Biomedical subjects

R J Baskin

Publications and source records attributed to R J Baskin.

At least 19 recordsLinked to original sources

PubMed1992

Rigorous analysis of light diffraction by a striated muscle fibre.

A rigorous theory describing the diffraction of light by a muscle fibre has been formulated. The basis of this analysis is the rigorous coupled-wave approach of T. K. Gaylord & M. G. Moharam (Proc. IEEE 73, 894 (1985)); however, we obtain here a closed-form analytical result that is both mathematically simple and physically easy to understand. We have compared our results on striated muscle fibres with the analytical results obtained by A. F. Huxley (Proc. R. Soc. Lond. B 241, 65 (1990)) using the normal mode approach, and with those obtained by R. A. Thornhill, N. Thomas & N. Berovic (Eur. Biophys. J. 20, 87 (1991)) using a multiwave first-order coupled-wave approximation. For an equivalent set of assigned fibre parameters, our results are consistent with these mentioned. Extension of this analysis to a fibre with different structures showed that the differences in diffraction efficiencies of different orders for a frog skeletal fibre and for an insect flight fibre are clear; the sensitivity to distinct structural organization of the fibre is very good.

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PubMed1992

Isolation of a sea urchin egg kinesin-related protein using peptide antibodies.

To understand the roles of kinesin and its relatives in cell division, it is necessary to identify and characterize multiple members of the kinesin superfamily from mitotic cells. To this end we have raised antisera to peptides corresponding to highly conserved regions of the motor domains of several known members of the kinesin superfamily. These peptide antibodies react specifically with the motor domains of kinesin and ncd protein, as expected, and they also react with several polypeptides (including kinesin heavy chain) that cosediment with microtubules (MTs) precipitated from AMPPNP-treated sea urchin egg cytosol. Subsequent fractionation of ATP eluates of these MTs yields a protein of relative molecular mass 330 x 10(3) that behaves as a complex of three polypeptides that are distinct from conventional kinesin subunits or fragments thereof. This complex contains 85 kDa and 95 kDa polypeptides, which react with our peptide antibodies, and a 115 kDa polypeptide, which does not. This triplet of polypeptides, which we refer to as KRP(85/95), binds to purified sea urchin egg tubulin in an AMPPNP-enhanced, ATP-sensitive manner and induces the formation of microtubule bundles. We therefore propose that the triplet corresponds to a novel sea urchin egg kinesin-related protein.

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PubMed1991

The molecular origin of birefringence in skeletal muscle. Contribution of myosin subfragment S-1.

The state of optical polarization of He-Ne laser light diffracted by single skinned frog skeletal muscle fibers has been determined after decoration of the thin filaments of rigor fibers with exogenous S-1. Light on the first diffraction order was analyzed using optical ellipsometry for changes occurring in total birefringence (delta nT) and total differential field ratio (rT) and the experimental results compared with theoretical predictions. Fibers were examined with SDS-gel electrophoresis and electron microscopy as independent assays of S-1 binding. The binding of S-1 to the thin filaments caused a significant increase in rT and a small but significant decrease in delta nT. Release of bound exogenous S-1 with magnesium pyrophosphate demonstrated that the effect of S-1 on the optical parameters was reversible and both electrophoresis and electron microscopy demonstrated the presence of S-1 specifically bound to the thin filaments. Model simulations based on the theory of Yeh, Y., and R. Baskin (1988. Biophys. J. 54:205-218) showed that the values of delta nT and rT were sensitive to the axial bonding angle of exogenous S-1 as well as to the volume fraction of added S-1. Analysis of the data in light of the model showed that an average axial S-1 binding angle of 68 degrees +/- 7 degrees best fit the data.

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PubMed1990

Diffraction ellipsometry studies of osmotically compressed muscle fibers.

Microstructural features of relaxed, skinned muscle fibers compressed with polyvinylpyrrolidone were examined by optical diffraction ellipsometry. This technique is sensitive to the optical anisotropy within the muscle, including that due to intrinsic properties of the protein molecules as well as that due to the regular arrangement of proteins in the surrounding medium. The change in polarization state of light after interacting with the muscle is described by the differential field ratio (DFR) and birefringence (delta n). Compression of single fibers (sarcomere length = 2.6 microns) with 0%-21% polyvinylpyrrolidone caused an increase of up to 23% and 31% for DFR and delta n, respectively. The largest increase in both parameters occurred at intermediate sarcomere lengths. Theoretical modelling of the results suggest that the average S-1 tilt angle may be reduced upon compression of the filament lattice. This is supported by experiments in which S-1 was enzymatically cleaved with alpha-chymotrypsin. Separate experiments comparing fibers with intact membranes and skinned fibers compressed to an equivalent lattice spacing showed little difference in DFR or delta n.

