Biomedical subjects

S B Marston

Publications and source records attributed to S B Marston.

At least 37 records · Page 2Linked to original sources

PubMed1998

Interaction of isoforms of S100 protein with smooth muscle caldesmon.

Interaction of S100a and S100b with duck gizzard caldesmon was investigated by means of native gel electrophoresis, fluorescent spectroscopy and disulfide crosslinking. Both isoforms of S100 interact with intact caldesmon and its C-terminal deletion mutant 606C (residues 606-756) with apparent Kd of 0.2-0.6 microM thus indicating that the S100-binding site is located in the C-terminal domain of caldesmon. The single SH group of duck gizzard caldesmon can be crosslinked to Cys-84 of the beta-chain or to Cys-85 of the alpha-chain of S100. Crosslinking of S100 reduces the inhibitory action of caldesmon on actomyosin ATPase activity. S100 reverses the inhibitory action of intact caldesmon and its deletion mutants 606C (residues 606-756) and H9 (residues 669-737) as effectively as calmodulin. S100a has higher affinity to caldesmon and is more effective than S100b in reversing caldesmon-induced inhibition of actomyosin ATPase activity. Although monomeric (calmodulin, troponin C) and dimeric (S100) Ca-binding proteins have different sizes and structures they interact with caldesmon in a very similar fashion.

Actomyosin↗

PubMed1997

Location and functional characterization of myosin contact sites in smooth muscle caldesmon.

Caldesmon interaction with smooth muscle myosin and its ability to cross-link actin filaments to myosin were investigated by the use of several bacterially expressed myosin-binding fragments of caldesmon. We have confirmed the presence of two functionally different myosin-binding sites located in domains 1 and 3/4a of caldesmon. The binding of the C-terminal site is highly sensitive to ionic strength and hardly participates in acto-myosin cross-linking, while the N-terminal binding site is relatively independent of ionic strength and apparently contains two separate myosin contact regions within residues 1-28 and 29-128 of chicken gizzard caldesmon. Both these N-terminal sub-sites are involved in the interaction with myosin and are predominantly responsible for the caldesmon-mediated high-affinity cross-linking of actin and myosin filaments, without affecting the affinity of direct acto-myosin interaction. Binding of caldesmon and its fragments to myosin or rod filaments revealed affinity in the micromolar range. We determined various stoichiometries at maximal binding, which depended on the ionic strength and the concentration of Mg2+ ions. At 30 mM NaCl and 1 mM Mg2+ the maximum stoichiometry was 4 moles of caldesmon (or caldesmon fragment) per mole of myosin. At 130 mM NaCl/1 mM Mg2+, or at 30 mM NaCl/5mM Mg2+ it decreased to about two caldesmon molecules bound per myosin, while remaining 4:1 for individual caldesmon fragments, suggesting that all binding sequences on myosin were still fully capable of interaction. A further increase in the Mg2+ concentration led to a substantial decrease in both the affinity and maximum stoichiometry of caldesmon and the fragments binding to myosin. We suggest that caldesmon-myosin interaction varies according to the conformation of caldesmon in solution, that caldesmon-binding sites on myosin are not well defined and that their accessibility is determined by spatial organization and is blocked by divalent cations like Mg2+.

Actins↗

PubMed1997

3-D image reconstruction of reconstituted smooth muscle thin filaments containing calponin: visualization of interactions between F-actin and calponin.

