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Biomedical subjects

R A Cross

Publications and source records attributed to R A Cross.

At least 19 recordsLinked to original sources
PubMed1992

Expression of the N-terminal domain of dystrophin in E. coli and demonstration of binding to F-actin.

The N-terminal head domain of human dystrophin has been expressed in soluble form and high yield in E. coli, allowing us to test the previously unconfirmed assumption that dystrophin binds actin. DMD246, the first 246 amino acid residues of dystrophin, binds F-actin in a strongly co-operative manner with a Hill constant of 3.5, but does not bind G-actin. Dystrophin heads are thus functionally competent actin-binding proteins. This result opens the way to identifying critical residues in the actin-binding site and encourages us that the other domains of dystrophin might also be treated as functionally autonomous modules, accessible to a similar approach.

Actins↗
PubMed1991

Scanning transmission electron microscopic mass determination of in vitro self-assembled smooth muscle myosin filaments.

We have measured the mass per unit length of in vitro self-assembled smooth muscle myosin filaments, using scanning transmission electron microscope darkfield images of freeze-dried samples. The measured values were integral multiples, usually 1, 2, 3 or 4, of 75 kDa per nm. The data corroborate an earlier proposal, that these filaments are built from monolayer sheets of molecules, each sheet having two antiparallel myosin molecules per 14.3 nm of its length.

Animals↗
PubMed1991

A folded (10 S) conformer of myosin from a striated muscle and its implications for regulation of ATPase activity.

Myosin from the striated adductor muscle of the scallop Pecten maximus is shown to fold into a compact 10 S conformer under relaxing conditions, as has been characterized for smooth and non-muscle myosins. The folding transition is accompanied by the trapping of nucleotide at the active site to give a species with a half-life of about an hour at 20 degrees C. Ca2+ binding to the specific, regulatory sites on a myosin head promotes unfolding to the extended 6 S conformer and activates product release by 60-fold. The unfolding transition, however, remains much slower than the contraction-relaxation cycle of scallop striated muscle and could not play a role in the regulation of these events. The dissociation of products from myosin heads in native thick filaments is Ca2(+)-regulated, but under relaxing conditions the nucleotide is released at least an order of magnitude faster than from the 10 S monomeric myosin, at a rate similar to that observed with heavy meromyosin. Thus, there is no evidence for any intermolecular interaction between neighbouring molecules in the filament analogous to the head-neck intramolecular interaction in the 10 S conformer. It is possible that the 10 S myosin state represents an inert form involved in the control of filament assembly during muscle growth and development. Removal of regulatory light chains or labelling the reactive heavy chain thiol of myosin prevents, or at least disfavours, formation of the folded 10 S conformer and allows separation of the modified protein from the native molecules.

Adenosine Triphosphate↗
PubMed1991

A nucleation--elongation mechanism for the self-assembly of side polar sheets of smooth muscle myosin.

Self-assembled filaments of smooth muscle myosin were observed by low dose electron microscopy to be flat side-polar sheets, in which the component molecules appeared straight and close-packed. Fraying experiments released small oligomers, in which molecules were staggered in parallel by about +/- 14 nm relative to two immediate neighbours, and were bound also to an antiparallel partner via a approximately 14 nm overlap at the very tip of the tail. We suggest a filament model which preserves these packing relationships. Adding stoichiometric amounts of MgATP to the filaments caused them to disassemble completely by progressive loss of material from their ends, at a limiting rate equivalent to about 2 monomers per second per end in physiological saline. The rate of the competing association reaction varied linearly with the monomer concentration, as determined in pressure-jump experiments. This suggests that myosin monomers, rather than dimers or higher oligomers, are the building blocks of these filaments. Shearing and annealing of assembled filaments appeared negligible on a time scale of a few hours. In consequence, filament number and filament length were dependent on the rate at which monomers were supplied to the assembly reaction, and on the number of filaments already present at the start of the assembly reaction.

Actin Cytoskeleton↗
PubMed1991

Self-assembly pathway of nonsarcomeric myosin II.

Cells need to control the location and timing of actomyosin-dependent force generation, and appear to do so in the first instance by regulating myosin filament self-assembly (Yumura and Fukui, 1985). The mechanism of the self-assembly is little understood. In vitro it is a true self-assembly, which requires a short domain at the C terminus of the myosin molecule. The availability of this domain appears suppressed by the folding of the molecule into a compact, looped state. In vitro, the rate at which these looped molecules unfold turns out to be a key determinant of filament number and filament length.

Actin Cytoskeleton↗
PubMed1991

Small angle X-ray scattering studies on myosin.

Small angle X-ray scattering (SAXS) is a potentially powerful method for obtaining structural information from biological molecules in solution. The use of this technique in the laboratory has hitherto been limited by the long exposures necessary to obtain patterns on photographic film. Multi-wire area detectors, due to their high efficiency and absence of noise, enable patterns to be collected much more rapidly, typically in 1-2 h for a typical protein using laboratory sources. This opens up the possibility of using the technique on a semi-routine basis for a wide variety of problems. We outline the use of SAXS to characterise a large conformational change of myosin.

