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D Mornet

Publications and source records attributed to D Mornet.

At least 91 records · Page 5Linked to original sources
PubMed1991

Ultrastructural localization of dystrophin in chicken smooth muscle.

We investigated the presence of dystrophin in gizzard smooth muscle by immunofluorescence assay, immunoblot detection and an immunogold electron microscopy technique. Western blot analyses, using antibodies raised against sequences 1173-1728 and 3357-3660 of the dystrophin molecule, revealed the presence of a major intact 400 kDa protein band and an immunofluorescence localization restricted to the periphery of the smooth muscle cells. We were able to precisely determine the dystrophin distribution along the plasmalemma whereas caldesmon molecules were present in the cytoplasm. The most commonly observed distance between two neighbouring dystrophin molecules suggested a self-associating arrangement. We discuss these findings in relation to the function of dystrophin in the smooth muscle cell structure.

Animals↗
PubMed1991

Turkey gizzard caldesmon: complete sequence of a C-terminal thrombic fragment that binds actin, tropomyosin and calmodulin.

We have determined, by protein chemistry methods, the complete amino acid sequence of a thrombic fragment, "CaD35", which contains the C-terminal 274 residues of turkey gizzard caldesmon. Residues 1-96 of CaD35 comprise an actin-binding subfragment which resembles the tropomyosin-binding segment of troponin T. Residues 111-128 and 255-272 may form basic, amphipathic helices that interact with calmodulin. Residues 251-253 (Ser-Ser-Ser) may be phosphorylated. Chymotryptic CaD35, obtained from chicken gizzard caldesmon, nearly identical in sequence to the turkey gizzard fragment, but contains an additional 32 amino acid residues at its N terminus.

Actins↗
PubMed1990

Structural study of gizzard caldesmon and its interaction with actin. Binding involves residues of actin also recognised by myosin subfragment 1.

The interaction between actin and caldesmon that is associated with the inhibition of actomyosin ATPase activity in smooth muscle has been studied using 1H-NMR spectroscopy. Binding studies using the intact molecules were complemented by the use of thrombic cleavage fragments of both turkey and chicken gizzard caldesmon as well as defined peptides of actin, in order to investigate the conformational properties of caldesmon and to localise regions of the primary structures that participate in protein-protein contacts. The binding of caldesmon is shown to involve distinct segments on the N-terminal region (residues 1-44) of actin, as previously observed for the inhibitory component of the thin filament of striated muscle, troponin I [Levine et al. (1988) Eur. J. Biochem. 153, 389-397]. The comparable structural properties of these tissue-specific inhibitors of actomyosin ATPase and the similarities in their mode of interaction at the N-terminal region of actin suggest common aspects to the structural mechanism for thin-filament regulation in smooth and striated muscle. Unlike the inhibitory interaction of troponin I, however, the binding of caldesmon to the N-terminal region of actin directly involves groups within residues 20-41 of actin that are also recognised by myosin subfragment 1. The complementary segment of caldesmon has been localised to a 15-kDa thrombic fragment (residues 483-578) derived from the N-terminal portion of a 35-kDa proteolytic cleavage product from the C-terminal of caldesmon whose interaction with actin is modulated by calmodulin. The results are discussed in relation to the calcium-mediated mechanism for thin-filament regulation in smooth and striated muscle.

Actins↗
PubMed1990

Complexes between 15 kDa caldesmon fragment and actin investigated by immuno-electron microscopy.

The regulatory system of smooth muscle thin filaments is thought to involve a major calcium-calmodulin and actin binding protein: caldesmon. A dissective approach was used to isolate a 35 kDa C-terminal fragment of the molecule and to produce antibodies reacting against both the intact and the 15 kDa N-terminal end of this parental fragment. While this purified 15 kDa caldesmon fragment demonstrates a weak actin association, we observed that it cross-links actin filaments into loose bundles. These structures were labelled with a selective antibody and showed regular periodic striation with repeats of approximately 40 nm. This work brings additional information to previous reports using an actin and calmodulin binding 25 kDa C-terminal fragment of the caldesmon molecule [(1989) J. Biol. Chem. 264, 2869-2875]. We demonstrate that a purified fragment corresponding to a sequence smaller than 96 amino acids, which contains no cystein residue, is able to interact with actin at a single site which is not the calmodulin modulated.

