muscle myosin light chain kinase. This peptide, Ala-Lys-Lys-Leu-Ser-Lys-Asp-Arg-Met-Lys-Lys-Tyr-Met-Ala-Arg-Arg-Lys-Trp- Gln-Lys-Thr-Gly, inhibited calmodulin-dependent activation of the smooth muscle myosin light chain kinase with an IC50 of 46 nM. At saturating concentrations of calmodulin, the 22-residue peptide inhibited myosin light chain and synthetic peptide substrate phosphorylation competitively with IC50 values of 2.7 and 0.9 microM, respectively. An 11-residue synthetic peptide analog, corresponding to part of the calmodulin-binding sequence in skeletal muscle myosin light chain kinase, Lys-Arg-Arg-Trp-Lys-Lys-Asn-Phe-Ile-Ala-Val, also competitively inhibited synthetic peptide substrate phosphorylation with a Ki of 1 microM. The competitive inhibitory activity of the calmodulin binding regions is similar to the apparent Km of 2.7 microM for phosphorylation of the 23-residue peptide analog of the smooth muscle myosin light chain and raises the possibility that the calmodulin binding region of the myosin light chain kinase may act as a pseudosubstrate inhibitor of the enzyme.     

Anomalous binding of MgADP to myosin of skeletal muscle

Binding of adenosine diphosphate to skeletal muscle myosin was studied using a range of concentrations from 0 to 2 mM. Up to 0.2 mM adenosine diphosphate two equivalent and independent nucleotide binding sites were detected, characterized by the single association constant of 5 x 10(4)M(-1). At greater adenosine diphosphate concentrations a decreasing binding capacity was noticed, bound nucleotide being essentially approximately 0.1 mol/mol at a 1-2mM adenosine diphosphate concentration. We tentatively propose that nucleotides act indirectly on myosin by promoting the perturbation of the solvent, which is supported by the fact that polyphosphates are known powerful kosmotropes.     

Cross-linking of actin to myosin subfragment 1 in the presence of nucleotides

Chemical cross-linking of actin to the 20K and 50K fragments of tryptically cleaved myosin subfragment 1 (S-1) by the zero-length cross-linking reagent 1-ethyl-3-[3-dimethylamino)propyl]carbodiimide (EDC) was used as a probe of the acto-S-1 interface in the presence of nucleotides. The course of the two reactions was monitored by measuring on sodium dodecyl sulfate (SDS)-polyacrylamide gels the time-dependent formation of the 20K-actin and 50K-actin cross-linked products. Both reactions were inhibited somewhat in the presence of MgADP, were slowed 3-4-fold in the presence of magnesium 5'-adenylyl imidodiphosphate (MgAMPPNP), and proceeded at least 7-fold slower with N,N'-p-phenylenedimaleimide (pPDM) modified S-1, as compared to the respective rates in the absence of nucleotides. However, neither the binding of the nucleotides MgADP and MgAMPPNP to S-1 nor the modification of S-1 by pPDM significantly changed the ratio of the cross-linking rates of actin to the 20K and 50K fragments. Similar to what was previously observed in the absence of nucleotides [Chen, T., Applegate, D., & Reisler, E. (1985) Biochemistry 24, 137-144], actin was cross-linked at an approximately 3-fold faster rate to the 20K fragment than to the 50K fragment under all reaction conditions tested. Thus, irrespective of the extent of acto-S-1 dissociation or the binding of nucleotides to acto-S-1, the 20K fragment remains the preferred cross-linking site for actin. These results show that the interaction of actin with each of the cross-linking sites on S-1 is not under selective or preferential control by nucleotides.     

