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O A Andreev

Publications and source records attributed to O A Andreev.

15 recordsLinked to original sources
PubMed1999

Interaction of the N-terminus of chicken skeletal essential light chain 1 with F-actin.

Skeletal myosin has two isoforms of the essential light chain (ELC), called LC1 and LC3, which differ only in their N-terminal amino acid sequence. The LC1 has 41 additional residues containing seven pairs of Ala-Pro, which form an elongated structure, and two pairs of lysines located near the N-terminus. When myosin subfragment-1 (S1) binds to actin, these lysines may interact with the C-terminus of actin and be responsible for the isoform specific properties of myosin. Here we employ cross-linking to identify the LC1 residues that are in contact with actin. S1 was reconstituted with various LC1 mutants and reacted with the zero-length cross-linker 1-ethyl-3-[3-dimethyl-aminopropyl]-carbodiimide (EDC). Cross-linking occurred only when actin was in molar excess over S1. Wild-type LC1 could be cross-linked through the terminal alpha-NH2 group, as well as via the two pairs of lysines. In a mutant ELC, where the lysines were deleted but two arginines were introduced near the N-terminus, the light chain could still be cross-linked via the terminal alpha-NH2 group. When the charge was reduced in the N-terminal region while retaining the Ala-Pro rich region, the mutant could not be cross-linked. These results suggest that as long as the N-terminus contains charged residues and an Ala-Pro rich extension, the binding between LC1 and actin can occur.

Actins↗
PubMed1998

Effect of ADP on binding of skeletal S1 to F-actin.

The proximity of skeletal myosin subfragment-1 (S1) to actin, and its orientation with respect to thin filaments of single muscle fibers, were compared in the presence and in the absence of ADP. The proximity was assessed by the efficiency of carbodiimide-induced cross-linking and the orientation by polarization of fluorescence of probes attached to the essential light chains. ADP made no difference in proximity or orientation when the molar ratio of S1 to actin was low or high. However, at the intermediate ratios, ADP made a significant difference. Strong dissociating agents, AMP-PNP and PPi, made significant differences at all ratios. To explain this behavior, it is unnecessary to invoke the ADP-induced "swinging" of the tail of S1. Rather, it is simply explained by the "two-state" model which we proposed earlier, in which S1 binds to one or to two actin protomers, depending on the saturation of the filaments with S1s. The dissociation induced by the ADP shifts the equilibrium between the two bound states. At high and low degrees of saturation, ADP is unable to significantly decrease the amount of S1 bound to F-actin. However, at intermediate saturation levels, ADP causes significantly more S1s to bind to two actins. These results suggest that the ADP-induced changes seen at the intermediate molar ratios are due to the dissociation-induced reorientation of S1.

Actins↗
PubMed1997

Interaction of the heavy and light chains of cardiac myosin subfragment-1 with F-actin.

The interaction of the heavy chain (HC) and the light chain (cdLC1) of cardiac S1 (cdS1) with F-actin was studied by cross-linking, Western blotting, and fluorescence polarization methods. Incorporation of cdLC1 in cross-linked products was examined by Western blots using the primary antibody against 71-74 residues of cdLC1. Cross-linking with zero-length, water-soluble reagent yielded three products with apparent molecular masses of 150, 160, and 210 kD. Like in the case of cross-linking of skeletal S1 with actin, these complexes included only HC of S1 and actin. The composition of the products were as follows: 150 kD, one HC of S1 cross-linked through a primary site (on the C-terminal of the 20-kD fragment) to the N-terminus of actin; 160 kD, one HC of S1 cross-linked through a secondary site (on the 50 kD fragment) to the N-terminus of actin; and 210 kD, one HC of S1 cross-linked through primary and secondary sites to two actins. Four additional products with apparent molecular masses of 66, 120, 185, and 235 kD contained cdLC1 and were identified as cdLC1 + actin, cdLC1 + HCS1, cdLC1 + actin + HCS1, and cdLC1 + two actins + HCS1, respectively. The same products were observed when cross-linking was performed in cardiac myofibrils incubated with cdS1. The production of cross-linked complexes of the heavy and light chain with actin decreased with an increase in the molar ratio of cdS1:actin. To test whether the orientation of myosin heads depended on a degree of occupation of thin filaments, myofibrils were irrigated with varying concentrations of cdS1. Fluorescence polarization measurements of cdS1 bound to individual I-bands revealed that the orientation depended on the concentration.