Actins↗
PubMed1990

New subfragment 1 of skeletal muscle myosin obtained by thrombin cleavage.

The head of the myosin molecule (i.e., subfragment 1 with a heavy chain of 95 kDa) is usually obtained by chymotryptic cleavage in the presence of a divalent cation chelator. In the present work, we used another specific proteolytic enzyme, thrombin, to produce a limited cut within the myosin molecule, resulting in a new species of N-terminal fragment. Treatment of skeletal muscle myosin yielded a 97-kDa split heavy chain associated with intact light chains, corresponding to a single cut. The ATPase activities of this new S-1 derivative were slightly affected by the breakdown. It recognized actin in an ATP-dependent manner, as expected, with an affinity 2-5 times higher than that of the usual chymotryptic S-1 preparation but with a very different electron microscopic pattern. Functional differences are noted, and we involve them more precisely in relation to possible structural aspects of the additional C-terminal segment extending the usual S-1 heavy chain from 95 to 97 kDa.

Actins↗
PubMed1990

Expression of human beta-myosin heavy chain fragments in Escherichia coli; localization of actin interfaces on cardiac myosin.

A cDNA clone coding for an internal fragment of slow-cardiac beta-myosin heavy chain was isolated from a lambda gt10 human skeletal muscle library. Six overlapping cDNA subclones, which span myosin heavy chain subregions and presumably interact with actin, were derived from this clone, fused to a beta-galactosidase vector and expressed in Escherichia coli. Three of the subclones were obtained by PCR (polymerase chain reaction) which enables gene or cDNA fragments to be amplified independently of preexisting restriction sites. Initially, various experiments were carried out using a long MHC (myosin heavy chain) fusion protein containing the 50 kDa-20 kDa connecting region, the whole 20 kDa region and the short subfragment 2 region. This MHC fusion protein was chemically or proteolytically cleaved in the same conditions as the native myosin molecule. Whole and truncated forms of the MHC fusion protein were separated on polyacrylamide gels, electroblotted on nitrocellulose sheets and renatured. They were then assayed in overlay experiments with F-actin and/or myosin light chains in solution. Specific antibodies were used to detect interactions between heavy chain fragments and F-actin or light chains. We thus observed that one long heavy chain fragment synthesized by E. coli behaved like proteolytic or chemical MHC preparations made from native myosin molecules. Two chymotryptic fragments of the MHC fusion protein, which are soluble at low ionic strength, cosedimented with F-actin in solution. Our results demonstrate that, in actin overlay experiments with whole fusion proteins, interactions seem to be due to the heavy chain fragment, not to the bacterial component. All interactions were non ATP-sensitive. We further investigated the possible participation of the six recombinant MHC fragments in contributing to the actomyosin interfaces on the 50 kDa-20 kDa regions of the human cardiac beta-MHC. The present procedure, which enables the synthesis of any MHC fragment independent of any protease site, is a powerful new tool for studying structure-function relationships within the myosin molecule family.

Actins↗
PubMed1990

Isolated dystrophin molecules as seen by electron microscopy.

Dystrophin, the protein product of the Duchenne muscular dystrophy locus [Hoffman, E. P., Brown, R. H., Jr., & Kunkel, L. M. (1987) Cell 51, 919-928], is expressed in striated and smooth muscles as well as in non-muscle tissues. Examination of its primary structure has revealed that the molecule is composed of four domains, three of which share many features with the membrane cytoskeletal proteins spectrin and actinin. Dystrophin has thus been predicted to adopt a rod shape [Koenig, M., Monaco, A. P. & Kunkel, L. M. (1988) Cell 53, 219-228]. In the present study, we describe its isolation from the chicken gizzard smooth muscle and present electron microscopic images of the molecule. Polyclonal antibodies were first prepared from a dystrophin fragment derived from the chicken skeletal muscle gene (residues 1173-1728). A dystrophin-enriched membrane preparation from chicken gizzard muscle was then purified by passing it through an affinity chromatography column made with the anti-dystrophin antibodies. Electron microscopy of isolated and rotatory-shadowed dystrophin molecules revealed that the lengths measured for the dystrophin monomers (175 +/- 15 nm) are compatible with a structural arrangement of the repeat sequence segments in triple-barrel alpha-helices connected by short-turn regions, as was earlier postulated for the repeat domains of spectrin and actinin. Electron microscopic images indicate that in addition the dystrophin molecules could present the same capacity of self-association in oligomeric structures as these cytoskeletal proteins and may thus be a part of a complex molecular meshwork essential to muscle cell function.

