Complexes of smooth muscle tropomyosin with F-actin studied by differential scanning calorimetry.

Differential scanning calorimetry (DSC) and light scattering were used to analyze the interaction of duck gizzard tropomyosin (tropomyosin) with rabbit skeletal-muscle F-actin. In the absence of F-actin, tropomyosin, represented mainly by heterodimers, unfolds at 41 degrees C with a sharp thermal transition. Interaction of tropomyosin heterodimers with F-actin causes a 2-6 degrees C shift in the tropomyosin thermal transition to higher temperature, depending on the tropomyosin/actin molar ratio and protein concentration. A pronounced shift of the tropomyosin thermal transition was observed only for tropomyosin heterodimers, and not for homodimers. The most pronounced effect was observed after complete saturation of F-actin with tropomyosin molecules, at tropomyosin/actin molar ratios > 1 : 7. Under these conditions, two well-separated peaks of tropomyosin were observed on the thermogram besides the peak of F-actin, the peak characteristic of free tropomyosin heterodimer, and the peak with a maximum at 45-47 degrees C corresponding to tropomyosin bound to F-actin. By measuring the temperature-dependence of light scattering, we found that thermal unfolding of tropomyosin is accompanied by its dissociation from F-actin. Thermal unfolding of tropomyosin is almost completely reversible, whereas F-actin denatures irreversibly. The addition of tropomyosin has no effect on thermal unfolding of F-actin, which denatures with a maximum at 64 degrees C in the absence and at 78 degrees C in the presence of a twofold molar excess of phalloidin. After the F-actin-tropomyosin complex had been heated to 90 degrees C and then cooled (i.e. after complete irreversible denaturation of F-actin), only the peak characteristic of free tropomyosin was observed on the thermogram during reheating, whereas the thermal transitions of F-actin and actin-bound tropomyosin completely disappeared. Therefore, the DSC method allows changes in thermal unfolding of tropomyosin resulting from its interaction with F-actin to be probed very precisely.

Use of stable analogs of myosin ATPase intermediates for kinetic studies of the "weak" binding of myosin heads to F-actin.

It is known that ternary complexes of myosin subfragment 1 (S1) with ADP and the Pi analogs beryllium fluoride (BeFx) and aluminum fluoride (AlF4-) are stable analogs of the myosin ATPase intermediates M* x ATP and M** x ADP x Pi, respectively. Using kinetic approaches, we compared the rate of formation of the complexes S1 x ADP x BeFx and S1 x ADP x AlF4- in the absence and in the presence of F-actin, as well as of the interaction of these complexes with F-actin. We show that in the absence of F-actin the formation of S1 x ADP x BeFx occurs much faster (3-4 min) than that of S1 x ADP x AlF4- (hours). The formation of these complexes in the presence of F-actin led to dissociation of S1 from F-actin, this process being monitored by a decrease in light scattering. The light scattering decrease of the acto-S1 complex occurred much faster after addition of BeFx (during 1 min) than after addition of AlF4- (more than 20 min). In both cases the light scattering of the acto-S1 complex decreased by 40-50%, but it remained much higher than that of F-actin measured in the absence of S1. The interaction of the S1 x ADP x BeFx and S1 x ADP x AlF4- complexes with F-actin was studied by the stopped-flow technique with high time resolution (no more than 0.6 sec after mixing of S1 with F-actin). We found that the binding of S1 x ADP x BeFx or S1 x ADP x AlF4- to F-actin is accompanied by a fast increase in light scattering, but it does not affect the fluorescence of a pyrene label specifically attached to F-actin. We conclude from these data that within this time range a "weak" binding of the S1 x ADP x BeFx and S1 x ADP x AlF4- complexes to F-actin occurs without the subsequent transition of the "weak" binding state to the "strong" binding state. Comparison of the light scattering kinetic curves shows that S1 x ADP x AlF4- binds to F-actin faster than S1 x ADP x BeFx does: the second-order rate constants for the "weak" binding to F-actin are (62.8 +/- 1.8) x 10(6) M-1 x sec-1 in the case of S1 x ADP x AlF4- and (22.6 +/- 0.4) x 10(6) M-1 x sec-1 in the case of S1 x ADP x BeFx. We conclude that the stable ternary complexes S1 x ADP x BeFx and S1 x ADP x AlF4- can be successfully used for kinetic studies of the "weak" binding of the myosin heads to F-actin.

