FRINK Linked Data Fragments server

Matches in SemOpenAlex for { <https://semopenalex.org/work/W1995214557> ?p ?o ?g. }

• W1995214557 endingPage "56" @default.
• W1995214557 startingPage "47" @default.
• W1995214557 abstract "Smooth muscle thin filaments are made up of actin, tropomyosin, caldesmon, and a Ca2+-binding protein and their interaction with myosin is Ca2+-regulated. We suggested that Ca2+ regulation by caldesmon and Ca2+-calmodulin is achieved by controlling the state of thin filament through a cooperative-allosteric mechanism homologous to troponin-tropomyosin in striated muscles. In the present work, we have tested this hypothesis. We monitored directly the thin filament transition between the ON and OFF state using the excimer fluorescence of pyrene iodoacetamide (PIA)-labeled smooth muscle αα-tropomyosin homodimers. In steady state fluorescence measurements, myosin subfragment 1 (S1) cooperatively switches the thin filaments to the ON state, and this is exhibited as an increase in the excimer fluorescence. In contrast, caldesmon decreases the excimer fluorescence, indicating a switch of the thin filament to the OFF state. Addition of Ca2+-calmodulin increases the excimer fluorescence, indicating a switch of the thin filament to the ON state. The excimer fluorescence was also used to monitor the kinetics of the ON-OFF transition in a stopped-flow apparatus. When ATP induces S1 dissociation from actin-PIA-tropomyosin, the transition to the OFF state is delayed until all S1 molecules are dissociated actin. In contrast, caldesmon switches the thin filament to the OFF state in a cooperative way, and no lag is displayed in the time course of the caldesmon-induced fluorescence decrease. We have also studied caldesmon and Ca2+-calmodulin-caldesmon binding to actin-tropomyosin in the ON and OFF states. The results are used to discuss both caldesmon inhibition and Ca2+-calmodulin-caldesmon activation of actin-tropomyosin. Smooth muscle thin filaments are made up of actin, tropomyosin, caldesmon, and a Ca2+-binding protein and their interaction with myosin is Ca2+-regulated. We suggested that Ca2+ regulation by caldesmon and Ca2+-calmodulin is achieved by controlling the state of thin filament through a cooperative-allosteric mechanism homologous to troponin-tropomyosin in striated muscles. In the present work, we have tested this hypothesis. We monitored directly the thin filament transition between the ON and OFF state using the excimer fluorescence of pyrene iodoacetamide (PIA)-labeled smooth muscle αα-tropomyosin homodimers. In steady state fluorescence measurements, myosin subfragment 1 (S1) cooperatively switches the thin filaments to the ON state, and this is exhibited as an increase in the excimer fluorescence. In contrast, caldesmon decreases the excimer fluorescence, indicating a switch of the thin filament to the OFF state. Addition of Ca2+-calmodulin increases the excimer fluorescence, indicating a switch of the thin filament to the ON state. The excimer fluorescence was also used to monitor the kinetics of the ON-OFF transition in a stopped-flow apparatus. When ATP induces S1 dissociation from actin-PIA-tropomyosin, the transition to the OFF state is delayed until all S1 molecules are dissociated actin. In contrast, caldesmon switches the thin filament to the OFF state in a cooperative way, and no lag is displayed in the time course of the caldesmon-induced fluorescence decrease. We have also studied caldesmon and Ca2+-calmodulin-caldesmon binding to actin-tropomyosin in the ON and OFF states. The results are used to discuss both caldesmon inhibition and Ca2+-calmodulin-caldesmon activation of actin-tropomyosin. The contractile system of smooth muscles is based on actomyosin interaction like all muscles; however, it is adapted for the maintenance of sustained isometric force and slow contraction. Vertebrate smooth muscle is a dual regulated muscle: activation of myosin by phosphorylation of light chains by myosin light chain kinase and its dephosphorylation by a phosphatase is the prime regulator of smooth muscle contractility. However it is well established that the activity of the thin filaments toward myosin is independently regulated by Ca2+ because native thin filaments isolated from smooth muscles confer a Ca2+-dependent regulation on unregulated myosin from skeletal or smooth muscle (1Marston S.B. Smith C.W.J. J. Musc. Res. Cell Motil. 