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C G dos Remedios

Publications and source records attributed to C G dos Remedios.

At least 19 recordsLinked to original sources
PubMed1992

Uncoupling of actin-activated myosin ATPase activity from actin binding by a monoclonal antibody directed against the N-terminus of myosin light chain 1.

The role of the N-terminal region of myosin light chain 1 (LC1) in actomyosin interaction was investigated using an IgG monoclonal antibody (2H2) directed against the N-terminal region of LC1. We defined the binding site of 2H2 by examining its cross-reactivity with myosin light chains from a variety of species and with synthetic oligopeptides. Our findings suggest that 2H2 is directed against the N-terminal region of LC1 which includes the trimethylated alanine residue at the N-terminus. In the presence of 2H2, the rate of actomyosin superprecipitation was reduced, although the extent was not. 2H2 caused a reduction in the Vmax of both myosin and chymotryptic S1(A1) actin-activated ATPase activity, while the Km appeared to be unaltered. The Mg(2+)-ATPase activity of myosin alone was also unaffected. Binding studies revealed that 2H2 did not prevent the formation of acto-S1 complex, either in the presence or in the absence of ATP, nor did it affect the ability of ATP to dissociate S1 from F-actin. Our findings suggest that the N-terminal region of LC1 is not essential for actin binding but is involved in modulating actin-activated ATPase activity of myosin.

Actins↗
PubMed1992

Models of the actin monomer and filament from fluorescence resonance-energy transfer.

We have developed algorithms for combining fluorescence resonance-energy transfer (FRET) efficiency measurements into structural models which predict the relative positions of the chemical groups used in FRET. We used these algorithms to construct models of the actin monomer and filament derived solely from FRET measurements based on seven distinct loci. We found a mirror-image pair of monomer models which best fit the FRET data. One of these models agrees well with the atomic-resolution crystal structure recently published by Kabsch et al. in Heidelberg [Kabsch, W., Mannherz, H. G., Suck, D., Pai, E. F. & Holmes, K. C. (1990) Nature 347, 37-44]. The root-mean-square deviation between this FRET model and the crystal structure was about 0.9 nm. Other macromolecular models assembled from FRET measurements are likely to have a similar resolution. The largest discrepancy was for the Cys10 locus which deviated 1.44 nm from the crystal position. We discuss the limitations of the FRET method that may have contributed to this discrepancy, and conclude that the Cys10 FRET data have probably located Cys10 incorrectly in the FRET monomer model. Using the FRET monomer models, we found three orientations in the filament which best fit the intermonomer FRET data. These orientations differ substantially from the atomic-resolution filament model proposed by the Heidelberg group [Holmes, K., Popp, D., Gebhard, W. & Kabsch, W. (1990) Nature 347, 44-49], largely because of the discrepancies in the Cys10 data. These data should probably be excluded from the analysis; however, this would leave too few measurements to assemble a filament model. In the near future, we hope to obtain additional FRET measurements to other actin loci so that the filament modelling can be done without the Cys10 data.

Actins↗
PubMed1992

Removing the two C-terminal residues of actin affects the filament structure.

We define conditions under which the two C-terminal residues of actin, Cys-374 and Phe-375, can be selectively removed by proteolysis with trypsin. This modification had little effect on the secondary structure of actin detected by Fourier-transform infrared spectroscopy. However, removing these residues caused small but significant decreases in the critical concentration of actin, in its ability to activate myosin ATPase, and in its interaction with tropomyosin and troponin. Removing residues 374-375 caused dramatic changes in the actin filament as seen by electron microscopy. The filaments had a much greater and more irregular curvature and were intertwined into disordered multifilament bundles. Removing 374-375 also significantly lowered the flow viscosity of filamentous-actin solutions. These data suggest an increase in the flexibility and fragility of the filament, supporting the idea that the C-terminus forms one of the major intermonomer contacts in the filament.

Actin Cytoskeleton↗
PubMed1991

Actin dynamics studied by solid-state NMR spectroscopy.

Solid-state nuclear magnetic resonance spectroscopy was used to study the motion of 2H and 19F probes attached to the skeletal muscle actin residues Cys-10, Lys-61 and Cys-374. The probe resonances were observed in dried and hydrated G-actin, F-actin and F-actin-myosin subfragment-1 complexes. Restricted motion was exhibited by 19F probes attached to Cys-10 and Cys-374 on actin. The dynamics of probes attached to dry cysteine powder or F-actin were very similar and the binding of myosin had little effect indicating that the local probe environment imposes the major influence on motion in the solid state. Correlation times determined for the solid state probes indicated that they were undergoing some rapid internal motion in both G-actin and F-actin such as domain twisting. The probe size influenced the motion in G-actin and appeared to sense monomer rotation but not in F-actin where segmental mobility and intramonomer co-ordination appeared to dominate.