Kinetics of interaction of myosin subfragment 1 isoforms with F-actin.

The interaction of the myosin subfragment-1 (S-1) isoforms containing different alkali light chains A1 and A2 with F-actin has been studied using the stopped-flow technique and sedimentation in an analytical ultracentrifuge. The data obtained suggest a different mode of attachment of the S-1 isoforms to F-actin filaments.

Polycondensation reactions of certain biologically essential molecules on mineral surfaces.

Methods of detecting extraterrestrial forms of organic matter should take into account the results of research in the effects of irradiation on the synthesis of biopolymers. Processes occurring on clay mineral surfaces and on the surfaces of minerals of different kinds are illustrated and the possible role of clays in prebiological evolution is discussed.

[The simultaneous synthesis of peptides and oligonucleotides on kaolinite with the participation of aminoacyladenylates]. / Odnovremennyi sintez peptidov i oligonukleotidov na kaolinite s uchastiem aminoatsiladenilatov.

A simultaneous synthesis of peptides (2-5 residues) and oligonucleotides (3-9 residues) has been carried out on caolinite matrix using amino acids and aminoacyladenylates as substrates. The rate of oligomer synthesis on mineral surface is higher than that in solution. The mechanism of synthesis has been described. The data has been discussed in connection with abiogenesis of two major types of biopolymers, proteins and nucleic acids.

[Structural and functional properties of irradiated nuclear membranes at the air-water phase interface]. / Strukturno-funktsional'nye svoistva obluchennykh iadernykh membran na granitse razdela faz vozdukh-voda.

The monolayer technique, the methods of electron microscopy and IR-spectroscopy were used to study isolated nuclear membranes of calf thymus cells. The data obtained permitted to study the relationship between structural disorders induced by irradiation of membranes, the changes in their functional status, and the role played by a lipid component of membranes in these processes.

[Study of radiation damage to nuclear membranes by IR spectroscopy and monolayer technics]. / Issledovanie radiatsionnykh narushenii iadernykh membran metodom IK-spektroskopii i monosloinoi tekhniki.

A study was made of the effect of X-radiation on nuclear membranes. Good resolution spectra of nuclear membrane of equal effective thickness were obtained by the method of internal reflection spectroscopy in the infrared region. The experiment indicated the presence of major characteristic bands of protein and phospholipid molecular groups of non-irradiated nuclear membranes. Conformational changes in the samples of the exposed (100 and 1000 Gy) lipoprotein complex resulted in some quantitative changes in the absorption intensities of the molecular groups of membranes. These data are consistent with the behaviour of nuclear membranes on the interphase, studied by the Langmur monolayer technique, and with the changes in the membrane morphology observed with the electron microscope.

[Kinetics of abiogenic peptide bond synthesis with aminoacyladenylate participation as condensing agents]. / Issledovanie kinetiki abiogennogo sinteza peptidnoi sviazi pri uchastii aminoatsiladenilatov v kachestve kondensiruiuschchikh agentov.

Kinetics of the process has been investigated employing differential spectroscopy technique. Possibility of synthesis of peptide bonds in aqueous solutions using aminoacyladenylates as condensing agents was shown. Analysis of the data obtained shows that the synthesis of peptide bond under the conditions investigated with the excess amounts of the second substrate follow the first order equation for irreversible reactions, the rate of the reaction decreasing with the increase in substrate concentrations. Velocity constants are determined for the first order reaction at different substrate concentrations. The data obtained are discussed in relation to the problem of abiogenic synthesis of biologically important macromolecules.