1985; 6: 669-708Crossref PubMed Scopus (122) Google Scholar, 2Marston S.B. Smith C.W.J. J. Musc. Res. Cell Motil. 1984; 5: 559-575Crossref PubMed Scopus (67) Google Scholar, 3Marston S.B. Biochem. J. 1986; 237: 605-607Crossref PubMed Scopus (12) Google Scholar). Smooth muscle thin filaments are made up of actin, tropomyosin, caldesmon, and a Ca2+-binding protein (CaBP) 2The abbreviations used are:CaBPCa2+-binding proteinPIA-tropomyosinpyrene iodoacetamide-labeled tropomyosinNEM-S1N-ethylmaleimide-treated S1CaMcalmodulinCaDcaldesmonKTequilibrium constant [ON]/[OFF]MDCCN-[2-(1-maleimidyl)ethyl]-7-(diethylamino)coumarin-3-carboxamidePIPES1,4-piperazinediethanesulfonic acidDTTdithiothreitol with a stoichiometry of 1 caldesmon and CaBP per two tropomyosin and fourteen actin monomers in native thin filaments; synthetic Ca2+-regulated thin filaments may be reconstituted from these components at similar ratios (4Marston S.B. Biochem. J. 1990; 272: 305-310Crossref PubMed Scopus (26) Google Scholar, 5Smith C.W. Pritchard K. Marston S.B. J. Biol. Chem. 1987; 262: 116-122Abstract Full Text PDF PubMed Google Scholar). There is substantial evidence that caldesmon-based regulation is involved in modulating smooth muscle Ca2+ sensitivity and relaxation (6Earley J.J. Su X. Moreland R.S. Circ. Res. 1998; 83: 661-667Crossref PubMed Scopus (71) Google Scholar, 7Malmqvist U. Arner A. Makuch R. Dabrowska R. Pflugers Archiv. 1996; 432: 241-247Crossref PubMed Scopus (33) Google Scholar, 8Burton D.J. Marston S.B. Pflugers Archiv. 1999; 437: 267-275Crossref PubMed Scopus (11) Google Scholar). Ca2+-binding protein pyrene iodoacetamide-labeled tropomyosin N-ethylmaleimide-treated S1 calmodulin caldesmon equilibrium constant [ON]/[OFF] N-[2-(1-maleimidyl)ethyl]-7-(diethylamino)coumarin-3-carboxamide 1,4-piperazinediethanesulfonic acid dithiothreitol Like skeletal muscle thin filaments, the thin filaments of smooth muscles are negatively regulated: the function of caldesmon binding to actin-tropomyosin is to inhibit the activity of a constitutively active filament and the function of Ca2+ binding to the CaBP is to reverse the inhibition. Caldesmon inhibition is cooperative with up to 14 actin monomers being inhibited by the binding of one caldesmon molecule to actin-tropomyosin; moreover, Ca2+ and CaBP (or calmodulin) interacting with caldesmon activate thin filaments to up to 150% of the activity of actin-tropomyosin rather than simply neutralizing the inhibitory effect of caldesmon (5Smith C.W. Pritchard K. Marston S.B. J. Biol. Chem. 1987; 262: 116-122Abstract Full Text PDF PubMed Google Scholar, 9Pritchard K. Marston S.B. Biochem. J. 1989; 257: 839-843Crossref PubMed Scopus (35) Google Scholar, 10Notarianni G. Gusev N.B. Lafitte D. Hill T.J. Cooper H.S. Derrick P.J. Marston S.B. J. Musc. Res. Cell Motil. 2000; 21: 537-549Crossref PubMed Scopus (14) Google Scholar). There has been considerable debate about the mechanism of smooth muscle thin filament regulation. We have consistently argued that the only model of regulation that can account for all the regulatory characteristics is a cooperative allosteric mechanism analogous to troponin-tropomyosin in striated muscle thin filaments (10Notarianni G. Gusev N.B. Lafitte D. Hill T.J. Cooper H.S. Derrick P.J. Marston S.B. J. Musc. Res. Cell Motil. 2000; 21: 537-549Crossref PubMed Scopus (14) Google Scholar, 11Marston S.B. Redwood C.S. J. Biol. Chem. 1993; 268: 12317-12320Abstract Full Text PDF PubMed Google Scholar, 12Marston S.B. Redwood C.S. J. Biol. Chem. 1992; 267: 16796-16800Abstract Full Text PDF PubMed Google Scholar, 13Marston S.B. Fraser I.D.C. Huber P.A.J. J. Biol. Chem. 1994; 269: 32104-32109Abstract Full Text PDF PubMed Google Scholar, 14Fraser I.D.C. Marston S.B. J. Biol. Chem. 1995; 270: 19688-19693Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar, 15Marston S. Burton D. Copeland O. Fraser I. Gao Y. Hodgkinson J. Huber P. Levine B. EL-Mezgueldi M. Notarianni G. Acta Physiol. Scand. 