Rabbit skeletal muscle myosin light chain kinase. The calmodulin binding domain as a potential active site-directed inhibitory domain

A synthetic peptide modeled after the calmodulin (CaM)-binding domain of rabbit skeletal muscle myosin light chain kinase, Lys-Arg-Arg-Trp-Lys5-Lys-Asn-Phe-Ile-Ala10-Val-Ser-Ala-Ala-+ ++Asn15-Arg-Phe-Glycyl amide (M5), inhibited the CaM-independent chymotryptic fragment of the enzyme, C35 (Edelman, A. M., Takio, K., Blumenthal, D. K., Hansen, R. S., Walsh, K. A., Titani, K., and Krebs, E. G. (1985) J. Biol. Chem. 260, 11275-11285), with a Ki of 3.2 +/- 2.1 microM. Inhibition was competitive with respect to the peptide substrate Lys-Lys-Arg-Ala-Ala5-Arg-Ala-Thr-Ser-Asn10-Val-Phe-Ala and was of the noncompetitive linear mixed type with respect to ATP. M5 and homologues with a serine residue substituted at positions 9, 13, or 14 were phosphorylated with the following order of preference: M5(Ser9) greater than M5(Ser13) much greater than M5(Ser14) greater than M5. The order of preference observed agreed with that predicted by comparison of the sequence of these peptides with the phosphorylation sites of myosin P-light chains. Both inhibition of C35 by M5 and phosphorylation of M5 and its serine-substituted homologues were severely curtailed by the addition of a stoichiometric excess of CaM over peptide. Thus, synthetic peptides modeled after the CaM-binding domain of skeletal muscle myosin light chain kinase can function as calmodulin-regulated active site-directed inhibitors of the enzyme.     

Specificity and orientation of (iodoacetamido)proxyl spin-labeled myosin subfragment 1 decorating muscle fibers: localization of protein-bound spin labels using SDS-PAGE

The nitroxide spin label (iodoacetamido)proxyl (IPSL) was specifically and rigidly attached to sulfhydryl 1 (SH1) on myosin subfragment 1 (S1). The specificity of this label for SH1 was demonstrated by using a technique where the spin label is localized on the electrophoresis-isolated proteolytic fragments of myosin using electron paramagnetic resonance (EPR). Studies of the rigidity of the probe on SH1 indicate that the IPSL is immobilized on the surface of S1 in the presence and absence of the nucleotides MgADP or MgATP. The EPR spectrum of muscle fibers decorated with IPSL-S1 shows that the IPSL-S1 rotates from its orientation in rigor upon binding MgADP. The angular displacement due to nucleotide binding is larger than that detected with the (maleimido)tempo spin label [Ajtai, K., French, A. R., & Burghardt, T. P. (1989) Biophys. J. 56, 535-541], demonstrating that the IPSL is oriented on the myosin cross-bridge in a manner that is favorable for detecting cross-bridge rotation during the rigor to MgADP state transition.     

C-terminal sites of caldesmon drive ATP hydrolysis cycle by shifting actomyosin itermediates from strong to weak binding of myosin and actin

Polarized fluorimetry technique and ghost muscle fibers containing tropomyosin were used to study effects of caldesmon (CaD) and recombinant peptides CaDH1 (residues 506-793), CaDH2 (residues 683-767), CaDH12 (residues 506-708) and 658C (residues 658-793) on the orientation and mobility of fluorescent label 1.5-IAEDANS specifically bound to Cys-707 of myosin subfragment-1 (S1) in the absence of nucleotide, and in the presence of MgADP, MgAMP-PNP, MgATPgammaS or MgATP. It was shown that at modelling different intermediates of actomyosin ATPase, the orientation and mobility of dye dipoles changed discretely, suggesting a multi-step changing of the myosin head structural state in ATP hydrolysis cycle. The maximum difference in orientation and mobility of the oscillator (4 degrees and 30%, respectively) was observed between actomyosin in the presence of MgATP, and actomyosin in the presence of MgADP. Caldesmon actin-binding sites C and B' inhibit formation of actomyosin strong binding states, while site B activates it. It is suggested that actin-myosin interaction in ATP hydrolysis cycle initiates nucleotide-dependent rotation of myosin motor domain, or that of its site for dye binding as well as the change in myosin head mobility. Caldesmon drives ATP hydrolysis cycle by shifting the equilibrium between strong and weak forms of actin-myosin binding.     