Acetone↗
PubMed1991

Effects of sulphydryl modification on skinned rat skeletal muscle fibres using 5,5'-dithiobis(2-nitrobenzoic acid).

1. The sulphydryl groups of skinned skeletal muscle fibres have been reacted with 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB) in order to determine whether the effects of modifications to the contractile proteins are reflected in changes in the physiological properties of the contractile apparatus and Ca(2+)-regulatory system. 2. Results obtained from fast-twitch and slow-twitch rat fibres which were treated with DTNB (10 mM, pH 8.6, 5 degrees C) for various periods of time under relaxing conditions showed that a major effect of the modification was to reduce the level of maximally Ca(2+)-activated force and fibre stiffness. Force and fibre stiffness were found to decline in proportion. Treatment with DTNB under these conditions did not cause a rise in force or fibre stiffness in relaxed fibres of either type. 3. The effects induced by DTNB under relaxing conditions were substantially reversed by exposure to the reducing agent dithiothreitol (DTT) (10 mM, pH 7.1, 23 degrees C). Force abolished by 30-35 s treatment with DTNB recovered after subsequent DTT treatment to 67 +/- 3% (mean +/- S.E.M., n = 4) in fast-twitch fibres and to 91 +/- 2% (n = 7) in slow-twitch fibres. These results were significantly different (t test, P less than 0.001) indicating that the level of force recovery depended upon the fibre type. 4. DTNB was found to affect not only the maximal Ca(2+)-activated force, but also the force-pCa (pCa = -log10[Ca2+]) relationships of the fibres in a complex, fibre-type specific way. DTT treatment partially reversed these DTNB effects. 5. The skinned fibre preparations reacted differently with DTNB under rigor conditions than under relaxing conditions, indicating that rigor modifies the reactivity of the functional sulphydryl groups to the thiol-targeted agents. 6. When superprecipitation assays (an in vitro analogue of fibre contraction) were carried out with recombined myofibrillar proteins which had been previously reacted with DTNB it was found that modification of myosin, but not modification of thin filament proteins, led to changes in the superprecipitation reaction. 7. Both the skinned fibre results and the superprecipitation results indicate that the effects of DTNB upon the fibre characteristics are primarily due to modifications of the sulphydryl groups of myosin. Therefore, these results show that myosin is not only involved in determining the ability of the contractile apparatus to develop force but also in determining the Ca(2+)-regulatory characteristics of the muscle fibre.

Animals↗
PubMed1991

[Detection of cardiac myosin light chain I by monoclonal antibody in the patients with acute myocardial infarction].

Plasma cardiac myosin light chain I(CMLCI) was quantified by competitive enzyme-linked immunosorbent assay(ELISA) using a monoclonal antibody in 42 patients with confirmed acute myocardial infarction (AMI). In comparison to the value from healthy individuals, plasma CMLCI levels were significantly elevated in 40 patients. In 28 of the 42 patients (66.7%), two major peaks were observed on time activity curves. The early peak (CMLCIp1) began a rapid rising within 4-12 hours and peaked at a mean of 25 hours following the onset of chest pain. The CMLCIp1 appeared statistically higher and earlier in patients with early infarct-related artery recanalization (IRAR) than those without IRAR, which was assessed by CPK peaking time, continuous ST segment monitoring on 12 lead ECG and symptoms. Thus, CMLCIp1 might be influenced by early IRAR. The late peak (CMLCIp2), which was composed of another gradual increase of plasma CMLCI level, occurred at a mean of 142 hours after AMI and remained elevated for about 7-10 days. The magnitudes of CMLCIp2 were correlated with the infarct size estimated by LVEF and LVWMS on cineventriculography and QRS scores on ECG. In the rest 14 patients only one peak was observed. It was suggested that CMLCIp1 could provide an early diagnosis of AMI and might be influenced by early IRAR after thrombolysis, while CMLCIp2 was a good later marker of extent of myocardial necrosis.

Adult↗
PubMed1990

Elevated MHC class I and II antigens in cultured human embryonic myoblasts following stimulation with gamma-interferon.