1998; 164: 401-414Crossref PubMed Scopus (57) Google Scholar). Alternative models have been proposed that include a role for mutually exclusive competitive binding of caldesmon or myosin heads to actin-tropomyosin in determining thin filament interaction with myosin. The original model of Sobue et al. (16Sobue K. Morimoto K. Inui M. Kanda K. Kakiuchi S. Biomed. Res. 1982; 3: 188-196Crossref Scopus (122) Google Scholar) proposed a purely competitive “flip-flop” mechanism, which was ruled out by measurements showing that caldesmon and S1·ADP·Pi could bind simultaneously to actin-tropomyosin (12Marston S.B. Redwood C.S. J. Biol. Chem. 1992; 267: 16796-16800Abstract Full Text PDF PubMed Google Scholar). However, several studies appeared to show a relationship between caldesmon inhibition and S1·ADP·Pi displacement (17Hemric M.E. Chalovich J.M. J. Biol. Chem. 1988; 263: 1878-1885Abstract Full Text PDF PubMed Google Scholar, 18Velaz L. Hemric M.E. Benson C.E. Chalovich J.M. J. Biol. Chem. 1989; 264: 9602-9610Abstract Full Text PDF PubMed Google Scholar, 19Chalovich J.M. Hemric M.E. Velaz L. Ann. N. Y. Acad. Sci. 1990; 599: 85-99Crossref PubMed Scopus (16) Google Scholar, 20Velaz L. Ingraham R.H. Chalovich J.M. J. Biol. Chem. 1990; 265: 2929-2934Abstract Full Text PDF PubMed Google Scholar, 21Fredricksen S. Cai A. Gafurov B. Resetar A. Chalovich J.M. Biochemistry. 2003; 42: 6136-6148Crossref PubMed Scopus (13) Google Scholar, 22Chalovich J.M. Sen A. Resetar A. Leinweber B. Fredericksen R.S. Lu F. Chen Y.-D. Acta Physiol. Scand. 1998; 164: 427-435Crossref PubMed Scopus (46) Google Scholar). We have criticized such experiments for using unphysiologically high concentrations of caldesmon relative to actin. Displacement models cannot account for the activating property of Ca2+-CaBP and Ca2+-calmodulin and are ruled out by the consistent observation that both caldesmon and calmodulin or CaBP remain bound to actin-tropomyosin under activating conditions (9Pritchard K. Marston S.B. Biochem. J. 1989; 257: 839-843Crossref PubMed Scopus (35) Google Scholar, 10Notarianni G. Gusev N.B. Lafitte D. Hill T.J. Cooper H.S. Derrick P.J. Marston S.B. J. Musc. Res. Cell Motil. 2000; 21: 537-549Crossref PubMed Scopus (14) Google Scholar, 23Smith C.W.J. Marston S.B. FEBS Lett. 1985; 184: 115-119Crossref PubMed Scopus (43) Google Scholar). Nevertheless, it remains possible that smooth muscle thin filament regulation involves mixed cooperative and competitive regulation or even an entirely novel process (24Chen Y. Chalovich J.M. Biophys. J. 1992; 63: 1063-1070Abstract Full Text PDF PubMed Scopus (16) Google Scholar). The critical test for any regulatory scheme is that it produces experimentally testable predictions that can rule out other models. The concerted allosteric-cooperative transition model proposes two states of actin-tropomyosin termed ON and OFF. Myosin weak binding complex (M·ADP·Pi) affinity is the same for both states but strong binding complexes can only be formed with thin filaments in the ON state. Crossbridge cycling and force production is thus only possible when filaments are in the ON state, therefore the activity of the thin filament depends on the proportion of actin-tropomyosin in the ON state. Caldesmon is proposed to act as an allosteric inhibitor by binding preferentially to the OFF state while Ca2+-CaBP-caldesmon activates thin filaments by binding preferentially to the ON state. This mechanism is fundamentally the same as the mechanism for troponin regulation of actin-tropomyosin filaments in striated muscles (25Hill T.L. Eisenberg E. Greene L.E. Proc. Natl. Acad. Sci. U. S. A. 1980; 77: 3186-3190Crossref PubMed Scopus (229) Google Scholar, 26McKillop D.F.A. Geeves M.A. Biochem. J. 1991; 279: 711-718Crossref PubMed Scopus (46) Google Scholar, 27Geeves M.A. Halsall D.J. Biophys. J. 1987; 52: 215-220Abstract Full Text PDF PubMed Scopus (34) Google Scholar). We have previously demonstrated caldesmon inhibition with no change in the binding of myosin subfragment 1 (S1) weak binding complexes (S1·ADP·Pi) and the cooperative binding of strong-binding complexes (S1·ADP and S1·AMP·PNP) to caldesmon-inhibited actin-tropomyosin (12Marston S.B. Redwood C.S. J. Biol. Chem. 1992; 267: 16796-16800Abstract Full Text PDF PubMed Google Scholar, 13Marston S.B. Fraser I.D.C. Huber P.A.J. J. Biol. Chem. 1994; 269: 32104-32109Abstract Full Text PDF PubMed Google Scholar). These findings are compatible with the proposed mechanism. More recently we have investigated thoroughly the effect of caldesmon on the elementary steps of the actomyosin ATPase (28Alahyan M. Webb M.R. Marston