Determination of the affinity of each component of a composite quaternary transition-state analogue complex of creatine kinase

Recombinant rabbit muscle creatine kinase (CK) was titrated with MgADP in 50 mM Bicine and 5 mM Mg(OAc)2, pH 8.3, at 30.0 degrees C by following a decrease in the protein's intrinsic fluorescence. In the presence of 50 mM NaOAc, but in the absence of added creatine or nitrate, MgADP has an apparent K(d) of 135 +/- 7 microM, and the total change in fluorescence on saturation (Delta%F) is 15.3 +/- 0.3%. Acetate was used as the anion in this experiment because it does not promote the formation of a CK.MgADP.anion.creatine transition-state analogue complex (TSAC) [Millner-White and Watts (1971) Biochem. J. 122, 727-740]. In the presence of 80 mM creatine, but no nitrate, the apparent K(d) for MgADP remains essentially unchanged at 132 +/- 10 microM, while Delta%F decreases slightly to 13.2 +/- 0.3%. In the presence of 10 mM nitrate, but no creatine, the apparent K(d) is once again essentially unchanged at 143 +/- 23 microM, but the Delta%F is markedly reduced to 4.2 +/- 0.2%. The presence of both 10 mM nitrate and 80 mM creatine during titration reduces the apparent K(d) for MgADP 10-fold to 13.7 +/- 0.7 microM, and Delta%F increases to 20.6 +/- 0.3%, strongly suggesting that the simultaneous presence of saturating levels of creatine and nitrate increases the affinity of CK for MgADP and promotes the formation of the enzyme*MgADP*nitrate*creatine TSAC. When the fluorescence of CK was titrated with MgADP in the presence of 80 mM creatine and fixed saturating concentrations of various anions, apparent K(d) values for MgADP of 132 +/- 10 microM, 25.2 +/- 1.3 microM, 18.8 +/- 0.9 microM, 13.7 +/- 0.7 microM, and 6.4 +/- 0.7 microM were observed as the anion was changed from acetate to formate to chloride to nitrate to nitrite, respectively. This is the same trend reported by Millner-White and Watts for the effectiveness of various monovalent anions in forming the CK.MgADP.anion.creatine TSAC. On titration of CK with MgADP in the presence of 80 mM creatine and various fixed concentrations of NaNO3, the apparent K(d) for MgADP decreases with increasing fixed concentrations of nitrate. A plot of the apparent K(d) for MgADP vs [NO3-] suggests a K(d) for nitrate from the TSAC of 0.39 +/- 0.07 mM. Similarly, titration with MgADP in the presence of 10 mM NaNO3 and various fixed concentrations of creatine gives a value of 0.9 +/- 0.4 mM for the dissociation of creatine from the TSAC. The data were used to calculate K(TDAC), the dissociation constant of the quaternary TSAC into its individual components, of 3 x 10(-10) M3. To our knowledge this is the first reported dissociation constant for a ternary or quaternary TSAC.     

Inhibition of nucleoside diphosphate kinase in rat liver mitochondria by added 3'-azido-3'-deoxythymidine

The effect of 3'-azido-3'-deoxythymidine on nucleoside diphosphate kinase of isolated rat liver mitochondria has been studied. This is done by monitoring the increase in the rate of oxygen uptake by nucleoside diphosphate (TDP, UDP, CDP or GDP) addition to mitochondria in state 4. It is shown that 3'-azido-3'-deoxythymidine inhibits the mitochondrial nucleoside diphosphate kinase in a competitive manner, with a Ki value of about 10 microM as measured for each tested nucleoside diphosphate. It is also shown that high concentrations of GDP prevent 3'-azido-3'-deoxythymidine inhibition of the nucleoside diphosphate kinase.     

Probing cross-bridge angular transitions using multiple extrinsic reporter groups.

