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M Anson

Publications and source records attributed to M Anson.

15 recordsLinked to original sources
PubMed2000

Comparative single-molecule and ensemble myosin enzymology: sulfoindocyanine ATP and ADP derivatives.

Single-molecule and macroscopic reactions of fluorescent nucleotides with myosin have been compared. The single-molecule studies serve as paradigms for enzyme-catalyzed reactions and ligand-receptor interactions analyzed as individual stochastic processes. Fluorescent nucleotides, called Cy3-EDA-ATP and Cy5-EDA-ATP, were derived by coupling the dyes Cy3.29.OH and Cy5.29.OH (compounds XI and XIV, respectively, in, Bioconjug. Chem. 4:105-111)) with 2'(3')-O-[N-(2-aminoethyl)carbamoyl]ATP (EDA-ATP). The ATP(ADP) analogs were separated into their respective 2'- and 3'-O-isomers, the interconversion rate of which was 30[OH(-)] s(-1) (0.016 h(-1) at pH 7.1) at 22 degrees C. Macroscopic studies showed that 2'(3')-O-substituted nucleotides had properties similar to those of ATP and ADP in their interactions with myosin, actomyosin, and muscle fibers, although the ATP analogs did not relax muscle as well as ATP did. Significant differences in the fluorescence intensity of Cy3-nucleotide 2'- and 3'-O-isomers in free solution and when they interacted with myosin were evident. Single-molecule studies using total internal reflection fluorescence microscopy showed that reciprocal mean lifetimes of the nucleotide analogs interacting with myosin filaments were one- to severalfold greater than predicted from macroscopic data. Kinetic and equilibrium data of nucleotide-(acto)myosin interactions derived from single-molecule microscopy now have a biochemical and physiological framework. This is important for single-molecule mechanical studies of motor proteins.

Actins↗
PubMed1996

Myosin motors with artificial lever arms.

The myosin head consists of a globular catalytic domain and a light chain binding domain (LCBD). The coupling efficiency between ATP hydrolysis and myosin-induced actin movement is known to decline as the LCBD is truncated or destabilized. However, it was not clear whether the observed alteration in the production of force and movement reflects only the mechanical changes to the length of the LCBD or whether these changes also affect the kinetic properties of the catalytic domain. Here we show that replacement of the LCBD with genetically engineered domains of similar rigidity and dimensions produces functional molecular motors with unchanged kinetic properties. The resulting single-chain, single-headed motors were produced in Dictyostelium discoideum and obtained after purification from a standard peptone-based growth medium at levels of up to 12 mg/l. Their actin motility properties are similar or greater than those of native myosin. Rates of 2.5 and 3.3 microm/s were observed for motor domains fused to one or two of these domains, respectively. Their kinetic and functional similarity to the extensively studied myosin subfragment 1 (S1) and their accessibility to molecular genetic approaches makes these simple constructs ideal models for the investigation of chemo-mechanical coupling in the myosin motor.

Actin Cytoskeleton↗
PubMed1995

Actomyosin kinetics and in vitro motility of wild-type Drosophila actin and the effects of two mutations in the Act88F gene.

Two missense mutations of the flight muscle-specific actin gene of Drosophila melanogaster, Act88F, assemble into normally structured myofibrils but affect the flight ability of flies and the mechanical kinetics of isolated muscle fibers. We describe the isolation of actin from different homozygous Act88F strains, including wild-type, an Act88F null mutant (KM88), and two Act88F single point mutations (E316K and G368E), their biochemical interactions with rabbit myosin subfragment 1 (S1), and behavior with rabbit myosin and heavy meromyosin in in vitro motility assays. The rabbit and wild-type Drosophila actins have different association rate constants with S1 (2.64 and 1.77 microM-1 s-1, respectively) and in vitro motilities (2.51, 1.60 microns s-1) clearly demonstrating an isoform-specific difference. The G368E mutation shows a reduced affinity for rabbit S1 compared with the wild type (increasing from 0.11 to 0.17 microM) and a reduced velocity in vitro (reduced by 19%). The E316K mutant actin has no change in affinity for myosin S1 or in vitro motility with heavy meromyosin but does have a reduced in vitro motility (15%) with myosin. These results are discussed with respect to the recently published atomic models for the actomyosin structure and our findings that G368E fibers show a reduced rate constant for delayed tension development and increased fiber stiffness. We interpret these results as possibly caused either by effects on A1 myosin light chain binding or conformational changes within the subdomain 1 of actin, which contains the myosin binding site. E316K is discussed with respect to its likely position within the tropomyosin binding site of actin.

Actins↗
PubMed1992

Temperature dependence and Arrhenius activation energy of F-actin velocity generated in vitro by skeletal myosin.