S.B. El-Mezgueldi M. J. Biol. Chem. 2006; 281: 19433-19448Abstract Full Text Full Text PDF PubMed Scopus (18) Google Scholar). We found that caldesmon had very little effect on the rate of S1 binding to actin-tropomyosin, acto-S1 dissociation by ATP and the rate of ADP release. The rate of phosphate release was drastically reduced. We suggested that caldesmon inhibition of phosphate release is caused by the thin filament being switched to an inactive (OFF) state. In this report we have tested the hypothesis that smooth muscle thin filaments change state in both inhibitory and activating conditions. To do this we have used the excimer fluorescence of pyrene iodoacetamide-labeled smooth muscle tropomyosin as a probe of the thin filament ON-OFF transition and studied caldesmon and Ca2+-calmodulin-caldesmon binding to actin-tropomyosin and have related this to the control of thin filament activity as measured by actin activation of myosin S1 Mg2+-ATPase. The results support our hypothesis both for caldesmon inhibition of actin-tropomyosin and for Ca2+-calmodulin-caldesmon activation of actin-tropomyosin and also permit us to determine values of the size of the cooperative unit and the rates of transition and equilibrium constants between the ON and OFF states. Proteins—Previously described methods were used to prepare rabbit skeletal muscle actin (29Straub F.B. Studies from the Institute of Medical Chemistry, University of Szeged. 1942; 2: 3-16Google Scholar), rabbit skeletal muscle myosin subfragment-1 (S1) (30Margossian S.S. Lowey S. Methods Enzymol. 1982; 85: 55-71Crossref PubMed Scopus (825) Google Scholar), sheep aorta, and chicken gizzard tropomyosin (5Smith C.W. Pritchard K. Marston S.B. J. Biol. Chem. 1987; 262: 116-122Abstract Full Text PDF PubMed Google Scholar), bovine brain calmodulin (31Gopalakrishna R. Anderson W.B. Biochem. Biophys. Res. Commun. 1982; 104: 830-836Crossref PubMed Scopus (718) Google Scholar), chicken gizzard caldesmon, and sheep aorta caldesmon (32Ansari S.N. EL-Mezgueldi M. Marston S.B. J. Musc. Res. Cell Motil. 2003; 24: 513-520Crossref PubMed Scopus (7) Google Scholar). For experiments using low actin concentrations (below 4 μm) actin was stabilized with phalloidin and used within 20 min of actin sample preparation (28Alahyan M. Webb M.R. Marston S.B. El-Mezgueldi M. J. Biol. Chem. 2006; 281: 19433-19448Abstract Full Text Full Text PDF PubMed Scopus (18) Google Scholar). S1 was treated with N-ethyl maleimide as described by Swartz and Moss (33Swartz D.R. Moss R.L. J. Biol. Chem. 1992; 267: 20497-20506Abstract Full Text PDF PubMed Google Scholar), and sheep aorta caldesmon was labeled with [14C]iodoacetamide as described by Smith et al. (5Smith C.W. Pritchard K. Marston S.B. J. Biol. Chem. 1987; 262: 116-122Abstract Full Text PDF PubMed Google Scholar). Because of the inability of native smooth muscle αβ-tropomyosin heterodimers to display excimer fluorescence, smooth muscle αα-tropomyosin homodimers were purified using chromatofocusing on a mono-P 5/20 column in an FPLC chromatography system as described (34Horiuchi K.Y. Wang Z. Chacko S. Biochemistry. 1995; 34: 16815-16820Crossref PubMed Scopus (15) Google Scholar, 35Sanders C. Burtnick L.D. Smillie L.B. J. Biol. Chem. 1986; 261: 12774-12778Abstract Full Text PDF PubMed Google Scholar). Skeletal muscle αβ-tropomyosin heterodimers and smooth muscle αα-tropomyosin homodimers were labeled at cysteine with N-(1-pyrenyl)iodoacetamide (Molecular Probes) (referred to in the text as PIA) as described by Lehrer et al. (36Ischii Y. Lehrer S.S. Biochemistry. 1987; 26: 4922-4925Crossref PubMed Scopus (27) Google Scholar). The purified proteins were dialyzed against 5 mm PIPES, 120 mm KCl, 2.5 mm MgCl2, 1 mm NaN3, 1 mm DTT, pH 7.1 (ATPase buffer) and stored at 4 °C. Protein concentrations were determined by the Lowry method. Pyrene iodoacetamide concentration was measured by absorbance at 344 nm (ϵ344 = 45.5 μm PIA/1 OD). Labeling ratio PIA/tropomyosin was 1.7–2.1 for smooth muscle tropomyosin homodimers (corresponding to 95% labeling of the 2 cysteines present in each homodimer) and 2.4–2.8 for skeletal muscle tropomyosin (86% labeling of the 3 cysteines present in each skeletal muscle heterodimer). Steady State ATPase and Binding Measurements—Actin-tropomyosin activation of S1 Mg2+-ATPase was assayed as described previously (32Ansari S.N. EL-Mezgueldi M. Marston S.B. J. Musc. Res. Cell Motil. 