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Biomedical subjects

B D Hambly

Publications and source records attributed to B D Hambly.

At least 19 recordsLinked to original sources
PubMed2001

Independent movement of the regulatory and catalytic domains of myosin heads revealed by phosphorescence anisotropy.

Inter- and intradomain flexibility of the myosin head was measured using phosphorescence anisotropy of selectively labeled parts of the molecule. Whole myosin and the myosin head, subfragment-1 (S1), were labeled with eosin-5-iodoacetamide on the catalytic domain (Cys 707) and on two sites on the regulatory domain (Cys 177 on the essential light chain and Cys 154 on the regulatory light chain). Phosphorescence anisotropy was measured in soluble S1 and myosin, with and without F-actin, as well as in synthetic myosin filaments. The anisotropy of the former were too low to observe differences in the domain mobilities, including when bound to actin. However, this was not the case in the myosin filament. The final anisotropy of the probe on the catalytic domain was 0.051, which increased for probes bound to the essential and regulatory light chains to 0.085 and 0.089, respectively. These differences can be expressed in terms of a "wobble in a cone" model, suggesting various amplitudes. The catalytic domain was least restricted, with a 51 +/- 5 degrees half-cone angle, whereas the essential and regulatory light chain amplitude was less than 29 degrees. These data demonstrate the presence of a point of flexibility between the catalytic and regulatory domains. The presence of the "hinge" between the catalytic and regulatory domains, with a rigid regulatory domain, is consistent with both the "swinging lever arm" and "Brownian ratchet" models of force generation. However, in the former case there is a postulated requirement for the hinge to stiffen to transmit the generated torque associated by nucleotide hydrolysis and actin binding.

Animals↗
PubMed2001

The regulatory domain of the myosin head behaves as a rigid lever.

The regulatory domain of the myosin head is believed to serve as a lever arm that amplifies force generated in the catalytic domain and transmits this strain to the thick filament. The lever arm itself either can be passive or may have a more active role storing some of the energy created by hydrolysis of ATP. A structural correlate which might distinguish between these two possibilities (a passive or an active role) is the stiffness of the domain in question. To this effect we have examined the motion of the proximal (ELC) and distal (RLC) subdomains of the regulatory domain in reconstituted myosin filaments. Each subdomain was labeled with a spin label at a unique cysteine residue, Cys-136 of ELC or Cys-154 of mutant RLC, and its mobility was determined using saturation transfer electron paramagnetic resonance spectroscopy. The mobility of the two domains was similar; the effective correlation time (tau(eff)) for ELC was 17 micros and that for RLC was 22 micros. Additionally, following a 2-fold change of the global dynamics of the myosin head, effected by decreasing the interactions with the filament surface (or the other myosin head), the coupling of the intradomain dynamics remained unchanged. These data suggest that the regulatory domain of the myosin head acts as a single mechanically rigid body, consistent with the regulatory domain serving as a passive lever.

Animals↗
PubMed2000

Growth arrest-specific gene 6 expression in proliferating rabbit vascular smooth muscle cells in vitro and in vivo.

Proliferation and migration of vascular smooth muscle cells (VSMCs) are involved in the processes of atherosclerosis and restenosis. The protein product of the growth arrest-specific gene 6 (Gas-6) has recently been identified as a ligand for the Axl/Rse/Mer tyrosine kinase receptor family, which may be involved in proliferation and migration of VSMCs. Here we show that Gas-6 gene expression is increased in proliferating VSMCs in tissue culture (2.5-fold increase by Northern blot) and following neointimal proliferation in a rabbit balloon-injury model (3-fold increase by Western blot). Neither platelet-derived growth factor (PDGF) nor thrombin stimulate the expression of Gas-6 in cultured VSMCs despite the ability of the PDGF, but not thrombin, to stimulate proliferation in growth-arrested cells. These data suggest a role for the Gas-6 regulatory system in VSMC proliferation, which may be a target for therapeutic interventions in the atherosclerotic process and restenosis after angioplasty.

Animals↗
PubMed2000

P2X (purinergic) receptor redistribution in rabbit aorta following injury to endothelial cells and cholesterol feeding.

