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R D Ludescher

Publications and source records attributed to R D Ludescher.

8 recordsLinked to original sources
PubMed1990

Time-resolved rotational dynamics of phosphorescent-labeled myosin heads in contracting muscle fibers.

We have measured the microsecond rotational motions of myosin heads in contracting rabbit psoas muscle fibers by detecting the transient phosphorescence anisotropy of eosin-5-maleimide attached specifically to the myosin head. Experiments were performed on small bundles (10-20 fibers) of glycerinated rabbit psoas muscle fibers at 4 degrees C. The isometric tension and physiological ATPase activity of activated fibers were unaffected by labeling 60-80% of the heads. Following excitation of the probes by a 10-ns laser pulse polarized parallel to the fiber axis, the time-resolved emission anisotropy of muscle fibers in rigor (no ATP) showed no decay from 1 microsecond to 1 ms (r infinity = 0.095), indicating that all heads are rigidly attached to actin on this time scale. In relaxation (5 mM MgATP but no Ca2+), the anisotropy decayed substantially over the microsecond time range, from an initial anisotropy (r0) of 0.066 to a final anisotropy (r infinity) of 0.034, indicating large-amplitude rotational motions with correlation times of about 10 and 150 microseconds and an overall angular range of 40-50 degrees. In isometric contraction (MgATP plus saturating Ca2+), the amplitude of the anisotropy decay (and thus the amplitude of the microsecond motion) is slightly less than in relaxation, and the rotational correlation times are about twice as long, indicating slower motions than those observed in relaxation. While the residual anisotropy (at 1 ms) in contraction is much closer to that in relaxation than in rigor, the initial anisotropy (at 1 microsecond) is approximately equidistant between those of rigor and relaxation.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1988

Rotational dynamics of the single tryptophan of porcine pancreatic phospholipase A2, its zymogen, and an enzyme/micelle complex. A steady-state and time-resolved anisotropy study.

The rotational dynamics of the single tryptophan of porcine pancreatic phospholipase A2 and its zymogen (prophospholipase A2) have been studied by polarized fluorescence using steady-state and time-resolved single-photon counting techniques. The motion of Trp-3 in phospholipase A2 consists of a rapid subnanosecond wobble of the indole ring with an amplitude of about +/- 20 degrees accompanied by slower isotropic rotation of the entire protein. The rotational correlation times for overall particle rotational diffusion are consistent with conventional hydrodynamic theory. When phospholipase A2 binds to micelles of n-hexadecylphosphocholine, the amplitude of the fast ring rotation decreases. The whole particle rotational correlation time of the enzyme/micelle complex is smaller than the minimum value calculated from hydrodynamic theory. A similar result is obtained for the micelle itself by using the lipophilic probe transparinaric acid. These low values for the particle correlation times can be understood by postulating that an isotropic motion of the fluorophore in the small detergent particles contributes to the angular reorientation of the fluorophore. The internal reorientational motion of the tryptophan in the zymogen, prophospholipase A2, is of larger amplitude than that observed for the enzyme; specifically, the proenzyme exhibits a motion with a significant amplitude on the nanosecond time scale. This additional freedom of motion is attributed to segmental mobility of the N-terminal residues of prophospholipase A2. This demonstrates that this region of the protein is flexible in the zymogen but not in the processed enzyme. The implications of these findings for the mechanism of surface activation of phospholipase A2 are discussed by analogy with a trypsinogen-trypsin activation model.

Animals↗
PubMed1988

Microsecond rotational dynamics of phosphorescent-labeled muscle cross-bridges.

We have measured the microsecond rotational motions of myosin heads in muscle cross-bridges under physiological ionic conditions at 4 degrees C, by detecting the time-resolved phosphorescence of eosin-maleimide covalently attached to heads in skeletal muscle myofibrils. The anisotropy decay of heads in rigor (no ATP) is constant over the time range from 0.5 to 200 microsecond, indicating that they do not undergo rotational motion in this time range. In the presence of 5 mM MgATP, however, heads undergo complex rotational motion with correlation times of about 5 and 40 microsecond. The motion of heads in relaxed myofibrils is restricted out to 1 ms, as indicated by a nonzero value of the residual anisotropy. The anisotropy decay of eosin-labeled myosin, extracted from labeled myofibrils, also exhibits complex decay on the 200-microsecond time scale when assembled into synthetic thick filaments. The correlation times and amplitudes of heads in filaments (under the same ionic conditions as the myofibril experiments) are unaffected by MgATP and very similar to the values for heads in relaxed myofibrils. The larger residual anisotropy and longer correlation times seen in myofibrils are consistent with a restriction of rotational motion in the confines of the myofibril protein lattice. These are the first time-resolved measurements under physiological conditions of the rotational motions of cross-bridges in the microsecond time range.

Animals↗
PubMed1988

Ionic effects on the rotational dynamics of cross-bridges in myosin filaments, measured by triplet absorption anisotropy.