2003; 24: 513-520Crossref PubMed Scopus (7) Google Scholar). Phosphate liberated following ATP hydrolysis was measured by the method of Taussky and Schorr (38Taussky H.H. Schorr E. J. Biol. Chem. 1953; 202: 675-685Abstract Full Text PDF PubMed Google Scholar). [14C]Caldesmon binding to actin-tropomyosin was measured by co-sedimentation as previously described (5Smith C.W. Pritchard K. Marston S.B. J. Biol. Chem. 1987; 262: 116-122Abstract Full Text PDF PubMed Google Scholar). Steady State Fluorescence Titrations—Steady state fluorescence measurements were obtained with a Fluoromax-2 photon counting fluorimeter in the ratio mode. Titrations were carried out with excitation at 343 nm and emission at 350 nm to monitor light scattering and 485 nm to monitor the excimer fluorescence of PIA-tropomyosin (37Mirza M. Robinson P. Kremneva E. Copeland O. Nikolaeva O. Watkins H. Levitsky D. Redwood C. El-Mezgueldi M. Marston S. J. Biol. Chem. 2007; 282: 13487-13497Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar). Before titrations, all proteins were clarified by centrifugation in a refrigerated bench top centrifuge for 10 min at 25,000 × g. All buffers used were filtered before use. The temperature was maintained at 25 °C by a circulating water bath. Stopped-flow Experiments—All transient kinetic measurements were performed on a Hi-Tech Scientific SF-61 double mixing stopped-flow system using a 100 watt Xe/Hg lamp and a monochromator for excitation wavelength selection as previously described (28Alahyan M. Webb M.R. Marston S.B. El-Mezgueldi M. J. Biol. Chem. 2006; 281: 19433-19448Abstract Full Text Full Text PDF PubMed Scopus (18) Google Scholar). Pyrene iodoacetamide fluorescence was excited at 364 nm and emission of excimer fluorescence was monitored through a 455 nm cut-off filter. Light scattering was observed at 90o to the incident beam using a UG-5 filter (light over 400 nm was cut off). The measurements were carried out in ATPase buffer at 20 °C unless otherwise stated (28Alahyan M. Webb M.R. Marston S.B. El-Mezgueldi M. J. Biol. Chem. 2006; 281: 19433-19448Abstract Full Text Full Text PDF PubMed Scopus (18) Google Scholar). The temperature of the stopped-flow machine was maintained within 0.1 °C during the course of the experiment. All buffers were filtered and all proteins were clarified by centrifugation in a refrigerated bench top centrifuge for 10 min at 25,000 × g just before use. Usually four to nine transients were collected and averaged. The data were then fitted to one or two exponentials by a non-linear least square curve fit using the software provided by Hi-Tech. The stated concentrations of reactants are those after mixing in the stopped-flow observation cell. Inhibition of Actin-Tropomyosin by Caldesmon and Activation by Caldesmon-Ca2+-Calmodulin—Fig. 1 illustrates the regulatory properties of reconstituted smooth muscle thin filaments. Addition of caldesmon to actin-smooth muscle tropomyosin resulted in inhibition to 6.6 ± 3.6% of the actin-tropomyosin-activated ATPase activity at low stoichiometry (Fig. 1A). 80% inhibition of ATPase was obtained with 0.074 ± 0.16 caldesmon added per actin (six preparations) in agreement with previous measurements (5Smith C.W. Pritchard K. Marston S.B. J. Biol. Chem. 1987; 262: 116-122Abstract Full Text PDF PubMed Google Scholar, 11Marston S.B. Redwood C.S. J. Biol. Chem. 1993; 268: 12317-12320Abstract Full Text PDF PubMed Google Scholar, 12Marston S.B. Redwood C.S. J. Biol. Chem. 1992; 267: 16796-16800Abstract Full Text PDF PubMed Google Scholar). Chicken gizzard and sheep aorta caldesmon were indistinguishable in this assay, and the inhibition was independent of temperature (20–37 °C) and KCl concentration. In suitable conditions (120 mm KCl, 37 °C), 10–20 μm Ca2+-calmodulin (Ca2+-CaM)-activated actin-tropomyosin, which had been inhibited by caldesmon. ATPase activity with saturating quantities of Ca2+-calmodulin was activated to 115 ± 2% of the uninhibited actin-tropomyosin ATPase (six preparations) (Fig. 1B). This level of activation is similar to that previously observed for Ca2+-calmodulin (9Pritchard K. Marston S.B. Biochem. J. 1989; 257: 839-843Crossref PubMed Scopus (35) Google Scholar, 39Marston S. Lehman W. Moody C. Smith C. Adv. Prot. Phosphatases. 