Calponin is a putative thin filament regulatory protein of smooth muscle that inhibits actomyosin ATPase in vitro. We have used electron microscopy and three-dimensional reconstruction to elucidate the structural organization of calponin on actin and actin-tropomyosin filaments. Calponin density was clearly delineated in the reconstructions and found to occur peripherally along the long-pitch actin-helix. The main calponin mass was located over sub-domain 2 of actin, and connected axially adjacent actin monomers by binding to the "upper" and "lower" edges of sub-domains 1 of each actin. When the reconstructions were fitted to the atomic model of F-actin, calponin appeared to contact actin near the N terminus and at residues 349 to 352 close to the C terminus of sub-domain 1 on one monomer. It also touched residues 92 to 95 of sub-domain 1 on the axially neighboring actin and continued up the side of this monomer as far as residues 43 to 48 of sub-domain 2. These positions are consensus binding sites for a number of actin-associated proteins and are also near to sites of weak myosin interaction. Calponin did not appear to block strong myosin binding sites on actin. In contrast to the calponin mass which appeared monomeric in reconstructions, tropomyosin formed a continuous strand of added density along F-actin. When added to tropomyosin-containing filaments, calponin caused a shift of tropomyosin away from sub-domain 1 towards sub-domain 3 of actin, exposing strong myosin-binding sites that were previously covered by tropomyosin. This structural effect is unlike that of troponin and therefore inhibition of actomyosin ATPase by calponin and troponin cannot be strictly analogous. The location of calponin would allow it to directly compete or interact with a number of actin-binding proteins.

Actins↗

PubMed1997

Troponin I and troponin T interact with troponin C to produce different Ca2+-dependent effects on actin-tropomyosin filament motility.

We have developed an in vitro motility assay to make a detailed quantitative analysis of Ca2+ control of skeletal-muscle troponin-tropomyosin control of actin-filament movement over immobilized myosin. Ca2+ regulates both filament velocity and the fraction of filaments that are motile. We have demonstrated that the two effects are due to separate interactions of troponin C with troponin I and troponin T. When 64nM of the complex actin-tropomyosin-troponin I-troponin C was added at pCa 5, more than 80% of filaments were moving and their velocity did not change. At pCa 9, more than 20% of the filaments were moving. When 20nM of the complex actin-tropomyosin-troponin T+troponin I+troponin C was added at pCa 5, filament motility remained high, whereas velocity increased. The 30% increase in velocity observed when troponin T was present was also observed when heavy meromyosin fragment 1 labelled with N-ethylmaleimide (NEM S-1) was added after actin-tropomyosin filaments. The NEM S-1 effect was not additive with the troponin T-dependent velocity increase. The pattern of motile behaviour is characteristic of myosin on silicone-treated glass and different from the behaviour on nitrocellulose-coated glass.

Actins↗

PubMed1997

Effects of two hypertrophic cardiomyopathy mutations in alpha-tropomyosin, Asp175Asn and Glu180Gly, on Ca2+ regulation of thin filament motility.

The functional properties of wild type alpha-tropomyosin expressed in E. coli with an alanine-serine N-terminal leader (AS-alpha-Tm) were compared with those of AS-alpha-Tm with either of two missense mutations (Asp175Asn and Glu180Gly) shown to cause familial hypertrophic cardiomyopathy (FHC). Wild type AS-alpha-Tm and AS-alpha-Tm(Asp175Asn) binding to actin was indistinguishable from rabbit skeletal muscle ab-tropomyosin whilst the affinity of AS-alpha-Tm(Glu180Gly) was about threefold weaker. In vitro motility assays were performed with AS-alpha-tropomyosin incorporated into skeletal muscle actin-rhodamine phalloidin filaments moving over skeletal muscle heavy meromyosin. Under relaxing conditions (pCa9), troponin added to actin filaments containing AS-alpha-tropomyosin or mutant tropomyosins resulted in normal switch-off, with a decrease in the fraction filaments moving from >80% to <20%. Under activating conditions (pCa5), troponin had a minor effect upon actin-AS-alpha-tropomyosin filament velocity (increased by 5 +/- 1%, n=10), whereas the velocity increased by 18 +/- 3% (n=7) with actin filaments containing AS-alpha-tropomyosin(Asp175Asn) and by 21 +/- 2% (n=8) with filaments containing AS-alpha-tropomyosin(Glu180Gly) (p<0.05 compared with AS-alpha-tropomyosin). Thus FHC mutations in alpha-tropomyosin produce detectable changes in the Ca2+-regulation of thin filaments, presumably via altered interaction with troponin.

Actins↗

PubMed1997

Mapping of contact sites in the caldesmon-calmodulin complex.