The effect of temperature on the velocity of rhodamine phalloidin-labelled F-actin moving in vitro on rabbit skeletal myosin has been studied. Translating actin filaments were visualized by epi-fluorescence in an inverted microscope, equipped with temperature control (+/- 0.2 K) of the stage and objective. Images were recorded in real time at magnifications of 400x or 160x by an intensified CCD camera on video tape. Motion of individual filaments was tracked by hand and velocities determined using frame times recorded simultaneously on the video tape. Velocity changed from 12 microns per second at 42 degrees C to 11 nm per second at 3 degrees C. The Arrhenius plot is non-linear, with the data following a cubic regression curve with no evident breaks or jumps. Data taken over the temperature range from single preparations followed the same curve for both heating and cooling; this indicates reversibility and absence of hysteresis. A hyperbolic model that smoothly translates with temperature between two asymptotic activation energies fits the data above 7 degrees C: these energies are 50(+/- 5) kJ per mole (Q10 = 1.9) at high temperatures and 289(+/- 29) kJ per mole (Q10 = 76.5) at low temperature with a transition temperature of 15.4(+/- 0.6) degrees C. These values are compared with other measurements made in vitro, in solution studies and on muscle fibres. An Arrhenius activation energy of 50 kJ per mole and a transition temperature of 15 degrees C are consistent with previous determinations but 289 kJ per mole is significantly greater than has been seen at low temperatures in other systems. This may indicate a different rate-limiting step in the kinetics of skeletal myosin driving actin filaments in vitro below 15 degrees C. Current determinations of the myosin "step-size" assume that the actin velocity is determined by the rate of ATP hydrolysis; the data confirm similar activation energies above 20 degrees C but they show that the temperature dependencies and activation energies are different at lower temperatures, implying uncoupling of the two processes.

Actins↗
PubMed1987

Signal processing technique to extract neuronal activity from noise.

A method of extracting extracellularly recorded action potentials from background electronic noise is described. Segments of traces containing stimulus-induced activity are Fourier transformed and the increase in the total power density over that of control noise segments is used as a measure of stimulus-induced neuronal activity. We show first, with observations from the amphibian visual system and mammalian auditory system, that our technique yields similar quantitative information to that obtained from the conventional spike counting method when the recording arrangement is optimal. Moreover, the size and centre of a visual receptive field can be determined even when the evoked action potentials are buried in the background noise. To investigate the potential of this technique further, we have used it to study the auditory responses in the amphibian midbrain. The power spectral density, we demonstrate here, is proportional to the stimulus intensity over a wide range, and varies systematically with stimulus frequency and the direction of sound source. Other possible applications of this technique, together with the theoretical basis for it, are discussed.

Action Potentials↗
PubMed1985

Acoustic vibration of the amphibian eardrum studied by white noise analysis and holographic interferometry.

The motion of the amphibian eardrum under free-field acoustic stimulation was investigated using time-averaged holography. We show that the amplitude is linearly related to sound pressure up to +/- 1000 nm. The frequency response of the eardrum shows broad resonance characteristics with a main peak between 1200-2200 Hz. The velocity of the tympanic membrane's motion at its resonance frequency matches the acoustic velocity of air particles. The resonance characteristics of the eardrum are also revealed by white noise stimulation. The power spectrum obtained by Fourier transformation of the autocorrelation of the response to noise resembles closely that obtained by holography.

Acoustic Stimulation↗
PubMed1978

Neurophysiological basis of directional hearing in amphibia.

Discrete regions of neural tissue along the anterior-posterior axis of the torus semicircularis act as resonators, each one broadly tuned to a different range of frequencies, and each one responding optimally to the incident sound from a different direction. In this way, a map of auditory space is represented in the midbrain.

Animals↗
PubMed1978

Dynamics of the amphibian middle ear.

Using laser speckle interferometry, we show that the directional information of acoustic signals is encoded in the motion of eardrum. Moreover, the frequency sensitivity of the auditory system is determined by the mechanical properties of the middle ear, which acts like a damped resonator.

Animals↗
PubMedDate not supplied

Muscarinic acetylcholine receptor subtype mRNA expression and ligand binding in the aged rat forebrain.

Previous studies indicate that a 20-30% decline in muscarinic acetylcholine receptor binding occurs in localized areas of rat brain during aging. In this study, reduced [3H]-quinuclidinyl benzilate binding was observed in striata from 24-25-month-old rats relative to 5-6-month-old animals using homogenate binding assays. To determine if the decline in receptor concentration occurs as a result of decreased receptor synthesis, the expression of the m1, m3, and m4 muscarinic receptor mRNAs as well as [3H]-QNB binding were determined in adjacent sections of young and old male rats using in situ hybridization and in vitro receptor autoradiography respectively. A significant decline in collective muscarinic receptor binding as assessed by [3H]-QNB was observed in the caudate putamen, olfactory tubercle, nucleus accumbens, and several frontal and parietal cortical areas. The only difference observed in muscarinic mRNA expression for any of the three subtypes examined was a decline in m1 hybridization in the olfactory tubercle. The results of this study demonstrate that the regional brain areas displaying age-related decreases in receptor binding do not correlate with those areas showing a decrease in muscarinic receptor expression. Apparently, the decline in muscarinic acetylcholine receptor density with age does not result from a decline in receptor gene expression.

Aging↗