1985; 2: 171-189Google Scholar). Under comparable conditions NEM-S1, which switches thin filaments to the ON state, activated actin-tropomyosin to 200% of actin-tropomyosin ATPase at a ratio of 0.15 NEM-S1:1 actin-tropomyosin (Fig. 1C). Detection of the ON/OFF Equilibrium by PIA-Tropomyosin Excimer Fluorescence Changes—We monitored the equilibrium between the ON and OFF states of actin-tropomyosin by the excimer fluorescence technique of Ischii and Lehrer (36Ischii Y. Lehrer S.S. Biochemistry. 1987; 26: 4922-4925Crossref PubMed Scopus (27) Google Scholar). Smooth muscle αα-tropomyosin dimers were covalently labeled with pyrene iodoacetamide at 1.9 ± 0.08 mol/mol (number of experiments = 10). Fig. 2A shows fluorescence emission spectra of reconstituted actin-tropomyosin. Monomer fluorescence peaks are at 385 and 410 nm and excimer fluorescence is at 480 nm. When S1 is added to actin to turn the filaments ON, the excimer fluorescence is increased, and when S1 is displaced from actin by adding Mg2+-ATP, the excimer fluorescence returns to the original level. The average increase in excimer fluorescence induced by S1 using smooth muscle PIA-αα-tropomyosin was 55.6 ± 1.5% (number of experiments = 10). Titration of actin-tropomyosin with increasing concentrations of S1 shows that the switch between states is cooperative: pyrene excimer fluorescence increased to a plateau at much lower S1 concentrations than S1 binding monitored by light scatter (Fig. 2B). The fraction of the thin filaments in the ON state, fon determined from the excimer fluorescence, was plotted against Fb, the fraction of S1 bound to the thin filament calculated from the light scattering signal. Fitting the data to the equation fon = 1 – (1 – Fb)n generates the cooperative unit size, n (Fig. 2C) as described by Geeves and Lehrer (68Geeves M.A. Lehrer S.S. Biophys. J. 1994; 67: 273-282Abstract Full Text PDF PubMed Scopus (174) Google Scholar). A mean value for n of 10 ± 1 was obtained from three separate experiments. Cooperativity of the OFF/ON transition of actin-tropomyosin can also be determined from the transient kinetics of S1 binding to actin-tropomyosin (Fig. 3). When S1 is in excess over actin, the excimer fluorescence transient is faster than the light scattering supporting the assumption that the 2 signals monitor 2 different processes. Increasing S1 concentration lead to a hyperbolic increase of the 2 observed rate constants (kobs fluorescence and kobs light scattering) indicating that each of the 2 signals is monitoring a 2-step reaction. The first step correspond to S1 binding to the thin filaments, while the second step correspond to different processes. In the case of light scattering (monitoring S1 binding), the second step corresponds to an isomerization to a strongly bound acto-S1 complex. In the absence of any added nucleotide, this usually plateaus around 250 s–1 (28Alahyan M. Webb M.R. Marston S.B. El-Mezgueldi M. J. Biol. Chem. 2006; 281: 19433-19448Abstract Full Text Full Text PDF PubMed Scopus (18) Google Scholar). In the case of the excimer fluorescence (monitoring the thin filament switching between the OFF and ON states), the second step correspond to the transition from the OFF to the ON state. The hyperbolic fit gave a maximum rate of 833 s–1 for the OFF to ON transition albeit, it is anticipated that this value contains a large error because the experimental data cover less than half of the entire curve. Nevertheless we can put a lower limit of 350 s–1 for the maximum rate of the OFF to ON transition (The highest experimentally determined value, Fig. 3B). The slopes in the initial part of the 2 curves depend on the rate of S1 binding. It was previously modeled that the ratio of the 2 slopes (kobs fluorescence/kobs light scattering) corresponds to the size of the cooperative unit n (68Geeves M.A. Lehrer S.S. Biophys. J. 1994; 67: 273-282Abstract Full Text PDF PubMed Scopus (174) Google Scholar). Using this kinetic technique n was found to be 10, which is the same as determined from the steady state titrations. Caldesmon and Ca2+-Calmodulin Switch Actin-Tropomyosin between the OFF and ON States—We used the change in pyrene excimer fluorescence to determine whether caldesmon would switch actin-tropomyosin to the OFF state. Caldesmon reduced pyrene excimer fluorescence by 