The expression of major histocompatibility complex (MHC) antigens on the surface of cultured human embryonic myoblasts was studied by fluorescence microscopy. Class I or II MHC antigens were detected by monoclonal antibodies (MoAb) recognizing monomorphic determinants on HLA-A, B and C (class I), or HLA-DP, DQ and DR (class II) and a secondary fluorescein-labelled sheep anti-mouse immunoglobulin (Ig) antibody. Myoblasts were simultaneously identified using a MoAb directed against myosin light chains 1 and 2 (MLC1 and MLC2) and a combination of biotin-labelled sheep anti-mouse Ig antibody and Texas Red labelled streptavidin. We found that myoblasts demonstrated only very weak expression of either class of MHC but that, in the presence of more than 100 units of gamma-interferon (gamma-IFN) for 56-72 h, both class I and II MHC antigen expression increased significantly. During this time, class I antigen increased more than class II and at lower gamma-IFN concentrations. The implications of these findings for myoblast transfer therapy in Duchenne muscular dystrophy patients is discussed.

Antibodies, Monoclonal↗
PubMed1990

A determination of the radial coordinate of Tyr-69 in F-actin using fluorescence energy transfer.

Fluorescence energy transfer was measured between Tyr-69 residues in an F-actin filament using 5-dimethylaminonaphthalene-1-sulfonyl chloride (DNS-Cl) as a fluorescence energy donor and 4-dimethylaminoazobenzene-4-sulfonyl chloride (DABSYL-Cl) as the acceptor. Both labels are covalently attached to Tyr-69 residues in an F-actin filament. Taking the helical structure of the F-actin filament into consideration, the radial coordinate of Tyr-69 was calculated to be in the range from 2.0 nm to 4.0 nm.

Actins↗
PubMed1989

The effect of the replacement of ADP with a photoaffinity ATP analogue, 2-azido-ADP, in F-actin on its function.

2-Azido-ATP, a photoaffinity ATP analogue, was incorporated into actin and the influence of the incorporation on the actin function was studied. The replacement of ADP with 2-azido-ADP in F-actin both before and after photocross-linking decreased appreciably the actin-activated S1-ATPase activity. Photocross-linked 2-azido-ADP-F-actin could be depolymerized by dialysis against a solution containing 0.1 mM CaCl2, 0.1 mM ATP and 1 mM Tris-HCl (pH 8.0). However, once it depolymerized, it lost very quickly the ability to polymerize even in the presence of a sufficient amount of ATP and Ca2+.

Actins↗
PubMed1988

Fluorescence quenching studies of fluorescein attached to Lys-61 or Cys-374 in actin: effects of polymerization, myosin subfragment-1 binding, and tropomyosin-troponin binding.

The resonance energy transfer between fluorescein-5-isothiocyanate (FITC) attached to Lys-61 and Co2+ bound to the high-affinity metal binding site was measured. The distance between FITC and Co2+ on the actin molecule was calculated to be either 1.9 nm, using the absorption spectrum of Co-EDTA or 2.8 nm, using the absorption spectrum of Co2+ bound to carboxypeptidase as a model spectrum of Co2+ bound to actin, respectively. The effects of the polymerization of actin and of the interaction of actin with myosin subfragment-1 (S1) on the solvent accessibility of the fluorescein molecule attached to Lys-61 or Cys-374 were measured. The accessibility of the probe at Lys-61 was reduced following polymerization and also appreciably reduced by interaction with S1. The accessibility of the probe attached to Cys-374 was affected to only a small degree. These results indicate that the Lys-61 residue is located close to an actin-actin contact region as well as being close to an S1 binding site, although it is not directly involved [Miki, M. (1987) Eur. J. Biochem. 164, 228-235]. The accessibility of the probe at Lys-61 was also decreased by the addition of the tropomyosintroponin complex, although the accessibility of the probe at Cys-374 was not affected at all. Thus, Lys-61 appears to be involved in the binding site of the regulatory proteins.

Actins↗
PubMed1988

Structure and function of contractile proteins in muscle fibres.

The structural unit of muscle has long been defined as the myofibril, a supramolecular assembly of a dozen or more proteins of which two, actin and myosin, comprise more than 75%. In the past 40 years since Albert Szent-Gyorgyi first described the contractile response from the complex of actin and myosin, knowledge of the structure and function of these contractile proteins has been substantially refined. This paper describes these new discoveries and identifies the problems which remain to be elucidated.