The interaction of intact calmodulin and its four tryptic peptides with deletion mutants of caldesmon was analysed by native gel electrophoresis, fluorescence spectroscopy and zero-length cross-linking. Deletion mutants H2 (containing calmodulin-binding sites A and B) and H9 (containing sites B and B') interacted with intact calmodulin to form complexes whose stoichiometries varied from 2:1 to 1:1. The N-terminal peptides of calmodulin (TR1C, residues 1-77, and TR2E, residues 1-90) bound H2 with higher affinity than H9. At the same time H2 was less effective than H9 in binding to the C-terminal peptides of calmodulin TR2C (residues 78-148) and TR3E (residues 107-148). The N-terminal peptides of calmodulin (TR1C and TR2E) could be cross-linked to intact caldesmon and its deletion mutants H2 and H9. The similarity in the primary structures of sites A and B' of caldesmon and our measurements of the affinities of H2 and H9 to calmodulin and its peptides strongly indicate an orientation of the protein complex where sites A and B' interact with the N-terminal domain of calmodulin, whereas site B interacts with the C-terminal domain of calmodulin. The spatial organization of contact sites in the caldesmon-calmodulin complex agrees with the earlier proposed two-dimensional model of interaction of the two proteins [Huber, El-Mezgueldi, Grabarek, Slatter, Levine and Marston (1996) Biochem. J. 316, 413-420].

Animals↗

PubMed1997

The inhibitory complex of smooth muscle caldesmon with actin and tropomyosin involves three interacting segments of the C-terminal domain 4.

We have designed a series of recombinant peptides derived from the C-terminus of human caldesmon (amino acids 663-793, domain 4) to determine the structural basis of the multiple-sited caldesmon-actin-tropomyosin interaction. All the recombinant peptides are able to bind to actin and inhibit actin-activated myosin ATPase activity; 1 mol of peptide is bound per actin for >90% inhibition. However, equivalent inhibition of actin-tropomyosin activation of myosin ATPase requires less than one peptide per seven actin to be bound. We have found two sequences, H2 (amino acids 683-767) and H2+12 (amino acids 683-779), from the center of domain 4 which potentiate actin-tropomyosin filament activity; i.e., their effect is opposite to caldesmon. Maximum potentiation correlates with one H2 or H2+12 bound per four actin. This effect is completely dependent upon the presence of tropomyosin on the actin filament. H2 and H2+12 also increase actin-tropomyosin filament velocity in the in vitro motility assay. If the H2 sequence is extended by 20 amino acids at the N-terminal end to the N-terminus of domain 4, the peptide becomes an inhibitor. If H2 is extended by 19 amino acids at its C terminus, it becomes a tropomyosin-dependent inhibitor, and with a further extension of 7 amino acids to reach the C-terminus of human caldesmon (H2+26), inhibition is more potent. We conclude that three regions in domain 4 of caldesmon contribute to tropomyosin-dependent inhibition of actomyosin ATPase: a central segment [747-767 (690-710 in the chicken sequence)], which is essential but not sufficient for tropomyosin-dependent inhibition of actomyosin ATPase; and two actin binding segments N-terminal and C-terminal to this segment, 663-682 (606-625) and 770-793 (713-737). If only the central segment is present (H2, H2+12), the actin-tropomyosin-caldesmon peptide complex is not inhibitory, and its properties resemble actin-tropomyosin-caldesmon-Ca2+ x calmodulin.

Actins↗

PubMed1997

Three-dimensional image reconstruction of reconstituted smooth muscle thin filaments: effects of caldesmon.