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PubMed1990

Photon correlation spectroscopy of the polarization signal from single muscle fibres.

Measurements of crossbridge dynamics have been obtained from the spectrum of diffracted light derived from single, skinned fibres of skeletal muscle. This technique combines optical ellipsometry with photon correlation spectroscopy of the intrinsic reporters from the muscle cell. The difference in the intensities of the two linearly polarized electric field components of diffracted light is autocorrelated in time, and the dynamics of optical anisotropy from the contributing units of the sarcomere are measured. In focusing on the fast-time dynamics, we detected two principal features: a rapidly relaxing temporal signal with a relaxation time of approximately 5 microseconds, and an oscillatory component which transforms into a broad but spectrally defined signal centred at around 370 kHz. Experiments were conducted to measure changes of these two signals upon relax-rigor transition and alpha-chymotrypsin degradation. These studies strongly suggest that the relaxational component of the signal is primarily due to myosin crossbridge motion; the 370 kHz spectral signal has as its likely source the myosin thick filament.

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PubMed1990

Crossbridge activity monitored from the state of polarization of light diffracted by activated frog muscle fibres.

The polarization properties of the first diffraction order have been measured when single frog fibres are illuminated by laser light. The relative difference in the amplitudes of the orthogonal electric field polarization components (differential field ratio) as well as their phase shift normalized by the pathlength (birefringence) have been obtained from fibres at rest and during fixed-end twitches and tetani. The differential field ratio decreased during contraction and the change during a single twitch averaged 69% of that during a companion tetanus. The birefringence of the first order averaged 2.80 +/- 0.59 x 10(-3) (mean +/- SD) at rest and the average decrease during a tetanus was 8.4% +/- 6.4%. The decrease in the differential field ratio upon activation was a decreasing function of sarcomere length, maximum at rest length and falling to zero at about 3.7 microns. Differences between the two first diffraction orders were observed for both the differentiated field ratio and the birefringence. At the time when force had risen to half the value reached at the end of the fast rise of tension, the change in the differential field ratio lead the tension by about 10-15 ms. The differential field ratio returned to its resting value after the fall of tension. The above results suggest that the differential field ratio is a sensitive indicator of intact fibre structure. The temporal lead in the differential field ratio with respect to tension rise supports models in which crossbridges initially attach in a non-force-producing state.

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PubMed1989

Sarcomere length behaviour along single frog muscle fibres at different lengths during isometric tetani.

A detailed investigation of sarcomere lengthening and shortening during fixed-end tetani has been made along frog muscle fibres stretched over a large range of sarcomere lengths. A variety of sources of error common in such measurements are quantitated and give an uncertainty in sarcomere length of about 53-62 nm. The difference in sarcomere length calculated from the left and right first orders at rest was 21 nm +/- 16 nm and this is suggested to be a measure of 'Bragg artefact'. The laser diffraction measurements showed that the shortening end regions decrease in size during contraction and that the magnitude of shortening is increased at greater fibre extensions. The average length change and sarcomere length of the central and end regions was 0.10 microns (2.85 microns) and 0.37 microns (2.66 microns), respectively. The sarcomere length of the end regions at the end of creep was regularly observed to be less than 2.1 microns. An unexpected finding was the occasional observation of striations in the transition zone between lengthening and shortening regions which remained nearly isometric during a period of tension rise during creep. Measurements of diffraction order linewidth do not suggest increased sarcomere length dispersion in these areas. A smooth transition from shortening to lengthening was always observed. Although our data are in general agreement with the models proposed by Morgan, Mochon and Julian (Biophys. J. 39 (1982) 189-96) and Edman and Reggiani (J. Physiol. (Lond.) 351 (1984) 169-98), specific differences which do exist are discussed.

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PubMed1989

Polarization states of diffracted light. Changes accompanying fiber activation.

