The identification of tryptophan residues responsible for ATP-induced increase in intrinsic fluorescence of myosin subfragment 1

ATP binding to myosin subfragment 1 (S1) induces an increase in tryptophan fluorescence. Chymotryptic rabbit skeletal S1 has 5 tryptophan residues (Trp113, 131, 440, 510 and 595), and therefore the identification of tryptophan residues perturbed by ATP is quite complex. To solve this problem we resolved the complex fluorescence spectra into log-normal and decay-associated components, and carried out the structural analysis of the microenvironment of each tryptophan in S1. The decomposition of fluorescence spectra of S1 and S1-ATP complex revealed 3 components with maxima at ca. 318, 331 and 339-342 nm. The comparison of structural parameters of microenvironment of 5 tryptophan residues with the same parameters of single-tryptophan-containing proteins with well identified fluorescence properties applying statistical method of cluster analysis, enabled us to assign Trp595 to 318 nm, Trp440 to 331 nm, and Trp 13, 131 and 510 to 342 nm spectral components. ATP induced an almost equal increase in the intensities of the intermediate (331 nm) and long-wavelength (342 nm) components, and a small decrease in the short component (318 nm). The increase in the intermediate component fluorescence most likely results from an immobilization of some quenching groups (Met437, Met441 and/or Arg444) in the environment of Trp440. The increase in the intensity and a blue shift of the long component might be associated with conformational changes in the vicinity of Trp510. However, these conclusions can not be extended directly to the other types of myosins due to the diversity in the tryptophan content and their microenvironments.     

Voltammetry as a virtual potentiometric sensor in modelling of a metal-ligand system and refinement of stability constants. Part 4. An electrochemical study of NiII complexes with methylene diphosphonic acid

The Ni(II)-MDP-OH system (MDP=methylene diphosphonic acid) and stability constants of complexes formed at ionic strength 0.15M at 298K were established by direct current polarography (DCP) and glass electrode potentiometry (GEP). The final M-L-OH model could only be arrived to by employing recent concept of virtual potentiometry (VP). VP-data were generated from non-equilibrium and dynamic DC polarographic technique. The VP and GEP data were refined simultaneously by software dedicated to potentiometric studies of metal complexes. Species distribution diagrams that were generated for different experimental conditions employed in this work assisted in making the final choice regarding the metal-ligand model. The model established contains ML, ML(2), ML(OH) and ML(OH)(2) with stability constants, as logbeta, 7.94+/-0.02, 13.75+/-0.02, 12.04 (fixed value), and 16.75+/-0.05, respectively. It has been demonstrated that virtual potential must be used in modelling operations (predictions of species formed) when a polarographic signal decreases significantly due to the formation of polarographically inactive species (or formation of inert complexes). The linear free energy relationships that included stability constant logK(1) for Ni(II)-MDP established in this work together with other available data were used to predict logK(1) values for Sm(III) and Ho(III) with MDP. The logK(1) values for Sm(III)-MDP and Ho(III)-MDP were estimated to be 9.65+/-0.10 and 9.85+/-0.10, respectively.     

Evidence for pre- and post-power stroke of cross-bridges of contracting skeletal myofibrils

We examined the orientational fluctuations of a small number of myosin molecules (approximately three) in working skeletal muscle myofibrils. Myosin light chain 1 (LC1) was labeled with a fluorescent dye and exchanged with the native LC1 of skeletal muscle myofibrils cross-linked with 1-ethyl-3-[3(dimethylamino) propyl] carbodiimide to prevent shortening. We observed a small volume within the A-band (∼10⁻¹⁵ L) by confocal microscopy, and measured cyclic fluctuations in the orientation of the myosin neck (containing LC1) by recording the parallel and perpendicular components of fluorescent light emitted by the fluorescently labeled myosin LC1. Histograms of orientational fluctuations from fluorescent molecules in rigor were represented by a single Gaussian distribution. In contrast, histograms from contracting muscles were best fit by at least two Gaussians. These results provide direct evidence that cross-bridges in working skeletal muscle assume two distinct conformations, presumably corresponding to the pre- and post-power-stroke states.     