Caldesmon inhibits actomyosin ATPase and filament sliding in vitro, and therefore may play a role in modulating smooth and non-muscle motile activities. A bacterially expressed caldesmon fragment, 606C, which consists of the C-terminal 150 amino acids of the intact molecule, possesses the same inhibitory properties as full-length caldesmon and was used in our structural studies to examine caldesmon function. Three-dimensional image reconstruction was carried out from electron micrographs of negatively stained, reconstituted thin filaments consisting of actin and smooth muscle tropomyosin both with and without added 606C. Helically arranged actin monomers and tropomyosin strands were observed in both cases. In the absence of 606C, tropomyosin adopted a position on the inner edge of the outer domain of actin monomers, with an apparent connection to sub-domain 1 of actin. In 606C-containing filaments that inhibited acto-HMM ATPase activity, tropomyosin was found in a different position, in association with the inner domain of actin, away from the majority of strong myosin binding sites. The effect of caldesmon on tropomyosin position therefore differs from that of troponin on skeletal muscle filaments, implying that caldesmon and troponin act by different structural mechanisms.

Actins↗

PubMed1996

The effects of smooth muscle calponin on the strong and weak myosin binding sites of F-actin.

We have investigated the mechanism of inhibition of the actomyosin MgATPase by the smooth muscle protein calponin. We have shown previously the specific interaction of calponin with Glu334 of actin (EL-Mezgueldi, M., Fattoum, A., Derancourt, J., and Kassab, R. (1992) J. Biol. Chem. 267, 15943-15951). This residue is within the sequence 332-334, which has been proposed to be an important part of the strong myosin binding site (Rayment, I., Holden, H. M., Whittaker, M., Yohn, C. B., Lorenz, M., Holmes, K. C., and Milligan, R. A. (1993) Science 261, 58-65). Therefore, we suggested that calponin will affect the strong binding actin-myosin interaction. To test this hypothesis we have investigated the effect of calponin on the strong binding of S-1.MgAMP-PNP (5'-adenylyl imidodiphosphate) and on the weak binding of S-1.MgADP.Pi to actin. We found that an inhibitory concentration of calponin decreased the binding of S-1. MgAMP-PNP to actin but had no effect on the binding of S-1.MgADP.Pi. Similar results were obtained with skeletal muscle and smooth muscle S-1. In competition experiments calponin was found to displace S-1. MgAMP-PNP and S-1.MgADP but not S-1.MgADP.Pi from the actin filament. S-1 displaced calponin from actin in the rigor state, in the presence of MgADP, and in the presence of MgAMP-PNP. We conclude that calponin inhibits the actin activated S-1 ATPase by blocking a strong S-1 binding site on actin and does not block the weak binding site.

Actins↗

PubMed1996

Multiple-sited interaction of caldesmon with Ca(2+)-calmodulin.

The binding of Ca(2+)- and Ba(2+)-calmodulin to caldesmon and its functional consequence was investigated with three different calmodulin mutants. Two calmodulin mutants have pairs of cysteine residues substituted and oxidized to a disulphide bond in either the N- or C-terminal lobe (C41/75 and C85/112). The third mutant has phenylalanine-92 replaced by alanine (F92A). Binding measurements in the presence of Ca2+ by separation on native gels and by carbodiimide-induced cross-linking showed a lower affinity for caldesmon in all the mutants. When Ca2+ was replaced by Ba2+ the affinity of calmodulin for caldesmon was further reduced. The ability of Ca(2+)-calmodulin to release caldesmon's inhibition of the actin-tropomyosin-activated myosin ATPase was virtually abolished by mutation of phenylalanine-92 to alanine or by replacing Ba2+ for Ca2+ in native calmodulin. Both cysteine mutants retained their functional ability, but the increased concentration needed for 50% release of caldesmon inhibition reflected their decreased affinity. Ca2+ -calmodulin produced a broadening in the signals of the NMR spectrum of the 10 kDa Ca(2+)-calmodulin-binding C-terminal fragment of caldesmon arising from tryptophans -749 and -779 and caused an enhancement of maximum tryptophan fluorescence of 49% and a 16 nm blue shift of the maximum. Ca(2+)-calmodulin F92A produced a change in wavelength of 4 nm but no change in maximum, whereas Ca(2+)-calmodulin C41/75 binding produced a decrease in fluorescence with no shift of the maximum. We conclude that functional binding of Ca(2+)-calmodulin to caldesmon requires multiple interaction sites on both molecules. However, some structural modification in calmodulin does not abolish the caldesmon-related functionality. This suggests that various EF hand proteins can substitute for the calmodulin molecule.