Actins↗
PubMed1996

Binding of S1(A1) and S1(A2) to F-actin.

The binding curve of myosin subfragment-1 (S1) to F-actin is not a simple hyperbola: at high concentrations of S1 the binding curve can be transformed into a linear plot ("normal" binding), but at small concentrations of S1 the binding complications deform the binding curve and produce nonlinear transforms ("anomalous" binding) [Andreev, O. A., & Borejdo, J. (1992) J. Muscle Res. Cell Motil. 13, 523-533]. This anomalous behavior may result either from the heterogeneity of S1 in regard to light chain isoforms or from the cooperativity between S1's. To distinguish between these possibilities we measured the affinity and the orientation of S1(A1) and S1(A2) with respect to F-actin. Affinity was measured in vitro by ultracentrifugation in the presence of F-actin, and orientation was measured in vivo by a combination of polarization of fluorescence and linear dichroism. We found that both the affinity and the orientation depended on the relative concentration of S1 isomer and actin: when S1 was in excess or was equimolar with actin (filament saturated with S1), each isomer bound F-actin with an affinity of 2 x 10(6) M-1 and was oriented approximately perpendicularly to the muscle axis. When actin was in excess (filament unsaturated with S1), each isomer bound F-actin with an affinity of 1.2 x 10(7) M-1 and was oriented more parallel to the muscle axis. S1(A1) and S1(A2) labeled on the light chain had different polarizations when bound to unsaturated filaments but had the same polarizations when bound to saturated filaments. These results excluded heterogeneity as a reason for anomalous binding and suggested that binding occurred with negative cooperativity. We think that the negative cooperativity occurs when saturation of actin filaments with heads leads to the lack of vacant adjacent sites on a filament and a consequent prevention of S1 binding to two actin protomers.

Actins↗
PubMed1995

Binding of heavy-chain and essential light-chain 1 of S1 to actin depends on the degree of saturation of F-actin filaments with S1.

The interaction of heavy-chain isoforms of myosin subfragment-1 with actin was examined by cross-linking with carbodiimide (EDC). The heavy chain of S1 could be cross-linked to a single actin molecule through sites on either 20 or 50 kDa proteolytic domains, resulting in complexes which migrated in an 8% polyacrylamide gel in the presence of Tricine buffer with an apparent molecular mass (M(app)) of 150 or 160 kDa, respectively. Cross-linking of S1 through both sites to two actins produced a complex migrating with an M(app) of 210 kDa. Cross-linking of the S1(A1) isoform [but not S1(A2)] to F-actin produced four additional peptides with M(app) values of 64, 160, 185, 210, and 235 kDa complexes was almost inhibited at a high degree of saturation while the inhibition of the 150 kDa product was relatively small. At a low degree of saturation, the ratio of 150 to 160 kDa complexes was 1. Cross-linking between the S1 isoforms and regulated F-actin was not affected by Ca2+. These data show that contact of the S1 to one actin protomer is through a site on the 20 kDa fragment and to the second actin protomer through the sites located on the 50 kDa fragment and on the essential light-chain 1. At nonphysiological conditions of full saturation of actin filaments with myosin heads, the binding of heavy chain at S1 and of A1 to the second actin could be almost abolished.

Actins↗
PubMed1995

Rigor cross-bridges bind to two actin monomers in thin filaments of rabbit psoas muscle.