19% and the quantity needed to fully switch the thin filament OFF, corresponded to the quantity needed to inhibit actin-tropomyosin activation of Mg2+-ATPase activity (Fig. 4A). The excimer fluorescence reached 80 ± 5% of the minimum level with 1:7 caldesmon added per actin. We confirmed that caldesmon switches actin-tropomyosin completely to the OFF state by comparison with skeletal muscle troponin in the absence of Ca2+: this induced the same decrease of excimer fluorescence of smooth muscle tropomyosin and subsequent addition of caldesmon had no effect on the excimer fluorescence (data not shown). We determined the effect of Ca2+-calmodulin upon actin-PIA-tropomyosin excimer fluorescence at 10 μm caldesmon where the inhibition of ATPase and decrease of excimer fluorescence was at a maximum (Fig. 4B). Calmodulin in the presence of activating Ca2+ concentrations switched the filaments toward the ON state. The maximum fluorescence was 30% greater than that of actin-tropomyosin (KT = 0.9 compared with 0.6 for actin-tropomyosin) and the concentration of Ca2+-calmodulin needed to give maximal increase in excimer fluorescence was in the same range as reversal of inhibition (Fig. 1). Addition of S1 to saturating actin-tropomyosin-caldesmon increased the fluorescence back up to the level of the fully ON filament (Fig. 5A). The transition was cooperative with an estimated cooperative unit size in the range 3–5. The further addition of caldesmon to actin-tropomyosin-S1 gave a decrease in fluorescence, which was only partial in the concentration range studied, where caldesmon affinity for actin is less than the S1 affinity (Fig. 5B). Because we have measured the fluorescence of the fully OFF state (the fluorescence plateau in the presence of caldesmon) and the fully ON state (the fluorescence plateau in the presence of S1) we can calculate the equilibrium constant KT for actin-tropomyosin. For smooth muscle αα-tropomyosin under our conditions KT was 0.60 ± 0.05 (number of experiments = 3). KT could be increased by increasing temperature or KCl concentrations. Kinetics of the ON-OFF Transition—We induced the transition from the ON to the OFF state using either ATP to dissociate pre-mixed actin-tropomyosin-S1 complex or caldesmon added to actin-tropomyosin. Fig. 6A shows light scattering and excimer fluorescence changes following acto-S1 dissociation by 40 μm ATP. The decrease in light scattering monitoring S1 dissociation from actin-tropomyosin is fast (130 s–1) and without delay. In contrast the excimer fluorescence transient representing the ON to OFF transition showed a lag (displayed as an upward curvature) before an exponential decrease (rate 67 s–1). The transition to the OFF state is delayed until all S1 molecules are dissociated from any single cooperative unit. If caldesmon is rapidly mixed with actin-tropomyosin, the excimer fluorescence decreased exponentially (Fig. 6B). The transient was best fit by a sum of 2 exponentials. The faster component represented about 75% of the total signal. Because the fluorescence change is very rapid (>400 s–1) and because of the relatively poor signal to noise ratio, there is a large error in the fitted rate constants; however, this rate is comparable with the value of >350 s–1 calculated for the transition from the OFF to the ON state (see section above for Fig. 3B). In contrast to the ATP induced acto-S1 dissociation, no lag is displayed in the time course of the caldesmon induced fluorescence decrease (Fig. 6B). These results suggest that each caldesmon binding to one actin switches the whole cooperative unit to the OFF state. Caldesmon Binding to the ON and OFF States of Actin-Tropomyosin—If actin-tropomyosin is fixed in either the ON or OFF state then predictions follow from the cooperative allosteric model that can uniquely distinguish it from other models. If actin-tropomyosin is in the ON state then caldesmon binding should be strongly inhibited and would become cooperative, conversely fixing actin-tropomyosin in the OFF state" @default.
• W1995214557 created "2016-06-24" @default.
• W1995214557 creator A5020794080 @default.
• W1995214557 creator A5056020498 @default.
• W1995214557 creator A5087260365 @default.
• W1995214557 creator A5090645838 @default.
• W1995214557 date "2008-01-01" @default.
• W1995214557 modified "2023-10-05" @default.