Adenosine Triphosphatases↗

PubMed1996

A simple method for automatic tracking of actin filaments in the motility assay.

A great deal of quantitative information about the actomyosin interaction can be obtained from the basic Kron and Spudich in vitro motility assay provided that care is taken to obtain consistency between experiments and that the data is examined comprehensively and not selectively. From observations of filament movement under a wide variety of conditions we have formulated the hypothesis that a large number of filaments moving over a short time period is indistinguishable from fewer filaments moving over a longer sequence of frames. This has been used to devise a simple automation of filament detection procedures. A sequence of images is digitized through a frame-grabber. If successive pairs of frames are compared the program will search for and detect the new position of every filament and show its vector on screen. Velocity is calculated and shown as a frequency histogram. The program regularly detects over 100 filaments moving in each pair of frames; usually a sequence of up to 15 pairs of frames are studied yielding 500-1000 vectors in total. The algorithm cannot deal with filaments that meet, cross or divide, however, when filaments are moving less than 2 microns between frames this is only a small proportion of the whole. The program outputs fraction of filaments motile, mean velocity with standard deviation and density of filaments (filaments microns-2). A cumulative frequency histogram gives an immediate visual indication of the performance of the population of filaments. Direct comparisons show that the data produced by automatic tracking is indistinguishable from manual tracking apart from the small apparent velocity of non-mobile filaments. The detection process takes about 5 min and requires little skill or judgement. This can lead to great increases in the rate of data analysis in motility work.

Actin Cytoskeleton↗

PubMed1996

Affinity and structure of complexes of tropomyosin and caldesmon domains.

The interaction of caldesmon domains with tropomyosin has been studied using x-ray crystallography and an optical biosensor. Only whole caldesmon and the carboxyl-terminal domain of caldesmon (CaD-4, chicken gizzard residues 597-756) bound to tropomyosin with greater than millimolar affinity at 100 and 150 microM salt. Under these conditions the affinities of whole caldesmon and CaD-4 were both in the micromolar range. Data from the x-ray studies showed that whole caldesmon bound to tropomyosin in several places, with the region of tightest interaction being at tropomyosin residues 70-100 and/or 230-260. Studies with CaD-4 revealed that this region corresponded to the strong binding site seen with whole caldesmon. Weaker association of other regions of caldesmon to tropomyosin residues 180-210 and 5-50 was also observed. The results suggest that the carboxyl-terminus of caldesmon binds tightly to tropomyosin and that other regions of caldesmon may interact with tropomyosin tightly only when they are held close to tropomyosin by the carboxyl-terminal domain. Four models are presented to show the possible interactions of caldesmon with tropomyosin.

Animals↗

PubMed1995

Location of smooth-muscle myosin and tropomyosin binding sites in the C-terminal 288 residues of human caldesmon.

We have produced nine recombinant fragments, H1 to H9, from a human cDNA that codes for the C-terminal 288 residues of caldesmon. The fragment H1, encompassing the 288 residues, is equivalent to domains 3 and 4 of caldesmon (amino acids 506-793 in human, 476-737 in the chicken gizzard sequence). It has been shown [Huber, Redwood, Avent, Tanner and Marston (1993) J. Muscle Res. Cell Motil. 14, 385-391] to bind to actin, Ca(2+)-calmodulin, tropomyosin and myosin. The fragments, H2 to H9, differ in length between 60 and 176 residues and cover the whole of domains 3 and 4 with many of the fragments overlapping. We have characterized the myosin and tropomyosin binding of these fragments. The binding of both tropomyosin and myosin is highly dependent on salt concentration, indicating the ionic nature of these interactions. The location of the myosin binding is an extended region encompassing the junction of domains 3/4 and domain 4a (residues 622-714, human; 566-657, chicken gizzard). Tropomyosin binds in a smaller region within domain 4a of caldesmon (residues 663-714, human; 606-657 chicken gizzard). We confirmed predictions based on sequence similarities of a tropomyosin binding site in domain 3 of caldesmon; however, this site bound to skeletal-muscle tropomyosin and had little affinity for the smooth-muscle tropomyosin isoform. None of the protein fragments H2-H9 retained the affinity of the parent fragment H1 for either myosin or tropomyosin. This indicates the need for several interaction sites scattered over an extended region to attain higher affinity. The regions interacting with caldesmon in both tropomyosin and myosin are coiled-coil structures. This is probably the reason for their shared interaction sites on caldesmon and their similar natures of binding.

