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G Holzwarth

Publications and source records attributed to G Holzwarth.

At least 19 recordsLinked to original sources
PubMed2004

Fast vesicle transport in PC12 neurites: velocities and forces.

Although the mechanical behavior of single-motor protein molecules such as kinesin has been carefully studied in buffer, the mechanical behavior of motor-driven vesicles in cells is much less understood. We have tracked single vesicles in neurites of PC12 cells with a spatial precision of +/-30 nm and a time resolution of 120 ms. Because the neurites are thin, long, straight, and attached to the surface of planar cover glasses, the velocity of individual vesicles could be measured for times as long as 15 s and distances as long as 15 mum. The velocity of anterograde vesicles was in most cases constant for periods of 1-2 s, then changed in a step-like fashion to a new constant velocity. The viscoelastic modulus felt by the vesicles within live PC12 cells was determined from the Brownian motion, using Mason's generalization of the Stokes-Einstein equation. From Stokes' law, the drag force at the smallest sustained velocity was 4.2+/-0.6 pN for vesicles of radius 0.30-0.40 mum, about half the maximum force which conventional kinesin can develop during bead assays in buffer. We interpret the observed velocity steps as changes of +/-1 or occasionally +/-2 in the number of active motor proteins dragging that vesicle along a microtubule. Assuming that the motor is conventional kinesin, which hydrolyzes one ATP per 8 nm step along the microtubule, the motor protein efficiency in PC12 neurites is approximately 35%.

Animals↗
PubMed2002

Forces required of kinesin during processive transport through cytoplasm.

The purpose of this paper is to deduce whether the maximum force, steplike movement, and rate of ATP consumption of kinesin, as measured in buffer, are sufficient for the task of fast transport of vesicles in cells. Our results show that moving a 200-nm vesicle in viscoelastic COS7 cytoplasm, with the same steps as observed for kinesin-driven beads in buffer, required a maximum force of 16 pN and work per step of 1 +/- 0.7 ATP, if the drag force was assumed to decrease to zero between steps. In buffer, kinesin can develop a force of 6-7 pN while consuming 1 ATP/step, comparable to the required values. As an alternative to assuming that the force vanishes between steps, the measured COS7 viscoelasticity was extrapolated to zero frequency by a numerical fit. The force required to move the bead then exceeded 75 pN at all times and peaked briefly to 92 pN, well beyond the measured capabilities of a single kinesin in buffer. The work per step increased to 7 +/- 5 ATP, greatly exceeding the energy available to a single motor.

Adenosine Triphosphate↗
PubMed1996

Real-time velocity of DNA bands during field-inversion gel electrophoresis.

The velocity v of bands of double-stranded, linear DNAs containing 48.5-5700 kbp was determined with 0.3 s resolution during field-inversion agarose gel electrophoresis (FIGE) for a broad range of the forward pulse period T+, keeping the duration of the backward pulse T- = T+/3. Within 0.6 s or less after the field changed sign from-to +, the velocity showed a sharp positive peak; a similar spike, but with negative velocity, occurred immediately after the field changed from + to -. For long pulses, the magnitude of this spike increased with M0.36, reaching ten times the steady-state velocity for M = 5.7 kbp. After this spike, the velocity dipped to 55-75% of its value in a steady field, then increased to a small secondary peak before reaching a steady-state plateau. The duration of the velocity trough, and the time of the small peak, increased as M1. For standard FIGE conditions (ratio of forward:reverse pulse duration, T+:T- = 3:1; equal forward and reverse field amplitudes, E+ = E-), the mobility mu = integral of vdt over a complete cycle was a minimum when E+ terminated at the end of the velocity trough. The minimum occurred because the velocity during E+ sampled primarily the trough, and because the backward velocity during E- was exceptionally large; the negative velocity spike was maximized when T+ terminated at the end of the velocity trough. Computer simulations of FIGE by Zimm (J. Chem. Phys. 1991, 94, 2187-2206) and by Duke and Viovy (J. Chem. Phys. 1992, 96, 8552-8563) generate real-time velocities that are in excellent agreement with our experimental data.