Animals↗
PubMed1989

Caldesmon structure and function: sequence analysis of a 35 kilodalton actin- and calmodulin-binding fragment from the C-terminus of the turkey gizzard protein.

We have determined the amino acid sequence of a 35 kDa proteolytic fragment ("CaD35") derived from the C-terminus of turkey gizzard caldesmon. This 239-residue peptide contains binding sites for actin and calmodulin. Residues 1-96 of CaD35 comprise "CaD15", an actin-binding subfragment which we previously showed to resemble the tropomyosin-binding segment of troponin T. The remainder of the CaD35 sequence shows no significant similarity to other proteins. Residues 111-128 may form a basic, amphipathic helix which interacts with calmodulin.

Actins↗
PubMed1989

Amino acid sequence of a 15 kilodalton actin-binding fragment of turkey gizzard caldesmon: similarity with dystrophin, tropomyosin and the tropomyosin-binding region of troponin T.

We have determined the amino acid sequence of a 15 kDa actin-binding fragment of turkey gizzard caldesmon. The 96-residue fragment contains 29 acidic and 29 basic residues, and is predicted to have an extended helical conformation stabilized by numerous internal salt bridges. CaD15 bears some resemblance to dystrophin, tropomyosin and several other proteins, but is most strikingly similar to the tropomyosin-binding segment of troponin T.

Actins↗
PubMed1989

Functional sequences of the myosin head.

Muscle contraction originates from the sliding of myosin filaments on actin filaments, the energy for which is supplied by the hydrolysis of adenosine-5-triphosphate (ATP) by myosin. The nucleotide first binds to the acto-myosin complex in the myosin head (or subfragment-1), producing a conformational change which induces actin dissociation. The release of phosphate (Pi) then allows a return to the strong actin-myosin association, corresponding to the rigor state. We discuss here certain controversial points arising from current concepts of the actin and nucleotide binding regions at the amino acid sequence level within the subfragment-1 heavy chain. We consider the actin and nucleotide binding regions to be two distinct sites (for each of these regions) one of which is shared competitively between actin and the nucleotide. In our model the cyclical actin-S1 association-dissociation steps correspond to different ATP, actin and ADP affinities for the same amino acid sequence of the S1 heavy chain, contributing alternatively to a single hydrolytic nucleotide site or a strong actin site. We propose the existence of a flexible segment that forms or dismantles the nucleotide or actin sites. The large region (amino acids 540-707) overlapping the actin-myosin interface appears to be the main flexible region of the S1 molecule and we propose that this particular sequence plays a key role in the dissociation pathway of the actin-myosin complex and in the conversion of chemical energy into the mechanical energy of contraction.

Actins↗
PubMed1988

The actin-myosin subfragment-1 complex stabilized by phenyldiglyoxal.

Nuclear magnetic resonance studies have revealed the importance of arginine residues in the actin-myosin interface (Moir, A. J. G., and Levine, B. A. (1986) J. Inorg. Chem. 27, 271-278). In the present study, we tested the involvement of these residues in the rigor complex between actin and subfragment-1 (S1) by chemical cross-linking experiments using phenyldiglyoxal. Two kinds of linkages were observed, one within the S1 heavy chain itself (120-kDa product) and the other between actin and the S1 heavy chain (200-kDa product). The phenyldiglyoxal had an effect similar to that of phenylglyoxal on S1 ATPase activities. We also show that phenyldiglyoxal (of 0.6-0.8 nm arm length) cross-links an arginine residue of the 50-kDa domain to one in the 20-kDa domain of the S1 heavy chain in the absence of actin or to an arginine in actin when actin is present. The presence of Mg2+, adenosine 5'-diphosphate or 5'-adenylyl imidodiphosphate suppressed the intermolecular linkage with actin, and favored the intramolecular cross-link, (i.e. between 50-kDa and 20-kDa fragments). We propose that the same arginyl residue in the N-terminal part of the 50-kDa domain can be cross-linked to a nearby arginine in either the 20-kDa domain or the actin molecule. In accordance with the amino acid sequence of each protein this also implies that the actin-myosin interaction involves arginine residues located either after residue 28 of the N-terminal part of actin, since this actin region is devoid of arginine residues, or in the N-terminal portion of the 50-kDa domain, i.e. between residues 239 and 455.