We have measured the rotational motion of myosin heads in synthetic thick filaments at 4 degrees C in the time range from 10(-7) to 10(-4) seconds, by measuring transient absorption anisotropy of an eosin probe attached to a reactive sulfhydryl on the myosin head. Under conditions that result in monomeric myosin (500 mM ionic strength), the anisotropy decay is independent of pH in the range from 7.0 to 8.2 and [Mg2+] in the range from 0.1 to 10 mM; the anisotropy decays bi-exponentially with correlation times of 0.4 and 2 microseconds to a constant value of 0.016. Under more physiological conditions (115 mM ionic strength), resulting in filament formation, the anisotropy decay is sensitive to both pH and [Mg2+]. The anisotropy at pH 8.2 and 0.1 mM-Mg2+ decays with correlation times of 0.5 and 3.8 microseconds to a constant limiting anisotropy of 0.038. When the [Mg2+] is increased to 10 mM, the correlation times are 0.6 and 5.7 microseconds and the limiting anisotropy value is 0.055. Identical changes in the anisotropy decay are caused by an increase in [H+] to pH 7.0, in the presence of 0.1 mM-Mg2+. Increasing the total ionic strength to 187 mM decreases the amplitude of the cation effects. These results provide direct evidence that the rotational dynamics of myosin heads in thick filaments are influenced by physiological concentrations of cations. The results are qualitatively consistent with the proposal that these and other ionic conditions regulate transitions between "spread" and "compact" cross-bridge conformations, but the quantitative results indicate that cross-bridges undergo large-amplitude microsecond rotations even under conditions where the compact state should predominate.

Eosine Yellowish-(YS)↗
PubMed1987

Time-resolved fluorescence anisotropy for systems with lifetime and dynamic heterogeneity.

The time dependence of the fluorescence anisotropy expected when a fluorophore exists in distinct environments having different fluorescence decay and motional behavior is illustrated by simulation calculations. A wide range of behavior is observed. The analysis of such decays in terms of the underlying physical parameters is also illustrated and discussed. In particular, the analysis of 'associated' heterogeneous behavior using a homogeneous environment model with complex motional behavior is evaluated. It is argued that anisotropy decays that exhibit a high initial anisotropy and that rise at long times must be due to a heterogeneous environment. Anisotropic rotor diffusion cannot give rise to behavior of this type. A similar conclusion is reached for anisotropies that exhibit downward curvature. On the other hand, anisotropy decays that are monotonically decreasing and have a positive second derivative at all times cannot be analyzed in a unique fashion and therefore an ambiguity exists in the interpretation of such data in terms of motional behavior.

Fluorescence Polarization↗
PubMed1985

Complex photophysics of the single tryptophan of porcine pancreatic phospholipase A2, its zymogen, and an enzyme/micelle complex.

The fluorescence emission of the single tryptophan in porcine pancreatic phospholipase A2, its zymogen, and a micellar complex of the enzyme with the nonhydrolyzable substrate analogue n-hexadecylphosphocholine has been studied by both steady-state and time-resolved techniques. Stern-Volmer quenching studies with acrylamide indicate that, both in the enzyme and in the zymogen, the tryptophan is exposed to solvent. Similar studies with ionic quenchers show that there is appreciable ionic character to the tryptophan environment. Single photon counting fluorescence measurements were performed using a high repetition rate synchronously pumped dye laser as a light source. When tryptophan fluorescence is collected with a broad-band (80-nm) emission filter, the decay kinetics in the enzyme and the zymogen require at least three, and often four, exponential terms for a proper description. The decay kinetics can be adequately described by three exponential terms when the fluorescence is collected at specific wavelengths by using narrow (10-nm) band-pass filters. The lifetimes are approximately constant across the emission band, but the amplitudes vary with the fraction of the long lifetime increasing at longer emission wavelengths. Formation of a complex between phospholipase A2 and micelles of n-hexadecylphospohocholine produces large changes in the tryptophan emission that are associated with transfer to a hydrophobic environment. The decay kinetics of tryptophan in the enzyme/micelle complex appears to require only two exponential terms. This is the first reported instance of fluorescence data from a single tryptophan protein requiring more than double-exponential decay kinetics. The results are discussed in terms of the range of environments sampled by the tryptophan residue and the resulting distribution of lifetimes.

Animals↗
PubMed1979

Production of petites by cell cycle mutants of Saccharomyces cerevisiae defective in DNA synthesis.

Mutations in two genes (cdc8 and cdc21) required for nuclear and mitochondrial DNA synthesis in Saccharomyces cerevisiae result in a 6- to 11-fold increase in the rate of mitotic segregation of petites at the permissive temperature. The defect in DNA replication and the increased rate of petite production result from the same mutation since the two phenotypes cosegregate and corevert. Most of the petites isolated from strains carrying mutations in cdc8 and cdc21 contain mtDNA. Therefore, the petites do not result simply from an underreplication of mitochondrial DNA. The mutation rates for nuclear and mitochondrial genes are the same in cdc8, cdc21 and their wild-type parent. Therefore the petites are unlikely to result from an increase in the rate of base pair substitution.

Cell Cycle↗