The mode of binding of myosin subfragment-1 (S1) to actin is known to depend on their molar ratio: when actin is in excess, S1 binds to two actin monomers within the actin filament, and when S1 is in excess or is equimolar with actin, each S1 binds to one actin monomer. Since in vertebrate striated muscle actin is in molar excess over myosin, we expect that in fibers each myosin head binds to two actin monomers. To test this idea, we compared the conformation of the heads in native muscle with the conformation of S1 in fibers that were loaded with either high (S1 equimolar with actin) or low (excess of actin over S1) concentration of extrinsic S1. Conformation was assessed by the accessibility of heads to trypsin (measured by the rate of trypsinolysis) and by their orientation with respect to the muscle axis (measured by a combination of polarization of fluorescence and linear dichroism). In muscle fibers loaded with a high concentration of S1, the region of the heavy chain of the myosin head at the junction of 20 and 50 kDa proteolytic fragments was readily digested by trypsin and its orientation was approximately perpendicular to an axis of a thin filament. In contrast, when muscle fibers were loaded with a low concentration of S1, the 20/50 kDa junction of S1 was protected from trypsinolysis and its orientation was more parallel with a filament axis. Native muscle in rigor behaved like muscle irrigated with a low concentration of S1, i.e. the 20/50 kDa junction of the myosin head was protected from trypsinolysis and the orientation of the heads was parallel to the filament axis. We conclude that in rigor rabbit psoas muscle each myosin head binds to two actin monomers in a thin filament, and that this binding is different from the binding of S1 to actin in equimolar solutions.

Actins↗
PubMed1995

Fluorescence polarization study of the rigor complexes formed at different degrees of saturation of actin filaments with myosin subfragment-1.

A serine residue located in the active site of myosin head (S1) was labelled by 9-anthroylnitrile, an amino group located in the central domain of S1 was labelled by 7-diethylamino-3-(4'-isothio-cyanato-phenyl)-4-methylcoumari n, a cysteine residue located near the C-terminus of S1 was labelled by 5-[2-((iodoacetyl)-amino)ethyl]-amino-naphthalene-1-sulfonic acid (1,5-IAEDANS) and a cysteine residue located near the C-terminus of the alkali light chain 1 was labelled with iodoacetamido-tetramethyl-rhodamine. Polarization of fluorescence of S1 was measured in solution (where it indicated the mobility of actin-bound S1) and in myofibrils (where it indicated orientation of probes) to check whether the anisotropy of S1 labelled at different positions depended on the molar ratio S1:actin. In solution, when increasing amounts of actin were added to a fixed amount of labelled S1 (i.e. when myosin heads were initially in excess over actin), anisotropy saturated at 1 mol of S1 per 1 mol of actin. When increasing amounts of S1 were added to a fixed amount of F-actin (i.e. when actin was initially in excess over S1), the anisotropy saturated at 1 mol of S1 per 2 mols of actin. In myofibrils, orientation of S1 was different when S1 was added at nanomolar concentration (intrinsic actin was in excess over extrinsic S1) then when it was added at micromolar concentration (excess of S1 over actin). The fact that the anisotropy of S1 labelled at different positions depended on the molar ratio excluded the possibility that changes were confined to one part of the cross-bridge and supports our earlier proposal that the two rigor complexes which S1 can form with F-actin differ globally in conformation.

Actins↗
PubMed1993

Structure of the 265-kilodalton complex formed upon EDC cross-linking of subfragment 1 to F-actin.