Bacteriophage T4↗
PubMed1996

Channel thickness variations could degrade resolution in electrophoresis.

Variations in the lateral dimensions of capillaries and ultrathin gels along their length could lead to a distortion of the sample zone because charged particles traveling near the wall must travel a longer distance under a smaller field than particles traveling along the centerline. The size of the effect is computed by solving Laplace's equation for the potential everywhere in a conducting slab with a discontinuity in the lateral discontinuity in the lateral dimensions and integrating the travel time along different streamlines.

Electrochemistry↗
PubMed1993

Two-dimensional motion of DNA bands during 120 degrees pulsed-field gel electrophoresis.

The position and velocity of a band of double-stranded, linear DNA from bacteriophage G were measured during 120 degrees pulsed-field gel electrophoresis, using a video micrometer. Both the x and y coordinates were determined simultaneously in the plane of a 1% agarose gel; x is the mean drift direction. For pulse durations T greater than the tube renewal time T*, the path traced by the band of 670 kb DNA in the xy plane was in remarkably good accord with that predicted by Southern's ratchet model. However, the measured instantaneous velocity vx showed a sharp backward spike each time the field changed direction, with amplitude about twice the mean drift velocity. This spike is not consistent with models which assume a constant curvilinear velocity of DNA in a tube, nor with the biased reptation model without fluctuations. The corresponding measurements of vy showed a sharp positive spike with amplitude more than 3 times the plateau velocity in the y direction; neither model predicted this. The sharp velocity spikes are consistent with the idea that, for T > T*, a large fraction of the DNA chains are stretched into U-shaped or herniated configurations. When the field changes direction, the arms of the U's and the hernias recoil rapidly in response to intramolecular DNA chain tension. Because the base of a U or hernia is fixed by gel fibers, the center of mass of the chain recoils backward every time the field changes direction.

Chemical Phenomena↗
PubMed1990

On the movement and alignment of DNA during 120 degrees pulsed-field gel electrophoresis.

The displacement per pulse of lambda, T4, and G DNA during pulsed-field agarose gel electrophoresis has been measured for a fine mesh of pulse durations T between 0.02 and 120 s. The slopes of these curves show that the DNA moves by two distinct processes, designated 1 and 2, depending upon the pulse duration T. Process 1 operates at short T and causes dx/dT to decrease gradually with increasing T. This process is independent of molecular weight M. Process 2 is effective at longer T and causes dx/dT to rise sharply in sigmoidal fashion at a value of T which increases as M1.2, finally reaching a plateau of 1.4 microns/s for E = 4 V/cm. The shape of the dx/dT curve and its dependence on M lead directly to 4 zones of separation in plots of mobility vs M for different T. The alignment of the 3 DNAs during PFGE was measured by fluorescence-detected linear dichroism for E between 4 and 10 V/cm. These results are used in developing a molecular understanding of the mobility data.

Coliphages↗
PubMed1989

The acceleration of linear DNA during pulsed-field gel electrophoresis.

The velocity and orientation of T4 and lambda DNA have been measured for the first 20 s during pulsed-field gel electrophoresis in order to clarify the DNA motions that occur. For a square pulse with field strength E = 10 V/cm, the velocity of lambda DNA increases gradually to 10.5 microns/s in 1.0 s, declines to 8.6 microns/s, and then rises to a plateau value of 9.3 microns/s after 4 s. T4 DNA behaves similarly, but more slowly. Parallel measurements of fluorescence-detected linear dichroism show that the DNA becomes substantially aligned with its chain axis parallel to the electrophoretic field E after the pulse is applied. The alignment also shows an overshoot, an undershoot, and a plateau comparable to those seen for velocity. When the field strength increases, both the velocity and the alignment reach their peaks more quickly. For all field strengths and both molecular weights, the velocity peak occurs when the molecular center of mass has moved 0.3 to 0.5 L, where L is the chain contour length. A qualitative model is provided.