Enkephalin analogs modified in the aromatic ring of the N-terminal tyrosine residue

Two analogs of Leu⁵-enkephalin, (1-O-methyltyrosine,5-leucine)-enkephalin and [1-(3′-amino)-tyrosine,5-leucine]-enkephalin, were synthesized by classical methods. Both analogs show high biological potency after injection into the lateral brain ventricle of the rat. In both cases substitution of the Tyr residue of enkephalin leads to a pronounced prolongation of analgesic action, as compared with the unsubstituted peptide.     

Modification of membranes by quercetin, a naturally occurring flavonoid, via its incorporation in the polar head group

Quercetin is a naturally occurring flavonoid that has a lot of beneficial properties to human health. In this report, using the spin label technique, the influence of quercetin on the fluidity of multilamellar DPPC liposomes was studied. The polarity of the environment preferred by quercetin was also examined by determining the dependence of the position of electronic absorption maxima on dielectric properties of different environments. Autofluorescence of quercetin was also used to examine its distribution in cells. An additional aim of the study was to find how quercetin presence affects human skin fibroblasts. The results showed that incorporation of quercetin at physiological pH into DPPC liposomes caused changes in the partition coefficient of the Tempo spin label between water and polar head group phases. By determining the electronic absorption maxima, we observed that the chromophore of quercetin is localized in the polar head region. Fluorescence microscopy of HSF cells showed quercetin presence in the membrane, cytoplasm and inside the nucleus. Ultrastructural observation revealed some changes, especially in membranous structures, after flavonol treatment. From the results we have concluded that quercetin present in the membrane and other structures can cause changes within cells crucial for its pharmacological activity.     

On the genetic and structural similarities between the squash seeds polypeptide trypsin inhibitors and wheat germ agglutinin

Summary In this report we propose the disulfide bridges alignment in the squash polypeptide trypsin inhibitors. The prediction is based on the extensive homology in the amino acid sequence between these inhibitors and a portion of the wheat germ agglutinin domains for which the position of the disulfide bridges are known.     

Electrophoresis and orientation of F-actin in agarose gels.

F-Actin was electrophoresed on agarose gels. In the presence of 2 mM MgCl2 and above pH 8.5 F-actin entered 1% agarose; when the electric field was 2.1 V/cm and the pH was 8.8, F-actin migrated through a gel as a single band at a rate of 2.5 mm/h. Labeling of actin with fluorophores did not affect its rate of migration, but an increase in ionic strength slowed it down. After the electrophoresis actin was able to bind phalloidin and heavy meromyosin (HMM) and it activated Mg2+-dependent ATPase activity of HMM. The mobility of F-actin increased with the rise in pH. Acto-S-1 complex was also able to migrate in agarose at basic pH, but at a lower rate than F-actin alone. The orientation of fluorescein labeled F-actin and of fluorescein labeled S-1 which formed rigor bonds with F-actin was measured during the electrophoresis by the fluorescence detected linear dichroism method. The former showed little orientation, probably because the dye was mobile on the surface of actin, but we were able to measure the orientation of the absorption dipole of the dye bound to S-1 which was attached to F-actin, and found that it assumed an orientation largely parallel to the direction of the electric field. These results show that actin can migrate in agarose gels in the F form and that it is oriented during the electrophoresis.     

Tension fluctuations in contracting myofibrils and their interpretation

Repulsive pressure has been measured as a function of lattice spacing in gels of tobacco mosaic virus (TMV) and in the filament lattice of vertebrate striated muscle. External pressures up to ten atm have been applied to these lattices by an osmotic stress method. Numerical solutions to the Poisson-Boltzmann equation in hexagonal lattices have been obtained and compared to the TMV and muscle data. The theoretical curves using values for k calculated from the ionic strength give a good fit to experimental data from TMV gels, and an approximate fit to that from the muscle lattice, provided that a charge radius for the muscle thick filaments of approximately 16 nm is assumed. Variations in ionic strength, sarcomere length and state of the muscle give results which agree qualitatively with the theory, though a good fit between experiment and theory in the muscle case will clearly require consideration of other types of forces. We conclude that Poisson-Boltzmann theory can provide a good first approximation to the long-range electrostatic forces operating in such biological gel systems.