• W1995214557 title "Role of Caldesmon in the Ca2+ Regulation of Smooth Muscle Thin Filaments" @default.
• W1995214557 cites W100430351 @default.
• W1995214557 cites W14251027 @default.
• W1995214557 cites W1490442168 @default.
• W1995214557 cites W1493031501 @default.
• W1995214557 cites W1496193127 @default.
• W1995214557 cites W1496962480 @default.
• W1995214557 cites W151431147 @default.
• W1995214557 cites W1520250133 @default.
• W1995214557 cites W1528202931 @default.
• W1995214557 cites W1535139255 @default.
• W1995214557 cites W1570910952 @default.
• W1995214557 cites W1573771296 @default.
• W1995214557 cites W1578846125 @default.
• W1995214557 cites W1582325019 @default.
• W1995214557 cites W1582902635 @default.
• W1995214557 cites W1598176996 @default.
• W1995214557 cites W165633160 @default.
• W1995214557 cites W1972223624 @default.
• W1995214557 cites W1973507916 @default.
• W1995214557 cites W1974381583 @default.
• W1995214557 cites W1978364582 @default.
• W1995214557 cites W1980096676 @default.
• W1995214557 cites W1994863671 @default.
• W1995214557 cites W2004546144 @default.
• W1995214557 cites W2008847227 @default.
• W1995214557 cites W2009988419 @default.
• W1995214557 cites W2017556218 @default.
• W1995214557 cites W2018418014 @default.
• W1995214557 cites W2025623060 @default.
• W1995214557 cites W2029568121 @default.
• W1995214557 cites W2034570151 @default.
• W1995214557 cites W2036451625 @default.
• W1995214557 cites W2039817964 @default.
• W1995214557 cites W2046919025 @default.
• W1995214557 cites W2048565997 @default.
• W1995214557 cites W2050993512 @default.
• W1995214557 cites W2051206485 @default.
• W1995214557 cites W2053236545 @default.
• W1995214557 cites W2053676046 @default.
• W1995214557 cites W2054113630 @default.
• W1995214557 cites W2059232882 @default.
• W1995214557 cites W2059782931 @default.
• W1995214557 cites W2063023178 @default.
• W1995214557 cites W2068055758 @default.
• W1995214557 cites W2068063551 @default.
• W1995214557 cites W2070017733 @default.
• W1995214557 cites W2073389372 @default.
• W1995214557 cites W2081756741 @default.
• W1995214557 cites W2091060891 @default.
• W1995214557 cites W2091127975 @default.
• W1995214557 cites W2091245707 @default.
• W1995214557 cites W2092187742 @default.
• W1995214557 cites W2092991423 @default.
• W1995214557 cites W2095686193 @default.
• W1995214557 cites W2105944799 @default.
• W1995214557 cites W2139292873 @default.
• W1995214557 cites W2140114315 @default.
• W1995214557 cites W2151571057 @default.
• W1995214557 cites W2224560775 @default.
• W1995214557 cites W2240970945 @default.
• W1995214557 cites W2261150478 @default.
• W1995214557 cites W2341106386 @default.
• W1995214557 cites W2767789822 @default.
• W1995214557 doi "https://doi.org/10.1074/jbc.m706771200" @default.
• W1995214557 hasPubMedId "https://pubmed.ncbi.nlm.nih.gov/17933868" @default.
• W1995214557 hasPublicationYear "2008" @default.
• W1995214557 type Work @default.
• W1995214557 sameAs 1995214557 @default.
• W1995214557 citedByCount "19" @default.
• W1995214557 countsByYear W19952145572012 @default.
• W1995214557 countsByYear W19952145572013 @default.
• W1995214557 countsByYear W19952145572014 @default.
• W1995214557 countsByYear W19952145572020 @default.
• W1995214557 countsByYear W19952145572021 @default.
• W1995214557 crossrefType "journal-article" @default.
• W1995214557 hasAuthorship W1995214557A5020794080 @default.
• W1995214557 hasAuthorship W1995214557A5056020498 @default.
• W1995214557 hasAuthorship W1995214557A5087260365 @default.
• W1995214557 hasAuthorship W1995214557A5090645838 @default.
• W1995214557 hasBestOaLocation W19952145571 @default.
• W1995214557 hasConcept C105702510 @default.
• W1995214557 hasConcept C12554922 @default.
• W1995214557 hasConcept C125705527 @default.
• W1995214557 hasConcept C134018914 @default.
• W1995214557 hasConcept C181199279 @default.
• W1995214557 hasConcept C185592680 @default.
• W1995214557 hasConcept C2776050358 @default.
• W1995214557 hasConcept C2779360546 @default.
• W1995214557 hasConcept C29688787 @default.

• next