Fumio Oosawa

Polarized fluorescence from ε-ADP incorporated into F-actin in a myosin-free single fiber: Conformation of F-actin and hanges induced in it by heavy meromyosin

Abstract Fluorescent ADP analog, e-ADP (1: N 6 -ethenoadenosine 5′-diphosphate), was incorporated into F-actin in a myosin-free ghost single fiber and polarized fluorescence measurements were performed under a microspectrophotometer to investigate the conformation of F-actin and the changes induced in it by heavy meromyosin and subfragment-1. Four components of polarized fluorescence were obtained by exciting the fiber with light polarized parallel and perpendicular to the long axis of the fiber and measuring the intensity of emission polarized parallel and perpendicular. From these data it was shown that F-actin in the fiber was not rigid but flexible, with a value for the elastic modulus for bending of 5.3 × 10 −17 dyn cm 2 . The angles of absorption dipole and emission dipole of bound e-ADP with the long axis of F-actin were both about 75 °. The binding of heavy meromyosin decreased the elastic modulus of F-actin by 30% and the angles of absorption and emission dipoles by 2.5 ° and 1.5 °, respectively. The molar ratios of bound heavy meromyosin and subfragment-1 to actin in the ghost fiber at saturation were 0.3 and 0.6, respectively, being smaller than those in solution.

The loose coupling mechanism in molecular machines of living cells

For a bacterial flagellar motor driven by a proton flux, a loose coupling mechanism has been proposed in which the movement of the proton is indirectly and loosely coupled with the rotation of the motor. This mechanism assures the efficient and smooth conversion of both electrical and chemical potential energies of the proton of the same order as the energy of the thermal fluctuation. Loose coupling has been also assumed for the proton ATPase. A proton flux produces a rotational movement of protein molecules and this movement promotes the synthesis of ATP. In the proposed mechanism, the number of protons necessary for the synthesis of one ATP molecule is not an integer but varies depending on the environmental condition. In the case of muscle, the coupling between the hydrolysis of ATP and the shortening was found to be extremely loose. It is likely that molecular machines in living cells often adopt a loose coupling mechanism in which the chemical reaction and the physical cycle have not always a definite one-to-one correspondence.

Electronmicroscopic investigation of the flexibility of f-actin

The contour lenghts and the end-to-end distances of a large number of F-actin filaments were measured in electronmicrographs. Preparation of F-actin for electron-microscopy was made at three different temperatures. The flexibility parameter or the elastic modulus for bending of F-actin was determined from the relation between the contour length and the end-to-end distance after statistical treatment. The value of the flexibility parameter obtained here showed good agreement with that obtained by the quasielastic light scattering at all temperatures examined. The flexibility of F-actin increased with raising temperature and with the binding of heavy meromyosin.

Coupling between flagellar motor rotation and proton flux in bacteria

The bacterial flagellar motor is driven by a flow of proton according to the electrochemical potential gradient across the membrane. For understanding efficient and smooth transformation of both electric and chemical potential energies of proton into a mechanical work, loose coupling has been assumed between the flow of protons and the rotation of the motor. In the mechanism proposed, two kinds of protein molecules arranged on a disc and on a ring surrounding the disc repeat fast and reversible reactions regulated by the binding of protons. Based on this mechanism, the relations of the torque generated in the motor and the flux of protons to the speed of rotation of the motor have been derived. The results of numerical calculations show reasonable agreement with most of the experimental results. Particular attention has been paid to the possibility of reverse transformation of free energy. It is expected that by rotating the motor in the reverse direction by an external force, the flux of protons is decre...

Conformational changes of F-actin-ε-ADP in thin filaments in myosin-free muscle fibers induced by Ca2+

Polarized fluorescence from F-actin-e-ADP in thin filaments reconstituted in a myosin-free single muscle fiber was measured at various concentrations of Ca2+. Four components of polarized fluorescence changed with increasing Ca2+ concentration at pCa values of around 7 to 6, concomitant with a change of the tension generated by the fiber irrigated with myosin in the presence of Mg-ATP. From analysis of observed values of the four components, it was found that the flexibility of the thin filament increased, or the elastic modulus for bending decreased from 5.7 × 10−17 dyn cm2 to 4.7 × 10−17 dyn cm2, when the pCa value decreased from 7 to 6. In the same range of pCa values, the angles of absorption and emission dipoles of e-ADP changed, suggesting a small rotation of the base-plane of e-ADP around an axis perpendicular to the F-actin axis.

Behavior of micro-organisms as particles with internal state variables

Abstract A theory is presented to describe the behavior of micro-organisms, bacteria and protozoa. Individual cells are regarded as particles having internal state variables. The change of each variable with time depends on the environmental condition. The velocity and the frequency of direction change of swimming cells are determined by the values of these variables. With this framework, the theory gives a method to connect the behaviour in a spatial gradient of the environment and the behaviour upon a change of the environment with time. Observed behaviors of bacteria and protozoa are understandable on the basis of simple rate equations for internal state variables and the product expressions for the velocity and the frequency of direction change as functions of these variables. Experimental data on the thermotaxis of paramecium are shown for comparison with the theoretical results.

A Loose Coupling Mechanism of Synthesis of ATP by Proton Flux in the Molecular Machine of Living Cells

A loose coupling mechanism is proposed for the molecular machine of living cells which performs the synthesis of ATP from ADP and inorganic phosphate by a proton flux across the membrane. The basic assumption is that a torque or rotational movement is produced in protein molecules by the proton flux and the ATP synthesis is promoted by the torque or rotational movement in the molecules. The first process is carried out by a mechanism similar to the bacterial flagellar motor where the coupling between the flow of protons and the rotation of the motor is loose. A structural model for the second process is also presented. The threshold of the potential difference of proton for the ATP synthesis can be low and the number of protons to synthesize one ATP molecule is not an integer but a variable number.

Fluctuation in living cells: effect of field fluctuation and asymmetry of fluctuation

The electric potential in living cells of paramecium shows large spontaneous fluctuation, which consists of basic fluctuation and spikelike fluctuation. The spikelike fluctuation triggers transient reversal of ciliary beating and causes discontinuous change of the swimming direction. A positive shift of the basic potential increases the probability of the spikelike fluctuation. The spike is generated by opening of electric field-sensitive channels in the cell membrane. A differential equation is proposed to describe the probabilistic behavior of these channels in a fluctuating electric field. The fluctuating field increases the average rates of open-close transitions of channels and shifts the average opening probability towards 1/2. The open-close fluctuation in an assembly of the channels has asymmetry with respect to time reversal. Free energy is continuously consumed for generation of the spikelike fluctuation. The proposed equation which contains fluctuating quantities in exponential terms has a definite physical basis and is useful for the analysis of stochastic processes in a fluctuating field.