The conventional model of force generation in muscle requires the presence of at least two different contact areas between the myosin head (S1) and the actin filament. It has been found that S1 has two sites available for carbodiimide cross-linking, but it is generally believed that the myosin head can be cross-linked to only one actin through either site. We provide here, for the first time, evidence that one S1 can be cross-linked to two separate actin molecules. The covalent complex of one S1 with two actins was found to have an apparent molecular mass of 265 kDa. The formation of the 265-kDa acto-S1 complex was strongly dependent on the ratio of S1 to actin. Limited tryptic digestion converted the 265-kDa product into the 240-kDa complex by releasing a 27-kDa N-terminal S1 fragment. Limited subtilisin digestion of the 265-kDa covalent acto-S1 complex yielded 29-, 93-, and 66-kDa peptides which corresponded to the 29-kDa N-terminal domain of S1, actin-44-kDa (central domain of S1) and actin-22-kDa (C-terminal domain of S1) complexes, respectively. These peptides could be generated only if a single S1 has been cross-linked to two separate actins. The 265-kDa acto-S1 complex (S1:actin ratio = 0.5) had 60% of the ATPase activity of the 175-185-kDa acto-S1 complex (S1:actin ratio = 1).(ABSTRACT TRUNCATED AT 250 WORDS)

Actins↗
PubMed1993

Two different rigor complexes of myosin subfragment 1 and actin.

Our previous titration and cross-linking experiments showed that myosin subfragment 1 (S1) can bind to one or two monomers in F-actin [Andreev, O. A., & Borejdo, J. (1991) Biochem. Biophys. Res. Commun. 177, 350-356; (1992a) J. Muscle Res. Cell Motil. 13, 523-533; (1992b) Biochem. Biophys. Res. Commun. 188, 94-101]. In the present work we used a sedimentation method to extend these studies to equilibrium binding and a stopped flow method to investigate its kinetics. Both equilibrium and kinetic data indicated the existence of two different rigor complexes. On the basis of these data we developed a model which suggested that binding of S1 to F-actin occurred in two steps: (i) initial rapid binding to one monomer of F-actin, A + M<==>A.M and (ii) a consequent slow binding to a neighboring monomer, A.M + A<==>A.M.A, where A stands for actin and M for myosin subfragment 1. The second reaction can proceed only if the neighboring actin site is unoccupied. The model fit the equilibrium and kinetic binding data with equilibrium constants K1 = 6 x 10(6) M-1 and K2 = 4 and kinetic constants k+1 = 10.5 x 10(6) M-1 s-1, k-1 = 1.75 s-1, k+2 = 0.8 s-1, and k-2 = 0.2 s-1, where the subscripts refer to the reactions i and ii. These results corroborate our hypothesis that myosin head can make two types of complexes with F-actin and support our speculation that during a power stroke in contracting muscle a myosin head may first bind to one and then to two actins.

Actins↗
PubMed1993

Polarization of fluorescently labeled myosin subfragment-1 fully or partially decorating muscle fibers and myofibrils.

Fluorescently labeled myosin heads (S1) were added to muscle fibers and myofibrils at various concentrations. The orientation of the absorption dipole of the dye with respect to the axis of F-actin was calculated from polarization of fluorescence which was measured by a novel method from video images of muscle. In this method light emitted from muscle was split by a birefringent crystal into two nonoverlapping images: the first image was created with light polarized in the direction parallel to muscle axis, and the second image was created with light polarized in the direction perpendicular to muscle axis. Images were recorded by high-sensitivity video camera and polarization was calculated from the relative intensity of both images. The method allows measurement of the fluorescence polarization from single myofibril irrigated with low concentrations of S1 labeled with dye. Orientation was also measured by fluorescence-detected linear dichroism. The orientation was different when muscle was irrigated with high concentration of S1 (molar ratio S1:actin in the I bands equal to 1) then when it was irrigated with low concentration of S1 (molar ratio S1:actin in the I bands equal to 0.32). The results support our earlier proposal that S1 could form two different rigor complexes with F-actin depending on the molar ratio of S1:actin.

Actins↗
PubMed1992

Two different acto-S1 complexes.