Actins↗
PubMed1988

A 35-kilodalton fragment from gizzard smooth muscle caldesmon that induces F-actin bundles.

Specific thrombin proteolysis of native 120-kDa gizzard caldesmon gave rise to a major cleavage into an N-terminal 90-kDa and a C-terminal 35-kDa fragment. Fluorescent labeling, cosedimentation, passage through an affinity column, and carbodiimide crosslinking with actin revealed that the 35-kDa purified segment of the molecule contains the actin and the calcium-calmodulin binding regions. Electron microscopic analysis of its actin complex demonstrated that the 35-kDa segment possesses the bundling properties of the intact molecule. Thus, a possible pathway for the expression of the caldesmon regulatory function during smooth muscle contraction would be a conformational change twisting the helicoïdal structure of the actin filament, which occurs when the 35-kDa caldesmon portion binds to it.

Actins↗
PubMed1988

Identification of a 15 kilodalton actin binding region on gizzard caldesmon probed by chemical cross-linking.

Fluorescent labeling, limited proteolysis, amino acid sequence determinations, affinity chromatography and specific chemical crosslinking were used to determine the smallest fragment of gizzard caldesmon that interacts with actin. The time course of cleavage with thrombin or submaxillaris arginase-C protease indicates that 90kDa and 35kDa fragments are the two major pieces of the 120kDa native protein. Amino acid sequence determination indicates that the 90kDa fragment is the N-terminal portion of the molecule. Further degradation gave rise to a 15kDa product whose N-terminal amino acid sequence was determined within the first 28 amino acids. Carbodiimide crosslinking with actin revealed that the 15kDa part of the molecule is probably not involved in the actin binding process but may participate in a twisting of the F-actin filament and be responsible of the caldesmon regulatory function during smooth muscle contraction.

Actins↗
PubMed1988

The internal crosslinking of the S1 heavy chain from smooth muscle probed by dibromobimane.

The reaction of the crosslinker dibromobimane has recently revealed a functionally important internal loop structure within the skeletal myosin S1 heavy chain where Cys-522 of the 50K domain and Cys-707 (SH1) of the 20K region are spatially juxtaposed. Here we have studied the possible relevance of this topological feature to the architecture and transducing activity of the myosin head in general, by extending the dibromobimane modification to smooth muscle myosin. Treatment of chicken gizzard myosin S1 with dibromobimane resulted in intramolecular crosslinking between the C-terminal 25K and central 50K segments of the S1 heavy chain. The data suggest a conservation at the 50K-25K interface of smooth muscle S1 heavy chain and the importance of the neighboring SH1 region, whose conformation may play an important role in energy transduction by the myosin head.

Adenosine Triphosphatases↗
PubMed1987

Comparative structure of the protease-sensitive regions of the subfragment-1 heavy chain from smooth and skeletal myosins.

The heavy chain fragments generated by restricted proteolysis of the smooth chicken gizzard myosin subfragment-1 (S-1) with trypsin, Staphylococcus aureus V8 protease, and chymotrypsin were isolated and submitted to partial amino acid sequencing. The comparison between the smooth and striated muscle myosin sequences permitted the unambiguous structural characterization of the two protease-vulnerable segments joining the three putative domain-like regions of the smooth head heavy chain. The smooth carboxyl-terminal connector is a serine-rich region located around positions 632-640 of the rabbit skeletal sequence and would represent the "A" site that is conformationally sensitive to the myosin 10 S-6 transition and to its interaction with actin (Ikebe, M., and Hartshorne, D. J. (1986) Biochemistry 25, 6177-6185). A third site which undergoes a nucleotide-dependent chymotryptic cleavage which inactivates the Mg2+-ATPase (Okamoto, Y., and Sekine, T. (1981) J. Biochem. (Tokyo) 90, 833-842, 843-849) was identified at Trp-31/Ser-32. It is vicinal to Lys-34 that is monomethylated in the skeletal heavy chain but not at all in the smooth sequence. However, the two trimethyl lysine residues present in the skeletal sequence are conserved in the same regions of the smooth S-1 and may play a general functional role in myosin. The smooth central 50-kDa segment could be selectively destroyed by a mild tryptic digestion in the absence of any unfolding agent, with a concomitant inhibition of the ATPase activities. This feature is in line with the proposed domain structure of the S-1 heavy chain and also suggests a relationship between the specific biochemical properties of the smooth S-1 and the particular conformation of its 50-kDa region.