Actins↗
PubMed1987

Spatial relationship between the nucleotide-binding site, Lys-61 and Cys-374 in actin and a conformational change induced by myosin subfragment-1 binding.

The spatial relationship between Lys-61, the nucleotide binding site and Cys-374 was studied. Lys-61 was labelled with fluorescein-5-isothiocyanate as a resonance energy acceptor, the nucleotide-binding site was labelled with the fluorescent ATP analogues epsilon ATP or formycin-A 5'-triphosphate (FTP) and Cys-374 was labelled with 5-(2-[(iodoacetyl)amino]ethyl)aminonaphthalene-1-sulfonic acid (1,5-IAEDANS) as a resonance energy donor. The distances between the nucleotide binding site and Lys-61 or between Lys-61 and Cys-374 were calculated to be 3.5 +/- 0.3 nm and 4.60 +/- 0.03 nm, respectively. (The assumption has been made in calculating these distances that the energy donor and acceptor rotate rapidly relative to the fluorescence lifetime.) On the other hand, when doubly-labelled actin with 1,5-IAEDANS at Cys-374 and FITC at Lys-61 was polymerized in the presence of a twofold molar excess of phalloidin [Miki, M. (1987) Eur. J. Biochem. 164, 229-235], the fluorescence of 1,5-IAEDANS bound to actin was quenched significantly. This could be attributed to inter-monomer energy transfer. The inter-monomer distance between FITC attached to Lys-61 in a monomer and 1,5-IAEDANS attached to Cys-374 in its nearest-neighbour monomer in an F-actin filament was calculated to be 3.34 +/- 0.06 nm, assuming that the likely change in the intra-monomer distance does not change during polymerization by more than 0.4 nm. One possible spatial relationship between Lys-61, Cys-374 and the nucleotide binding site in an F-actin filament is proposed. The effect of myosin subfragment-1 (S1) binding on the energy transfer efficiency was studied. The fluorescence intensity of AEDANS-FITC-actin decreased by 30% upon interaction with S1. The fluorescence intensity of AEDANS-FITC-actin polymer in the presence of phalloidin increased by 21% upon interaction with S1. The addition of ATP led to the fluorescence intensity returning to the initial level. Assuming that the change of fluorescence intensity can be attributed to conformational change in the actin molecule induced by S1 binding, the intra-monomer distance was reduced by 0.4 nm and the inter-monomer distance was increased by 0.2 nm.

Actins↗
PubMed1987

Fluorescence resonance energy transfer between sites in G-actin. The spatial relationship between Cys-10, Tyr-69, Cys-374, the high-affinity metal and the nucleotide.

Intramonomer fluorescence resonance energy transfer spectroscopy was employed to investigate the spatial relationship between labels attached to the residues Cys-10, Tyr-69, Cys-374, the high-affinity metal binding site and the nucleotide binding site in G-actin. The separation between the fluorescence donor 5-(dimethylamino)naphthalene-1-sulphonyl (Dns) chloride (dansyl chloride) used to label Tyr-69 and the acceptor 4-dimethylaminophenylazophenyl-4'-maleimide (DABM) used to label Cys-374 was found to be 3.6 nm. The distance separating Dns on Tyr-69 from DABM on Cys-10 was found to be 2.7 nm. The distance separating the acceptor DABM bound to Cys-374 from the fluorescence donor formycin A 5'-triphosphate (FTP) occupying the nucleotide binding site was determined to be 3.0 nm. A slightly larger separation was determined between the FTP site and DABM attached to Cys-10. In this case a value of 3.2 nm was obtained. The distance separating Dns on Tyr-69 from Co2+ in the high-affinity metal binding site was determined to be 1.1 nm. Finally, the separation of FTP, now acting as donor, from the Dns molecule attached to Tyr-69 and acting as the acceptor was determined to be 2.1 nm. The likely relationship between these label sites on actin is represented by a model which is used to assist in the determination of the actin structure, with particular reference to the environment of the metal and nucleotide binding sites.

Actins↗
PubMed1987

Interaction of phalloidin with chemically modified actin.