Bacteriophage lambda↗
PubMed1987

Transient orientation of linear DNA molecules during pulsed-field gel electrophoresis.

The transient orientation of lambda DNA and lambda-DNA oligomers has been measured during pulsed field gel electrophoresis. The DNA becomes substantially aligned parallel to the electric field E. In response to a single rectangular pulse, orientation shows an overshoot with a peak at 1 second, then a small undershoot, and finally a plateau. When the field is turned off, the orientation dissipates in two distinct exponential phases. Field inversion leads to periods of orientation with intervening periods of reduced orientation as the chains reverse direction. Field inversion pulses applied to linear oligomers of lambda-DNA show that orientation responses slow down but increase in amplitude as molecular weight increases, for a given field. Because DNA stretching and alignment parallel to E are expected to correlate with DNA velocity, the velocity in response to a pulsed field is also expected to exhibit an overshoot.

DNA↗
PubMed1977

Multistranded helix in xanthan polysaccharide.

The extracellular polysaccharide xanthan is shown by electron microscopy to be an unbranched, probably double-stranded fiber 4 nanometers wide and 2 to 10 micrometers long when native. Denaturation yields a single strand only 2 nanometers wide and 0.3 to 1.8 micrometers long. Renatured xanthan shows short unraveled regions with two or three strands arranged in a right-handed twist.

Microscopy, Electron↗
PubMed1976

Conformation of the extracellular polysaccharide of Xanthomonas campestris.

The solution conformation of the extracellular polysaccharide of the bacterium Xanthomonas campestris is examined by optical rotation, viscometry, and potentiometric titration. Measurements of optical rotation vs. temperature for solutions of the polysaccharide at low ionic strength reveal a sharp transition to a denatured structure which is reversible if sufficient salt is present. The temperature Tm at the transition midpoint increases as log (Na+) or log (Ca2+). Viscosity-temperature profiles substantiate a structural change of the polysaccharide at Tm. The intrinsic viscosity of the native molecule at zero shear rate exceeds 5000 ml/g. This high figure is indicative of a stiff chain. The viscosity of the native molecule is relatively insensitive to salt, whereas the denatured molecule collapses if salt is present. Hydrogen-ion titration shows that the pKapp of the COO- groups of the polymer decreases from 3.2 in 0.01 M NaC1 to 2.6 in 0.2 M NaC1. All these data suggest that the native polysaccharide possesses ordered secondary structure stabilized by nonionic interactions outweighing the repulsion between adjacent COO- groups.

Hydrogen-Ion Concentration↗
PubMed1976

Backbone conformational change in the A to B Transition of deoxyribonucleic acid.

Infrared linear dichroism studies of A-and B-DNA films reveal six bands between 2800 and 3000 cm-1 which must arise from deoxyribose and thymine methyl CH stretching motions. The band at 2890 is perpendicularly polarized in A-DNA but parallel polarized in B-DNA. This band most probably originates in the C'(5)H2 symmetric stretch; the polarization flip is consistent with the structural change occurring at C'(5) during the A to B transition, according to models derived from x-ray work.

DNA↗
PubMed1976

Heterogeneity in the conformation of different protein fractions from the human erythrocyte membrane.

We have isolated 5 families of proteins from human red blood cell membranes and characterized their secondary structure by ultraviolet circular dichroism measurements. The protein families were prepared by selective solubilization from ghosts under nondenaturing conditions. We find that the intact ghost has a mean alpha-helix fraction of 0.37, whereas a low-ionic-strength extract (bands 1, 2, 5, "spectrin") has a substantially higher helix fraction, 0.55. Further extraction of the ghosts with para-chloromercuribenzoate yields bands 2.1, 4.1, 4.2, and 6; their helix content is only 0.17. Finally, the major intrinsic protein, band 3, was solubilized by a non-ionic detergent. Its helix fraction is 0.38.

Blood Proteins↗