The redistribution of purinergic P2X receptor subunits (P2X(1) to P2X(7)) within the rabbit aorta wall three weeks after endothelial balloon injury/cholesterol feeding was examined. P2X(1) receptor cluster density was elevated in the media following balloon injury/cholesterol feeding by about 30% and these clusters appeared on smooth muscle cells throughout the greatly expanded neointima but they did not change significantly on the endothelial cells following balloon injury. P2X(4) clusters were found in high density throughout the media and in very high density in the enlarged neointima following balloon injury, particularly on the endothelial cells where the density increased about 10-fold after balloon injury. P2X(5) clusters were found in high density in the media of normal aorta but with little change following balloon injury. P2X(3), P2X(6) and P2X(7) cluster density was low in normal aorta and remained unchanged following balloon injury. All receptor subunits were found on endothelial cells. It is suggested that the release of ATP from damaged endothelial cells and from smooth muscle cells sufficient to activate P2X(4) receptors may contribute to neointimal proliferation.

Animals↗
PubMed1999

Expression and localisation of stanniocalcin 1 in rat bladder, kidney and ovary.

Bony fish use the glycoprotein hormone stanniocalcin (STC) to counteract hypercalcaemia. This is achieved through dual mechanisms involving gill calcium uptake inhibition and stimulation of renal inorganic phosphate reabsorption. Human STC (hSTC-1) shows considerable homology with both rat and mouse STC (mSTC) and their mRNA is expressed in a wide range of tissues. In fish, STC is produced by endocrine glands known as the corpuscles of Stannius but in mammals the widespread expression is suggestive of a paracrine rather than an endocrine role. In order to determine the distribution and strucutral characteristics of hSTC-1, the recombinant protein was expressed in bacteria, purified by metal-ion affinity chromatography, and a study was made of the likely epitopes for raising an antibody. This novel hSTC-1 antibody was used to test the purification protocol. Since the role of mammalian STC is largely unknown, the specific distribution of STC needed to be addressed. To test the specificity of the antibody, sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE)/Western blotting was undertaken in homogenised rat bladder, ovary and kidney.

Amino Acid Sequence↗
PubMed1998

EPR and CD spectroscopy of fast myosin light chain conformation during binding of trifluoperazine.

The conformations of isolated rabbit fast myosin light chains (LCs) were modified using trifluoperazine (TFP), the hydrophobic calmodulin inhibitor. CD spectroscopy showed that TFP altered secondary structural content of the LCs, with half-maximal effects at TFP concentrations of approximately 14-50 microM, which is within the range required to alter muscle fiber contraction in both agonistic and antagonistic ways [Kurebayashi, N. & Ogawa, Y. (1988) J. Physiol. 403, 407-424]. EPR spectroscopy provided structural information from paramagnetic probes on C-terminal domain surfaces. In the absence of TFP, tauR (rotational correlation time) was 1.6 ns for both alkali light chains (ALCs) and 1.8 ns for light chain 2 (LC2). This was faster than expected for proteins of this size (approximately 10 ns). TFP progressively recruited the probes into populations with tauR sevenfold to 12-fold slower, with half-maximal effects at a TFP concentration of approximately 370-800 microM. The differences probably indicate that CD spectroscopy detects changes in protein conformation due to 'specific' TFP binding at the LC hydrophobic core, while less specific binding at higher TFP concentrations is required to effect conformational changes on the protein surfaces near the paramagnetic probes. TFP binding was generally not cooperative. Comparative sequence analysis between calmodulin, troponin C, and myosin LCs indicated considerable conservation between residues expected to bind TFP.

Amino Acid Sequence↗
PubMed1998

Evaluation of the risks of using an oversized balloon catheter in the human infrarenal abdominal aorta.