Based on change in anisotropy of fluorescently labelled S1 and on increase in turbidity of acto-S1 complex when S1 bound to F-actin, we reported previously that depending on the molar ratio of S1 to actin two different complexes of actin monomer (A) and myosin subfragment 1 (S1) could be formed: A1*S1 (one actin with one S1) and A2*S1 (two actins with one S1). Here we extend these findings to F-actin labelled with pyrene and cross-linked to S1 with 1-ethyl-3-(3-dimethyl-aminopropyl)carbodiimide (EDC). The fluorescence of pyrene F-actin decreased with increase in S1 concentration and reached saturation at a molar ratio of S1 to actin of either 0.5 or 1.0, depending on whether S1 was added slowly (5 min) or quickly (10-20 s between additions). Incubation of A2*S1 complex in excess of S1 for > 1 h caused a shift in equilibrium towards the A1*S1 complex. The A2*S1 complexes were not formed at high S1 to actin ratios (> 1.0) owing to competition between heads. Crosslinking experiments showed that the formation of EDC crosslinked products, 175-185 kDa doublet and 265 kDa band, depended on the ratio S1 to actin. To assess the relative ratio of S1 and actin in crosslinked products, we labelled S1 and F-actin with different fluorescent probes (5-IAF and IATR). The S1 to actin ratio was proportional to the ratio of intensities of fluorescence of labelled S1 and actin. The S1 to actin ratio in 265 kDa product was two times smaller than in 175-185 kDa doublet (which is believed to be A1*S1 complex) and therefore 265 kDa band corresponded to A2*S1. Transition between two types of binding may be important to understanding how muscle contracts.

Actin Cytoskeleton↗
PubMed1991

The myosin head can bind two actin monomers.

Force impulse is thought to be generated in muscle when myosin head (S-1), while weakly bound to actin filament, undergoes orientational change to form a strong (rigor) bond with actin. There is ample evidence that this bond involves interaction of 1 myosin head with 1 actin monomer. However, X-ray diffraction data of muscle decorated with S-1, as well as recently proposed model of the thin filaments, suggested that each S-1 molecule interacted with two actin monomers. We reinvestigated this controversy and found that the stoichiometry of acto-S-1 bond depended on the relative amounts of actin and myosin present during titrations: when increasing amounts of actin were added to a fixed amount of S-1 (i.e. when myosin heads were initially in excess over actin), the saturating stoichiometry was 1 mol of S-1 per 1 mol of actin. However, when increasing amounts of S-1 were added slowly to a fixed amount of F-actin (i.e. when actin was initially in excess over S-1), the stoichiometry at saturation was 1 mol of S-1 per 2 mols of actin. The ability of S-1 to bind either one or two actin monomers suggests a way that force could be generated during muscle contraction.

Actins↗
PubMedDate not supplied

[Effect of polyamines on the thermal resistance of Escherichia coli cells in heat shock].

Polyamines intensify the effect of a heat shock on Escherichia coli M-17 cells. The lethal effect of polyamines rises in the spermidine--spermine series as their concentration and the duration of heat action is increased. The effect of polyamines on the cells subjected to a heat shock is not associated with the activity of amine oxidases and, apparently, does not destabilize the membranes. As was demonstrated using electron microscopy, the cells undergo morphological changes in the presence of polyamines in the course of a heat shock; in particular, electron-dense regions appear in the nucleoid zone, presumably, due to DNA conformational rearrangements during the shock in the presence of polyamines.

Cell Nucleus↗
PubMedDate not supplied

[Accuracy of determining sarcomere lengths in contracted muscle by laser diffraction method].

The method of laser diffraction by cross-striated muscle was used to investigate changes of sarcomere lengths during contraction. The development of muscle tension was recorded simultaneously with length measurings. The changes of the sarcomere length were determined from angular displacement between the left (+1)- and right-(-1)-first order maximums. The absolute error is less than 0.65 micrometer for the sarcomere lengths 1.8--2.8 micrometers and for the lengths 2.8--3.3 micrometers it is less than 0.1 micrometer. The resolution of length changes is under 0.003 micrometer over the whole range. It was shown that not only the positions of (+1)- and (-1))-diffraction maximums were displaced, but the position of zero order diffraction maximum might be changed. We explained this phenomenon in terms of form changes of the illuminated region of the fiber.

Animals↗