15N- and 2H-substituted maleimido-TEMPO spin label ([15N,2H]MTSL) and the fluorescent label 1,5-IAEDANS were used to specifically modify sulfhydryl 1 of myosin to study the orientation of myosin cross-bridges in skeletal muscle fibers. The electron paramagnetic resonance (EPR) spectrum from muscle fibers decorated with labeled myosin subfragment 1 ([15N,2H]MTSL-S1) or the fluorescence polarization spectrum from fibers directly labeled with 1,5-IAEDANS was measured from fibers in various physiological conditions. The EPR spectra from fibers with the fiber axis oriented at 90 degrees to the Zeeman field show a clear spectral shift from the rigor spectrum when the myosin cross-bridge binds MgADP. This shift is attributable to a change in the torsion angle of the spin probe from cross-bridge rotation and is observable due mainly to the improved angular resolution of the substituted probe. The EPR data from [15N,2H]MTSL-S1 decorating fibers are combined with the fluorescence polarization data from the 1,5-IAEDANS-labeled fibers to map the global angular transition of the labeled cross-bridges due to nucleotide binding by an analytical method described in the accompanying paper [Burghardt, T. P., & Ajtai, K. (1992) Biochemistry (preceding paper in this issue)]. We find that the spin and fluorescent probes are quantitatively consistent in the finding that the actin-bound cross-bridge rotates through a large angle upon binding MgADP. We also find that, if the shape of the cross-bridge is described as an ellipsoid with two equivalent minor axes, then cross-bridge rotation takes place mainly about an axis parallel to the major axis of the ellipsoid. This type of rotation may imitate the rotation motion of cross-bridges during force generation.     

Characterization of the Ca2+ coordination site regulating binding of Ca2+ channel inhibitors d-cis-diltiazem, (+/-)bepridil and (-)desmethoxyverapamil to their receptor site in skeletal muscle transverse tubule membranes

Ca2+ inhibits (-)[3H]desmethoxyverapamil, d-cis-[3H]diltiazem and (+/-)[3H]bepridil binding to skeletal muscle transverse-tubule membranes with a half-maximum inhibition constant, K0.5 = 5 +/- 1 microM. This value is close to that of the high affinity Ca2+ binding site which controls the ionic selectivity of the Ca2+ channel found in electrophysiological experiments suggesting that the Ca2+ coordination site which regulates the ionic selectivity is also the one which alters binding of the Ca2+ channel inhibitors investigated here. Ca2+ and (-)D888 bind to distinct sites. Occupation of the Ca2+ coordination site decreases the affinity of (-)D888 for its receptor by a factor of 5. Other divalent cations have the same type of inhibition behavior with the rank order of potency Ca2+ (K0.5 = 5 microM) greater than Sr2+ (K0.5 = 25 microM) greater than Ba2+ (K0.5 = 50 microM) greater than Mg2+ (K0.5 = 170 microM).     

Affinity for MgADP and force of unbinding from actin of myosin purified from tonic and phasic smooth muscle

Smooth muscle is unique in its ability to maintain force at low MgATP consumption. This property, called the latch state, is more prominent in tonic than phasic smooth muscle. Studies performed at the muscle strip level have suggested that myosin from tonic muscle has a greater affinity for MgADP and therefore remains attached to actin longer than myosin from phasic muscle, allowing for cross-bridge dephosphorylation and latch-bridge formation. An alternative hypothesis is that after dephosphorylation, myosin reattaches to actin and maintains force. We investigated these fundamental properties of smooth muscle at the molecular level. We used an in vitro motility assay to measure actin filament velocity (nu(max)) when propelled by myosin purified from phasic or tonic muscle at increasing [MgADP]. Myosin was 25% thiophosphorylated and 75% unphosphorylated to approximate in vivo conditions. The slope of nu(max) versus [MgADP] was significantly greater for tonic (-0.51 +/- 0.04) than phasic muscle myosin (-0.15 +/- 0.04), demonstrating the greater MgADP affinity of myosin from tonic muscle. We then used a laser trap assay to measure the unbinding force from actin of populations of unphosphorylated tonic and phasic muscle myosin. Both myosin types attached to actin, and their unbinding force (0.092 +/- 0.022 pN for phasic muscle and 0.084 +/- 0.017 pN for tonic muscle) was not statistically different. We conclude that the greater affinity for MgADP of tonic muscle myosin and the reattachment of dephosphorylated myosin to actin may both contribute to the latch state.