Animals↗

PubMed1995

In vitro motility analysis of smooth muscle caldesmon control of actin-tropomyosin filament movement.

We have used the in vitro motility assay to investigate the effect of caldesmon on the movement of actin-tropomyosin filaments over thiophosphorylated smooth muscle myosin and skeletal muscle heavy meromyosin. Using either motor, incorporation of up to 8 nM caldesmon inhibited filament movement by decreasing the proportion of filaments motile from > 85% to < 30%. There was a minimal effect on filament attachment and a modest decrease in motile filament velocity in this concentration range. The reduction in the proportion of filaments motile could be completely reversed by incorporation of an excess of calmodulin at pCa 4.5. The expressed C-terminal fragment, 606C, which retains caldesmon's inhibitory capacity but does not bind to myosin, decreased the proportion of filaments motile but had no effect on velocity. We conclude that the velocity reduction by whole caldesmon is due to actin-myosin cross-linking. A significant decrease in filament attachment was observed when caldesmon was added to an excess over actin (> 10 nM). In the absence of tropomyosin, addition of an excess of caldesmon caused a similar decrease in the filament density, but there was no effect on the proportion of filaments that were motile. Our results demonstrate that caldesmon can switch actin-tropomyosin from motile to non-motile states without controlling velocity of movement or weak binding affinity and show the inhibitory action of caldesmon in the motility assay to be functionally indistinguishable from that reported for troponin.

Actin Cytoskeleton↗

PubMed1995

In vitro motility analysis of actin-tropomyosin regulation by troponin and calcium. The thin filament is switched as a single cooperative unit.

In striated muscles, contractility is controlled by Ca2+ binding to the regulatory protein complex troponin, which is a component of the thin filaments. Troponin is an allosteric inhibitor acting on tropomyosin to switch the thin filament between "on" and "off" states. We have used an in vitro motility assay to examine troponin regulation of individual actin-tropomyosin filaments moving over immobilized skeletal muscle heavy meromyosin. The most striking observation is that the actintropomyosin filament appears to be regulated as a single unit. At pCa 9.0, addition of up to 4 nM troponin causes the proportion of filaments motile to decrease from > 85% to 20% with no dissociation of the filaments from the heavy meromyosin surface or change in velocity. Increasing Ca2+ concentration causes the filaments to be switched back on with half-maximal increase in the proportion of filaments motile at pCa 5.8-6.0 and a modest increase in filament velocity. This is an "all or none" process in which an entire filament, up to 15 microns long, switches rapidly as a single cooperative unit. Thus, the effect of Ca2+ upon the thin filament is to recruit motile filaments.

Actins↗

PubMed1995

The binding of distinct segments of actin to multiple sites in the C-terminus of caldesmon: comparative aspects of actin interaction with troponin-I and caldesmon.

Thin-filament-based regulation of the contractile response is considered to involve the interaction of actin with troponin-I in striated muscle and the interaction of actin with caldesmon in smooth muscle. The nature of the interaction with actin of these inhibitory proteins has been studied by proton magnetic resonance spectroscopy using segments of caldesmon and troponin-I which mimic their functional properties. Caldesmon is shown to interact with two distinct sites on the N-terminal residues 1-44 of actin subdomain 1 with corresponding contacts on caldesmon domain 3 and domain 4 at its C-terminus. We demonstrate that, whereas inhibition by the troponin-I fragment (residues 96-117) is effected by its interaction with the N-terminal region of actin, the separate inhibitory ability of different regions of the C-terminus of caldesmon (domains 4a and 4b) is mediated by interaction with noncontiguous segments on subdomain 1 of actin. Our studies of the spatial relationship of these actin contacts on caldesmon further suggest that one molecule of caldesmon may associate with two actin monomers. The demonstrated interactive nature of these caldesmon attachments to distinct regions of actin is relevant to the mechanism of calcium modulation of inhibition of actomyosin ATPase by caldesmon.