Measurement of the state of optical polarization of light diffracted from single, skinned and intact fibers of anterior tibialis muscle from Rana pipiens revealed a dependence upon rigor, activation, and sarcomere length (SL) change. Changes in total birefringence, delta nT, and differential field ratio value, rT, were determined. In a relaxed, skinned fiber the total birefringence value, delta nT, decreases as sarcomere length is increased from 2.1 microns to approximately 2.8-3.0 microns. From there it increases significantly to a value of approximately 1.8 x 10(-3) at a sarcomere length of 3.6 microns. The differential field ratio, rT, also shows a biphasic response to increasing sarcomere length, first exhibiting a rapid decrease over shorter SL and leveling out after the SL is beyond 3.0 microns. In comparison, relaxed intact fibers change substantially less upon sarcomere length change, showing little change in birefringence and a small bi-phasic change in rT. Skinned fibers were activated using a solution that has the same ionic strength as the relaxing solution and allows repeatable, and sustained activation. A decrease in both delta nT and rT was observed upon fiber activation. The decrease in delta nT and rT was slightly larger at shorter sarcomere lengths than at longer lengths. Relaxed fibers placed in rigor showed changes in delta nT and rT similar to those observed in activated fibers. These results are consistent with the hypothesis that, after activation, a significant portion of the thick filament cross-bridges rotate towards the actin filament resulting in redistribution of the interfilament mass content. They are also consistent with an average orientation of crossbridges in the overlap region different from that in the nonoverlap region.

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PubMed1988

Theory of optical ellipsometric measurements from muscle diffraction studies.

A theory of optical ellipsometry describing the complete phase shift and ellipticity of light diffracted from a single muscle fiber is developed. We show that both the phase shift information, described commonly by the birefringence of the fiber, and the ellipticity information, described by the differential polarizability ratio, are necessary to provide a complete picture of the complex contributions to the total optical anisotropy spectra from a diffraction pattern derived from the striated muscle cell. Both form and intrinsic contributions play significant roles in either the birefringence measurement or the differential field ratio measurement. However, we show that their relative weights in these two measured quantities are different, and measuring both of these parameters is necessary to obtain a more complete assessment of the cross-bridge structure and dynamics. The theoretical results have been tested for three different situations: solvent index matching, passive stretch of a resting fiber, and cross-bridge changes under isometric conditions. Comparisons between experimental data and simple model calculations provide much information regarding cross-bridge orientation and structure.

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PubMed1987

Optical ellipsometry on the diffraction order of skinned fibers. pH-induced rigor effects.

The polarization properties of light diffracted from single-skinned fibers of skeletal muscles have been examined under conditions in which the bathing solution pH and the ionic strength are changed. For fibers in the relaxed state, we observe large decreases in both the total depolarization signal, r, and the total diffraction birefringence signal, delta nT, upon pH change from 7.0 to 8.0 at normal ionic strength. However, if the ionic strength is raised, then the r-value change as the pH changes from pH 7.0 to pH 8.0 is much smaller. If the rigor state is achieved at pH 8.0, and 0 mM ATP under either of the ionic strength conditions, the fiber can still be stretched. Rigor stiffness for this state is only approximately 20% that of the value of the stiffness at pH 7.0 rigor. Electron micrographs obtained under this pH 8.0 rigor state show that the overlap region can be decreased upon stretching the fiber, signifying a different kind of weaker-binding rigor state. Optically, the weaker-binding rigor state has a lower depolarization signal and larger form birefringence than the strong-binding rigor state. To convert from one type of rigor state (pH 7.0) to the other rigor state (pH 8.0), or vice versa, the fiber must first be relaxed. Apparently, either of the rigor states can block the full impact of the pH effect.

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PubMed1986

Light diffraction patterns and sarcomere length variation in striated muscle fibers of Limulus.

Light diffraction patterns produced by Limulus striated muscle fibers were examined. Segments of fibers were glycerinated, fixed or bathed in relaxing solution. Profiles of the intensity of a diffracted order vs. the angle of incidence of the laser beam often exhibited narrow peaks with the fiber at rest length. The incident angles at which the intensity of left and right orders is greatest are used to calculate the sarcomere length, supporting the notion that regions of the fiber are organised into Bragg reflecting planes. These profiles developed subpeaks and broadened upon stretch of the fiber. The broad angle scan profiles are suggested to result from a decrease in the regular packing of myofibrils as the fiber is lengthened. The angular width of the subpeaks is used to estimate the thickness of clusters of myofibrils. The variation in sarcomere length along the fiber, as determined by the 0th to 1st diffraction order spacing, was dependent upon the fiber preparation. Glycerinated fibers and those bathed in relaxing solution showed more variation than fixed fibers. The variation of sarcomere length is compared to the variation in thick filament lengths in Limulus reported by Dewey et al. (1982). These results are compared to those obtained from frog fiber segments.