Modification of Tyr-69 with tetranitromethane impairs the polymerizability of actin in accordance with the previous report [Lehrer, S. S. and Elzinga, M. (1972) Fed. Proc. 31, 502]. Phalloidin induces this chemically modified actin to form the same characteristic helical thread-like structure as normal F-actin. The filaments bind myosin heads and activate the myosin ATPase activity as effectively as normal F-actin. When a dansyl group is introduced at the same point [Chantler, P. D. and Gratzer, W. B. (1975) Eur. J. Biochem. 60, 67-72], phalloidin still induces the polymerization. The filaments bind myosin heads and activate the myosin ATPase activity. These results indicate that Tyr-69 is not directly involved in either an actin-actin binding site or the myosin binding site on actin. Moreover, the results suggest that phalloidin binds to actin monomer in the presence of salt and its binding induces a conformational change in actin which is essential for polymerization, or that actin monomer fluctuates between in unpolymerizable and polymerizable form while phalloidin binds to actin only in the polymerizable form and its binding locks the conformation which causes the irreversible polymerization of actin. Modification of Tyr-53 with 5-diazonium-(1H)tetrazole blocks actin polymerization [Bender, N., Fasold, H., Kenmoku, A., Middelhoff, G. and Volk, K. E. (1976) Eur. J. Biochem. 64, 215-218]. Phalloidin is unable to induce the polymerization of this modified actin nor does it bind to it. Phalloidin does not induce the polymerization of the trypsin-digested actin core. These results indicate that the site at which phalloidin binds is involved in polymerization and the probable conformational change involved in polymerization may be modulated through this site.

Actins↗
PubMed1987

Fluorescence resonance energy transfer measurements of distances in actin and myosin. A critical evaluation.

The contractile proteins actin and myosin are of considerable biological interest. They are essential for muscle contraction and in eukaryotic cells they play a crucial role in most contractile phenomena. Over the years since the first fluorescence resonance energy transfer (FRET) paper appeared, an extensive body of literature has accumulated on this technique using actin, myosin and the actomyosin complex. These papers are reviewed with several aims in mind: we assess the reliability and consistency of intra- and inter-molecular distances measured between the fluorescent probes attached to specific sites on these proteins; we determine whether the measurements can be assembled into an internally consistent model which can be fitted to the known dimensions of the actomyosin complex; several of the FRET distances are consistent with the available structural data from crystallographic and electron microscopic dimensions; the modelled FRET distances suggest that the assumed value of the orientation factor (k2 = 2/3) is reasonable; we conclude that the model has a predictive value, i.e. it suggests that a small number of the published dimensions may be incorrect and predicts the magnitude of a larger number of measurements which have not yet been reported; and finally (vi) we discuss the contribution of FRET determinations to the current debate on the molecular mechanism of contraction.

Actins↗
PubMed1986

Fluorescence resonance energy transfer between the nucleotide binding site and Cys-10 in G-actin and F-actin.

Intramonomer fluorescence resonance energy transfer between the donor epsilon-ATP bound to the nucleotide site and the acceptor N-(4-dimethylamino-3,5-dinitrophenyl)maleimide (DDPM) or 4-dimethylaminophenyl-azophenyl-4'-maleimide bound to Cys-10 in G-actin was measured. The donor-acceptor distance was calculated to be about 40 A. The intermonomer energy transfer in F-actin occurring between epsilon-ADP and DABMI was also measured. The radial coordinate of Cys-10 was calculated to be 25 A based on the helical symmetry of F-actin and the recently calculated radial coordinate of the nucleotide binding site in F-actin i.e. 25 A (Miki, M., Hambly, B. and dos Remedios, C.G. (1986) Biochim. Biophys. Acta 871, 137-141). (The assumption has been made in calculating these distances that the energy donor and acceptor rotate rapidly relative to the fluorescence lifetime.) Corresponding distances separating the donor nucleotide in one monomer from acceptors on Cys-10 in the first and second nearest neighbours in F-actin are 39-40 A and 41-43 A.

Actins↗
PubMed1986

Fluorescence energy transfer between Cys-10 residues in F-actin filaments.

Fluorescence energy transfer was measured between Cys-10 residues in an F-actin filament using 5-[2-((iodoacetyl)amino)-ethyl]aminonaphthalene-1-sulphonic acid (1,5-IAEDANS) as a fluorescence energy donor and 4-dimethylaminophenylazophenyl-4'-maleimide (DABMI) as the acceptor. Both labels were covalently attached to Cys-10 residues in an F-actin filament. Taking the helical structure of the F-actin filament into consideration, the radial coordinate of Cys-10 was calculated to be 23 A. This corresponds to a distance between adjacent sites along the long pitch helix of 56.1 A and along the genetic helix of 53.3 A.

Actins↗