Actins↗

PubMed1995

Caldesmon mRNA splicing and isoform expression in mammalian smooth-muscle and non-muscle tissues.

The recent determination of the genomic sequence of human caldesmon indicates that eight caldesmon mRNA species could be generated by selection of exon 1 or 1', exon 3a or 3ab and/or exon 4. We used reverse transcriptase PCR to determine which transcripts were produced in human, rabbit and sheep artery, vein, lung, intestine, kidney and liver. In all tissues the same three transcripts were present: exons 1'-2-3a-5-6...13, exons 1'-2-3a3b-5-6-...13 and exons 1'-2-3a3b-4-5-6...13. Exon 1 was not present and exon 4 was only present when exon 3b was also present. Three protein isoforms of caldesmon can be distinguished by electrophoresis on high-porosity 6% polyacrylamide gel: 130 kDa, 120 kDa and 70 kDa. The 70 kDa isoform lacks the sequence encoded by exon 3b. We investigated whether the two high-molecular-mass isoforms correspond to the presence and absence of exon 4 using an antiserum specific to the sequence encoded by exon 4. Western-blotting and immunoprecipitation experiments showed that both the 130 kDa and the 120 kDa isoforms were expressed with and without the exon 4 sequence. We therefore propose that the molecular-mass heterogeneity arises from additional first exons, possibly with separate promoter regions, which have not yet been characterized in the genomic sequence.

Animals↗

PubMed1994

Smooth muscle caldesmon controls the strong binding interaction between actin-tropomyosin and myosin.

We have demonstrated that caldesmon does not alter the affinity of weak binding actomyosin complexes when it inhibits actin-tropomyosin activation at physiological ratios (1 per 14 actins), and we proposed that it acts upon the strong binding complexes in the same way that troponin-tropomyosin does. We therefore compared the effect of caldesmon, caldesmon fragments, and troponin upon the interaction of the strongly bound complexes S-1.ADP, S-1.adenylyl imidodiphosphate (AMP.PNP), and N-ethylmaleimide-treated myosin subfragment-1 (NEM-S-1) with actin-tropomyosin. In 0.17 M ionic strength buffer [14C]iodoacetamide-labeled S1.ADP bound to actin-smooth muscle tropomyosin with no evidence of cooperativity; Kd = 0.8 +/- 0.3 microM (n = 5). Inhibitory concentrations of sheep aorta caldesmon or rabbit skeletal muscle troponin made the binding highly cooperative. At low levels of saturation the apparent Kd was 10-40 microM with 10 microM caldesmon and 8-20 microM with 6 microM troponin; at > 50% saturation the binding was indistinguishable from actin-tropomyosin alone. A similar result was obtained for the binding of [14C]iodoacetamide-labeled S-1.AMP.PNP to actin-smooth muscle tropomyosin at 0.03 M ionic strength (Kd = 0.47 +/- 0.05 microM). Binding was slightly cooperative and became highly cooperative in the presence of inhibitory concentrations of troponin, caldesmon, and the human caldesmon fragments H7 (amino acids 622-767) and H9 (amino acids 726-793). We conclude that caldesmon and troponin both act as allosteric effectors of the "on"/"off" equilibrium of actin-tropomyosin. 0.1 NEM-S-1/actin potentiated actin-smooth muscle tropomyosin activation of myosin MgATPase 7-fold at 0.03 M ionic strength. Caldesmon inhibited the ATPase in the presence and absence of 0.5 microM NEM-S-1. NEM-S-1 reactivated actin-tropomyosin, which had been inhibited by troponin, caldesmon, H7, or H9. This is compatible with opposing effects of NEM-S-1 and caldesmon or troponin upon the actin-tropomyosin on/off equilibrium.

Actins↗