OBJECTIVES: To evaluate the effects on the aortic wall of balloon dilatation as utilised in deployment of stent-graft devices during endoluminal repair of infrarenal abdominal aortic aneurysm. METHODS: Large dilatation balloons were expanded within the aorta of 41 cadavers. Testing was done to evaluate the effect of differing degrees of balloon oversizing, at pressures in the range of 0.15-2.5 atm. The aorta was then open for macroscopic inspection. RESULTS: In group 1 (mild atherosclerosis) no macroscopic abnormalities were detected with up to 6 mm oversized balloon. In group 2 (moderate atherosclerosis) fracture of atherosclerotic plaque occurred in seven of 14 aortas (50%) with 2.5 mm-4mm oversized balloon. In group 3 (severe atherosclerosis) fracture of atherosclerotic plaque occurred in six of seven (85%) with 2.5 mm to 4 mm oversized balloon and rupture of the aorta occurred at 6 mm oversizing. CONCLUSIONS: This study suggests that balloon overdilatation of the aorta by 2 mm, at pressures less than 2 atmospheres, allows safe deployment even in the presence of severe atheroma. Larger amounts of overdilatation are relatively safe in mildly atherosclerotic aorta. Aortic rupture is unlikely with overdilatation up to 6 mm, especially in less calcified vessels.

Adolescent↗
PubMed1997

Separation of tumor necrosis factor alpha isoforms by two-dimensional polyacrylamide gel electrophoresis.

The mouse macrophage cell-line RAW264.7, stimulated with lipopolysaccharide, was used as a model for the study of the production of tumor necrosis factor (TNF) isoforms. TNF is synthesised initially as a 26 kDa transmembrane precursor, which is then processed enzymatically by a protease to release a mature molecule of 17 kDa. Dose-dependent production of transmembrane TNF was assessed by fractionation of cell membranes and Western blot analysis followed by autoradiography and densitometry. Isoforms of both the precursor and mature molecules were separated using two-dimensional (2-D) electrophoresis with immobilised pH gradient 3-10 linear gels as the first dimension. After radiolabelling of cells with 35S, both cell-associated and supernate-associated TNF isoforms were immunoprecipitated. A large number of protein spots were visualised on the 2-D gel map, for both the transmembrane and mature TNF species, more than have been detected previously using one-dimensional sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE). The likelihood that these putative isoforms were the result of differential glycosylation was tested by preincubating the cells with tunicamycin. This had the effect of reducing the number of protein spots, notably the higher molecular weight species. There were a number of precursor TNF isoforms that were unchanged upon tunicamycin treatment and these presumably reflect protein modifications other than glycosylation.

Animals↗
PubMed1996

Distance measurements near the myosin head-rod junction using fluorescence spectroscopy.

We reacted a fluorescent probe, N-methyl-2-anilino-6-naphthalenesulfonyl chloride (MNS-Ci), with a specific lysine residue of porcine cardiac myosin located in the S-2 region of myosin. We performed fluorescence resonance energy transfer (FRET) spectroscopy measurements between this site and three loci (Cys109, Cys125, and Cys154) located within different myosin light-chain 2s (LC2) bound to the myosin "head". We used LC2s from rabbit skeletal muscle myosin (Cys125), chicken gizzard smooth muscle myosin (Cys109), or a genetically engineered mutant of chicken skeletal muscle myosin (Cys154). The atomic coordinates of these LC2 loci can be closely approximated, and the FRET measurements were used to determine the position of the MNS-labeled lysine with respect to the myosin head. The C-terminus of myosin subfragment-1 determined by Rayment et al. ends abruptly after a sharp turn of its predominantly alpha-helical structure. We have constructed a model based on our FRET distance data combined with the known structure of chicken skeletal muscle myosin subfragment-1. This model suggests that the loci that bracket the head-rod junction will be useful for evaluating dynamic changes in this region.

Allosteric Site↗
PubMed1995

Measuring protein self-association using pulsed-field-gradient NMR spectroscopy: application to myosin light chain 2.