Amino Acid Sequence↗
PubMed1986

Identification of polyphosphate recognition sites communicating with actin sites on the skeletal myosin subfragment 1 heavy chain.

Using the thrombin-cut [68-30 kilodalton (kDa)] myosin subfragment 1 (S-1) whose heavy chain has been selectively split within the central 50-kDa region, at Lys-560, with concomitant specific alterations of the ATPase and actin binding properties [Chaussepied, P., Mornet, D., Audemard, E., Derancourt, J., & Kassab, R. (1986) Biochemistry 25, 1134-1140; Chaussepied, P., Mornet, D., Barman, T., Travers, F., & Kassab, R. (1986) Biochemistry 25, 1141-1149], we have isolated and renatured the COOH-terminal 30-kDa fragment associated with the alkali light chains by the procedure recently described [Chaussepied, P., Mornet, D., Audemard, E., Kassab, R., Goodearl, J., Levine, B., & Trayer, I. P. (1986) Biochemistry 25, 4540-4547]. The 30-kDa peptide preparation was found to exhibit a crucial feature of the native S-1; namely, it interacts with F-actin in an adenosine 5'-triphosphate (ATP)-dependent manner. Studies by ultracentrifugation, turbidity measurements, and chemical cross-linking experiments showed that the acto-30-kDa peptide complex was dissociated almost completely by the gamma-phosphoryl group containing ligands ATP, 5'-adenylyl imidodiphosphate, and pyrophosphate, to a lesser extent by ADP, and not at all by AMP and inorganic phosphate. The maximal dissociating effect is operating with the thrombic 30-kDa entity, whereas the 22-kDa fragment produced by staphylococcal protease is only slightly dissociated. In contrast, the tryptic 20-kDa fragment binds irreversibly to actin.(ABSTRACT TRUNCATED AT 250 WORDS)

Actins↗
PubMed1986

Modification of the actin interface of skeletal myosin subfragment-1 by treatment with dibromobimane.

Recently, by treating the head portion of skeletal myosin subfragment-1 (S1) with the bifunctional agent dibromobimane, we introduced an intramolecular covalent cross-link which resulted in the stabilisation of an internal loop in the heavy chain structure of the head [Mornet et al. (1984) Proc. Natl Acad. Sci. USA 82, 1658-1662]. In order to define the functional properties of this new S1 conformational state, we have first determined the experimental conditions for the optimum modification of S1 by dibromobimane. We finally settled on a 60% yield of cross-linked S1. Because the modification occurs between the 50-kDa and the 20-kDa tryptic heavy chain fragments which have been postulated to be involved in the interaction of native S1 with actin, we have investigated the association of dibromobimane-treated S1 with actin, using chemical cross-linking of their rigor complex with 1-ethyl-3-[3-(dimethylamino)propyl] carbodiimide. The cross-linked species obtained were analyzed by polyacrylamide gel electrophoresis and compared with those known for unmodified S1. The carbodiimide-catalyzed linkage between actin and dibromobimane-modified S1 led to a singlet protein band migrating with an apparent molecular mass of 155 kDa, in contrast to the usual doublet bands of 175 kDa and 185 kDa produced with native S1. This result suggests that a change has occurred at the actin interface on the dibromobimane-treated S1 heavy chain. The covalent complex generated by carbodiimide cross-linking between actin and dibromobimane-modified S1 (27-kDa + 50-kDa + 20-kDa fragments) was submitted to chemical hydrolysis with hydroxylamine. The nature of the products identified is consistent with the conclusion that the internal freezing of the heavy chain structure by dibromobimane induces the loss of the ability to cross-linkage of the actin site on the 20-kDa domain but does not affect the conformation of the second site on the 50-kDa segment, which becomes the unique actin region cross-linkable by actin.

Actins↗