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PubMed1986

Optical depolarization changes in single, skinned muscle fibers. Evidence for cross-bridge involvement.

Optical ellipsometry studies of single, skinned muscle fibers conducted on the diffraction orders have yielded spectra that are sensitive to the state of the fiber. The linearly polarized light field vector becomes elliptically polarized as it passes through the fiber and may be collected at the diffraction orders. Fibers that have been subjected to extraction of myosin (0.6 M KCl) retain a weak diffraction pattern and exhibit a substantially decreased depolarization of incident linearly polarized light. A significant decrease in polarization is seen in skinned fibers that are subject to an increase in pH from 7.0 to 8.0. This increase in pH results in a decrease of approximately 30% in the depolarization angle of single fibers. The major decrease in depolarization angle that we observe at pH 8.0 is consistent with the notion that as cross-bridges move out from the shaft of the thick filament, their ability to cause depolarization of the incident linearly polarized light decreases. This interpretation is also consistent with the work of Ueno and Harrington where the decrease in the ability to cross-link S-1 and S-2 to the thick filament at pH 8.2 suggests cross-bridge movement away from the thick filament. A large decrease in birefringence, seen after treatment of skinned fibers with alpha-chymotrypsin, appears to be related to the breakdown of myosin into rod, S-1, heavy meromyosin, and light meromyosin.

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PubMed1985

Depolarization spectrum of diffracted light from muscle fiber. The intrinsic anisotropy component.

The depolarization signal of the diffraction patterns from muscle fibers includes information that differs from that of transmission birefringence experiments. Although both the birefringence studies and the phase shift studies of Yeh et al. (Yeh, Y, and G. Pinsky, 1983, Biophys. J., 42:83-90; Yeh, Y., M. E. Corcoran, R. J. Baskin, and R. L. Lieber, 1983, Biophys. J., 44:343-351) include inseparable intrinsic and form contributions, the present analysis shows that the magnitude of the E-field components of diffracted light is affected only by the intrinsic contribution. We have analyzed the amplitude portion of the data of which the phase shift portion had previously been reported (Yeh, Y., M. E. Corcoran, R. J. Baskin, and R. L. Lieber, 1983, Biophys. J., 44:343-351). For the relaxed-to-rigor transition, these field amplitudes also exhibit changes when ATP concentration is decreased. The observed decrease in optical depolarization upon rigor is consistent with the idea that optically anisotropic elements move away from the myosin thick filament under such conditions.

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PubMed1979

Cross-linking of the (Ca2+ + Mg2+)-ATPase protein.

The addition of cupric-1,10,-phenanthroline, a cross-linking catalyst, to sarcoplasmic reticulum membranes caused protein sulfhydryl groups to form disulfide bridges. Following a short exposure to the catalyst (15 s, 22 degrees C) most of the protein was in a dimeric form (Mr = 248 000). Longer exposure times resulted in the formation of trimers, tetramers and other oligomers too large to enter the gel. At low temperatures (4 degrees C) dimer formation predominates even for exposure times as long as 5 min. Cross-linking in the presence of 7.5 mM Triton X-100 (a concentration that resulted in clearing of the membrane suspension and thus solubilization of the membrane components) showed the appearance of a considerable dimer fraction, however, most of the (Ca2+ + Mg2+)-ATPase protein appeared as a monomer. Following 1 min of cross-linking at 22 degrees C, freeze-etched membranes showed no alteration in the number or appearance of 80 A intramembranous particles. Thus extensive cross-linking of the (Ca2+ + Mg2+)-ATPase protein can occur without disruption of the normal position of the intramembrane portion of the molecule.

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PubMed1979

Cross-linking of the (Ca2+ + Mg2+)-ATPase protein.