At the millimolar concentrations required for structural studies, NMR spectra of the calcium-binding protein myosin light chain 2 (MLC2) showed resonance line widths indicative of extensive self-association. Pulsed-field-gradient (PFG) NMR spectroscopy was used to examine whether MLC2 aggregation could be prevented by the zwitterionic bile salt derivative 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS). PFG NMR measurements indicated that CHAPS was capable of preventing MLC2 self-association, but only at concentrations well above the critical micelle concentration of approximately 7.5 mM. CHAPS was most effective at a concentration of 22.5 mM, where the apparent molecular mass of MLC2 corresponded to a protein monomer plus seven molecules of bound detergent. The resolution and sensitivity of 2D 15N-1H HSQC spectra of MLC2 were markedly improved by the addition of 25 mM CHAPS, consistent with a reduction in aggregation following addition of the detergent. The average amide nitrogen T2 value for MLC2 increased from approximately 30 ms in the absence of CHAPS to approximately 56 ms in the presence of 25 mM CHAPS. The results of this study lead us to propose that PFG NMR spectroscopy can be used as a facile alternative to conventional techniques such as analytical ultracentrifugation for examining the self-association of biological macromolecules.

Animals↗
PubMed1994

Evidence for an F-actin like conformation in the actin:DNase I complex.

We demonstrate that a ribose modified analogue of ATP, TNP-ATP, can exchange with a resident nucleotide in F-actin, but fails to bind to G-actin. TNP-ATP is also able to bind to actin in the actin:DNase I complex, suggesting that the nucleotide binding site in the actin:DNase I complex adopts a conformation similar to that found in F-actin. This result is consistent with the hypothesis that the two major domains of actin on either side of the cleft are able to "flex" or move relative to each other in G-actin, but that this flexing motion is limited as a consequence of either polymerisation or DNase I binding. F-actin, in which approximately 80% of the bound nucleotide is TNP-ADP, appears to be functionally similar to native ADP-F-actin. It can superprecipitate with myosin and, following regulation with troponin-tropomyosin, exhibits a Ca(2+)-sensitivity during superprecipitation. Sonication induced nucleotide exchange in regulated F-actin was not sensitive to the presence of Ca2+ which argues against a significant conformational change in the vicinity of the nucleotide binding site during Ca(2+)-sensitive thin filament regulation.

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↗
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

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

Localization of the phalloidin and nucleotide-binding sites on actin.

Phalloidin was found to block nucleotide exchange in F-actin, without interfering with nucleotide hydrolysis. This inhibition of nucleotide exchange occurs under conditions in which monomers are able to exchange. The distance separating a fluorescent chromophore attached to phalloidin from the nucleotide on actin was determined using fluorescence resonance energy-transfer spectroscopy. They are separated by less than 1.0 nm. Added confirmation of the close proximity of phalloidin to nucleotide was obtained by extracting a small peptide-ATP complex from an actin digest. The peptide comprises residues 114-118, which are from the same region as the residues that others have shown to crosslink to phalloidin [Vandekerckhove et al. (1985) EMBO J. 4, 2815-2818]. The results suggest that phalloidin has two major effects. It traps actin monomers in a conformation which appears to be distinct from G-actin and it stabilizes the structure of F-actin, an event accompanied by the trapping of ADP.

Actins↗
PubMed1986

Extraction of myosin light chains and actin from bovine cardiac muscle acetone powder.

A joint preparation of the two myosin light chains and actin from bovine cardiac muscle acetone powder is described. There is a significant improvement in the ease of purification, while the yield of the myosin light chains equals the best yields obtained from the use of established techniques. The actin yield greatly exceeds that obtained in an earlier published report.

Acetone↗
PubMed1986

Fluorescence energy transfer between nucleotide binding sites in an F-actin filament.

Fluorescence energy transfer between nucleotide binding sites in an F-actin filament was measured using 1-N6-ethenoadenosine diphosphate (epsilon-ADP) as a fluorescent donor and 2'(or 3')-O-(2,4,6-trinitrophenyl)adenosine 5'-diphosphate (TNP-ADP) as an acceptor, both of which were bound to F-actin. Taking into consideration the helical structure of the F-actin filament, the radial coordinate of the nucleotide binding site was calculated to be 25 A, which corresponds to a distance between these sites along the long-pitch helix of 56.3 A and along the genetic helix of 56.7 A.

Actins↗