Animals↗
PubMed1990

The functional properties of full length and mutant chicken gizzard smooth muscle caldesmon expressed in Escherichia coli.

Wild type chicken gizzard caldesmon (756 amino acids) was expressed in a T7 RNA polymerase-based bacterial expression system at a yield of 1 mg pure caldesmon per litre bacterial culture. A mutant composed of amino acids 1-578 was also constructed and expressed. The wild type and mutant caldesmon were purified and compared with native chicken gizzard caldesmon. Native and wild type expressed caldesmon were indistinguishable in assays for inhibition of actin-tropomyosin activation of myosin ATPase, reversal of inhibition by Ca2(+)-calmodulin and binding to actin, actin-tropomyosin, Ca2(+)-calmodulin, tropomyosin and myosin. The mutant missing the C-terminal 178 amino acids had no inhibitory effect and did not bind to actin or Ca2(+)-calmodulin. It bound to tropomyosin with a 5-fold reduced affinity and to myosin with a greater than 10-fold reduced affinity.

Animals↗
PubMed1990

Structural predictions for the central domain of dystrophin.

The amino acid sequence of dystrophin indicates that the molecule has globular N- and C-terminal domains separated by a long central rod domain. The central rod contains multiple repeats, about 100 amino acids long and of variable length. These diverge sufficiently in sequence that, in previous studies, only 14 of the most similar repeats have been aligned and analysed in any detail. We show here that a heptad pattern of hydrophobic residues is preserved across all repeats. Using the heptad pattern together with a consensus sequence template, we identified and aligned 25 repeats in the dystrophin rod sequence. Each repeat consists of a constant-length core helix of 54 residues, coupled via a short linker to a weakly conserved variable-length helix, and then via a second linker to the next core. The variable-length helix appears truncated in repeats 10 and 13 and extended in repeats 4 and 20. The extension of repeat 20 is particularly interesting since it corresponds to a hotspot of dystrophy-inducing mutations. Detailed modelling suggests that the classical Speicher-Marchesi [(1984) Nature 311, 177-180] model for spectrin may not be appropriate to dystrophin without some modification. We propose that whilst the repeating structural motif in dystrophin is probably a bead of triple coiled coil, this bead is twice as massive as, and out of phase with, those proposed for spectrin. Our model raises the possibility that the rod domain of dystrophin may confer elasticity on the molecule. Deletions which truncate this region would then reduce the extensibility of the molecule without affecting actin crosslinking, consistent with their typically producing the relatively benign Becker phenotype of muscular dystrophy.

Actinin↗
PubMed1989

Parallel modulation of brush border myosin conformation and enzyme activity induced by monoclonal antibodies.

Monoclonal antibodies binding to distinct epitopes on the tail of brush border myosin were used to modulate the conformation and state of assembly of this myosin. BM1 binds 1:3 of the distance from the tip of the tail to the head and prevents the extended-tail (6S) monomer from folding into the assembly-incompetent folded-tail (10S) state, whereas BM4 binds to the tip of the myosin tail, and induces the myosin to fold into the 10S state. Thus, at physiological ionic strength BM1 promotes and BM4 blocks the assembly of the myosin into filaments. Using BM1 and BM4 together, we were able to prevent both folding and filament assembly, thus locking myosin molecules in the extended-tail 6S monomer conformation at low ionic strength where they normally assemble into filaments. Using these myosin-antibody complexes, we were able to investigate independently the effects of folding of the myosin tail and assembly into filaments on the myosin MgATPase. The enzymatic activities were measured from the fluorescent profiles during the turnover of the ATP analogue formycin triphosphate (FTP). Extended-tail (6S) myosin molecules had an FTPase activity of 1-5 X 10(-3) s-1, either at high ionic strength as a monomer alone or when complexed with antibody, or at low ionic strength as filaments or when maintained as extended-tail monomers by the binding of BM1 and BM4. Folding of the molecules into the 10S state reduced this rate by an order of magnitude, effectively trapping the products of FTP hydrolysis in the active sites.

Animals↗
PubMed1988

Active site trapping of nucleotide by smooth and non-muscle myosins.

The folded 10 S monomer conformation of smooth muscle myosin traps the hydrolysis products ADP and Pi in its active sites. To test the significance of this, we have searched for equivalent trapping in other conformational and assembly states of avian gizzard and brush border myosins, using formycin triphosphate (FTP) as an ATP analogue. When myosin monomers were in the straight-tail 6 S conformation, the hydrolysis products were released at about 0.03 s-1. Adoption of the folded 10 S monomer conformation reduced this rate by more than 100-fold, effectively trapping the products FDP and Pi in the active sites. This profound inhibition of product release occurred only on formation of the looped tail monomer conformation. In vitro-assembled myosin filaments released products at a comparable rate to free straight-tail 6 S monomers, and smooth muscle heavy meromyosin, which lacks the C-terminal two-thirds of the myosin tail, also did not trap the products in this way. Phosphorylation of the myosin regulatory light chain had no effect on the rate of product release from straight-tail 6 S myosin monomers or from myosin filaments. Rather, it allowed actin to accelerate product release. Phosphorylation acted also to destabilize the folded monomer conformation, causing the recruitment of molecules from the pool of folded monomers into the myosin filaments. The two processes of contraction and filament assembly are thus both controlled in vitro by light-chain phosphorylation. A similar linked control in vivo would allow the organization of myosin in the cell to adapt itself continuously to the pattern of contractile activity.