The addition of cupric-1,10,-phenanthroline, a cross-linking catalyst, to sarcoplasmic reticulum membranes caused protein sulfhydryl groups to form disulfide bridges. Following a short exposure to the catalyst (15 s, 22 degrees C) most of the protein was in a dimeric form (Mr = 248,000). Longer exposure times resulted in the formation of trimers, tetramers and other oligomers too large to enter the gel. At low temperatures (4 degrees C) dimer formation predominates, even for exposure times as long as 5 min. Cross-linking in the presence of 7.5 mM Triton X-100 (a concentration that resulted in clearing of the membrane suspension and thus solubilization of the membrane components) showed the appearance of a considerable dimer fraction, however, most of the (Ca2+ + Mg2+)-ATPase protein appeared as a monomer. Following 1 min of cross-linking at 22 degrees C, freeze-etched membranes showed no alteration in the number or appearance of 80 A intramembranous particles. Thus extensive cross-linking of the (Ca2+ + Mg2+)-ATPase protein can occur without disruption of the normal position of the intramembrane portion of the molecule.

Adenosine Triphosphatases↗

PubMed1979

Light diffraction study of single skeletal muscle fibres.

Light diffraction patterns from isolated frog semitendinosus muscle fibers were examined. When transilluminated by laser light, the muscle striations produce a diffraction pattern consisting of a series of lines that are projected as points onto an optical detector by a lens system. Diffraction data may be sequentially stored every 18 ms for later processing by digital computer systems. First- and second-order diffraction line intensities were examined from intact, chemically skinned, and glycerinated single fibers. The diffraction line intensities demonstrated a strong length dependence upon passive stretch from reference length to 3.6 micrometer. The first-order intensity linearly increased an average of 15-fold over the range examined. The magnitude of the second order intensity was less than the first order and showed an exponential rise with increasing length. Both first- and second-order intensities decreased upon muscle activation. Data from chemically skinned and glycerinated single fibers were not significantly different from intact fibers, indicating that the membrane structure has little effect upon the diffraction phenomenon in muscle. Theoretical model systems are examined in an attempt to find the basis of these results. Neither an analysis based on a diffraction grating with variable spacing nor the unit cell model of Fujime provides an explanation for the observed length dependency of intensity. Though the origin of the intensity decrease upon stimulation is not known, we have suggested that it could result from lateral misalignment of myofibrils and can occur upon activation.

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PubMed1979

Stereologic analysis of dystrophic chicken muscle.

Stereologic methods have been used to estimate the volume and surface densities of sarcoplasmic reticulum (SR) and T tubules of normal and dystrophic chicken pectoralis muscle fibers. The surface and volume densities of the T system in dystrophic muscle fibers showed large increases compared with normal muscle fibers; the surface and volume densities of the SR showed large decreases. In addition, the SR and T system in dystrophic fibers undergo changes in shape. The tubules of the free SR become much narrower; the T system becomes dilated and vesiculated. Dystrophic fibers, on the average, are much larger than normal but maintain the same sarcolemmal surface/fiber volume ratio as normal fibers. Alterations in the surface and volume densities of the dystrophic sarcotubular system may account for some of the altered contractile properties of these muscles.

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PubMed1978

Dynamic light scattering characterization of the detergent-free, delipidated (Ca2+ + Mg2+)-ATPase from sarcoplasmic reticulum.

Dynamic light scattering studies have been conducted on the delipidated and detergent-removed (Ca2+ + Mg2+)-ATPase protein assemblies. Specific characterization of the state of aggregation and the extent of conformation change upon delipidation and detergent removal has been made. The results show that the prominent species are dimers and tetramers of very globular nature, with axial ratios of less than 2 : 1. The hydrodynamic radii of the dimers and the tetramers are, respectively, 57.5 A and 74.5 A. The globular nature of these observed entities differ from the delipidated ATPase proteins recently obtained (LeMaire, M., Jorgensen, K.E., Roigaard-Petersen, H. and Moller, J.V. (1976) Biochemistry 15, 5805--5812. Present results suggest that upon the removal of detergents from the lipid-free ATPase protein assembly, only a rather limited degree of aggregation takes place. Such a condition is consistent with models of the membrane protein system which has limited regions of hydrophobic contact. Oligomeric assemblies with aqueous channels is a possible active Ca2+ transport model consistent with results of the present data, as well as the data from several other recent studies.

Adenosine Triphosphatases↗