Adenosine Triphosphate↗
PubMed1988

Spatial pattern of myosin phosphorylation in contracting smooth muscle cells: evidence for contractile zones.

We have purified a polyclonal antibody by affinity chromatography which binds specifically to the phosphorylated form of the regulatory light chain (Mr = 20,000) of smooth muscle myosin. This antibody does not stain relaxed, permeabilized smooth muscle cells isolated from guinea pig taenia coli. However, when these cells were stimulated to contract with CaCl2 (100 microM) and ATP (1 mM), the immunofluorescence staining was localized in a series of transverse bands. This distribution of activated myosin appears to reflect an underlying structural organization of the smooth muscle cell cytoskeleton into mechanically coupled contractile zones.

Animals↗
PubMed1987

Salt dependent dimerisation of caldesmon.

Using analytical gel filtration (FPLC) we show here that avian gizzard caldesmon (chain molecular mass 150 kDa) self-associates to form end-to-end dimers. Increasing salt concentration promotes dimerisation: at 150 mM KCl, about 40% of the caldesmon was dimeric. Freshly gel filtered caldesmon had an actin gelating activity which decreased with increasing ionic strength. At 150 mM KCl, caldesmon at a 1:90 molar ratio to actin doubled the low shear viscosity of F-actin. Sixfold less filamin was required to produce the same effect.

Animals↗
PubMed1986

Solubility-determining domain of smooth muscle myosin rod.

Chymotryptic digestion of chicken gizzard light meromyosin (LMM) produced a 72 kDa core fragment, which was fully soluble at 150 mM KCl, pH 6.5-7.5. The fragment showed weak self-association at 50 mM KCl. The homology of the N-terminus amino acid sequence of this fragment with the sequence of the rabbit skeletal myosin rod suggested that the N-terminus of the core fragment originated 5 kDa from the hinge common to both smooth and skeletal myosin rod. Sedimentation experiments indicated that the domain specifying the insolubility of the intact LMM was 13 kDa long. Progressive proteolytic shortening of this region produced LMM fragments of progressively increasing solubility. Electron microscopy of segments formed from full-length LMM and from LMM core suggested that this 13 kDa domain specified the 43 nm parallel and antiparallel molecular overlaps characteristic of self-assembled intact myosin.

Amino Acid Sequence↗
PubMed1986

Caldesmon is an elongated, flexible molecule localized in the actomyosin domains of smooth muscle.

A rapid purification procedure has been developed for the isolation of caldesmon from hog stomach smooth muscle utilizing a KI extract of washed myofibrils as source material. On SDS-PAGE this mammalian caldesmon showed a closely-spaced doublet around 155 kd. By low-angle rotary shadowing caldesmon was shown to be an elongated, highly flexible molecule which tends to form end-to-end dimers that are structurally very similar to filamin. When added to F-actin solutions caldesmon increased the high-shear viscosity considerably, but by an extent that depended on sample preparation. The effect was shown to be due to caldesmon and not to a trace contaminant by its full reversibility after addition of a monospecific caldesmon antibody. Recent investigations have shown that in smooth muscle two structurally distinct domains can be distinguished: an actomyosin domain and an actin-intermediate filament domain. Immunocytochemistry of ultrathin sections of smooth muscle at the light and electron microscope level revealed that caldesmon is present in the actomyosin domain. Caldesmon is thus a potential regulator of the actomyosin system in smooth muscle.

Actins↗
PubMed1986

ATP-linked monomer-polymer equilibrium of smooth muscle myosin: the free folded monomer traps ADP.Pi.

In vitro and at physiological ionic strength, unphosphorylated smooth muscle myosin filaments dissolve on addition of ATP, forming folded (10S) myosin monomers. By following the fate of ATP and the time course of filament disassembly we have established details of the mechanism of this process. Myosin filaments first bind and hydrolyse 2.0 mol/mol of ATP before significant filament dissolution occurs. Following dissolution, the hydrolysis products ADP.Pi are retained on the heads of the folded myosin monomers, and are released so slowly (half time approximately 100 min at 100 mM KCl) as to be effectively trapped. The straight (6S) conformation of myosin, stable at greater than 225 mM KCl, did not exhibit this product trapping, and neither did myosin filaments held under conditions which disfavour ATP-induced disassembly. The implications of these results for filament stability in vivo are discussed in terms of a simple, testable model for smooth muscle myosin self-assembly.

Adenosine Diphosphate↗