Evaluation of the sliding distance in shortening muscles and in polymerizing actin from Hill's force-velocity equation.

The relationship derived earlier between the sliding distance, Deltal(m), and a/P(0), the characteristic parameter of Hill's force-velocity equation for muscle contraction, was re-formulated in order to get a more general relationship which can be applied also to other biological mechano-chemical energy converters: alpha x Deltal(m)=phi (0)(a/P(0))Deltal(m)=-Deltag where Deltag is the free energy change accompanying the hydrolysis of one ATP molecule while alpha and phi (0) are, respectively, the average forces developed by a myosin head-actin complex which are responsible for shortening and for isometric tension generation. These two molecular forces are different in magnitude and in nature and it is demonstrated that alpha , not phi (0), is the true contractile force. The values of alpha and of phi (0) have been calculated for three muscles.

The simultaneous collapse of both the swinging crossbridge theory of muscle contraction and the in vitro motility essays.

In the early seventies we discovered that isolated, active, myosin fragments can induce movement and tension generation by actin filaments in both in vitro and in vivo systems, employing a variety of techniques. It was not in line with the domineering swinging crossbridge theory of muscle contraction. We then proposed an hydrodynamic mechanism which explained our results and was applied to muscle contraction and to other biological engines.

A new outlook on the energetics of muscle contraction.

Analysis of experimental data on two muscles demonstrates that, in contracting striated muscle, the total rate of ATP splitting, nu(t) (number of ATP molecules split per active myosin head per second), comprises of three separate components: nu(p), which is required for the generation of the contractile force P which is equal to the external load; nu(v) which is devoted to the development of the velocity of shortening V; and nu(w), which is responsible for the production of the mechanical power (PV). Nu(p) is proportional to P and nu(v) to V, which means that the sliding distance is independent of P. The mechanical power was found to be equal to the free energy change associated with the hydrolysis of nu(w), which means that the thermodynamic efficiency of the power-producing component is practically 100%.

Do the bacterial flagellar motor and ATP synthase operate as water turbines?

Despite much progress in the study of the rotary motors ATP synthase (F0F1) and the bacterial flagellar motor, we still cannot answer the most basic and simple question, how the random thermal movement of H+ (or Na+) ions down a pH (or Na+) gradient spins the rotors. I suggest consideration of the possibility that the motors operate like water turbines, i.e.

Are rotors at the heart of all biological motors?

Biological motors are generally divided into two classes: 1) rotary motors. These include ATP synthase (F0-F1) and the bacterial flagellar motor which are driven by proton and Na+ gradients., 2) linear motors.

Critical review of the swinging crossbridge theory and of the cardinal active role of water in muscle contraction.

A critical analysis is presented of the experimental findings that led to the sliding filament model and to its offspring--the swinging (by rotating or tilting) crossbridge theory of muscle contraction (SCBT). Several principles that have been taken for granted implicitly and explicitly by the creators of these dogmas are discussed. The failure of numerous efforts to verify predictions of the SCBT, particularly the idea that the myosin molecules undergo a major conformational change, is critically reviewed.

Phosphatidylserine directs differential phosphorylation of actin and glyceraldehyde-3-phosphate dehydrogenase by protein kinase C: possible implications for regulation of actin polymerization.

The phospholipid-dependent protein kinase C is implicated in the regulation of cellular motility and energy metabolism. Phosphatidylserine, a main cofactor of protein kinase C, is involved in the regulation of glyceraldhehyde-3-phosphate dehydrogenase, which as actin, was shown to be phosphorylated by purified protein kinase C. Here, we study the effect of phosphatidylserine on the enzyme-substrate interaction of protein kinase C with glyceraldhehyde-3-phosphate dehydrogenase and actin.

Interaction of purified protein kinase C with key proteins of energy metabolism and cellular motility.

The phospholipid dependent protein kinase C is involved in regulation of cellular motility and energy metabolism. To study a possible direct interaction of protein kinase C with cellular motility and energy metabolism, we used purified rat brain protein kinase C to phosphorylate key proteins of these systems. Protein kinase C phosphorylates with comparable stoichiometry the G- but not the F- form of muscle and brain actin, the key protein of cellular motility.

The role of water in the mechanism of muscular contraction.

Twenty-five years after its proposal, the swinging theory of muscular contraction, in which the majority of scientists in the field have blindly believed, has not yet been verified. Rapidly growing experimental evidence indicates that the myosin heads do not swing. It is time to look for an alternative mechanism.

The molecular basis of chemomechanical coupling in muscle and in other biological engines.

It is argued that the force driving muscular shortening (psi) differs from that (phi) responsible for rigor tension generation. psi is associated with ATP-induced dissociation of actomyosin (a.m.

Translational motion of actin filaments in the presence of heavy meromyosin and MgATP as measured by Doppler broadening of laser light scattering.

Intensity fluctuations of laser light scattering were utilized in order to follow enhancement of translational motion of the actin-heavy meromyosin (HMM) complex in extremely dilute solutions accompanied by the hydrolysis of MgATP. Such enhancement was anticipated on the basis of the idea that active streaming along actin filaments should be associated with their mechanochemical reactivity. Native tropomyosin was added in order to stabilize actin in its filamentous form, thus allowing the reduction of actin concentration below 50 micrograms/ml to enable free movement of neighboring filaments and yet give a reliable signal.

Evaluation of mechanical parameters of the mechanochemically 'active state' of actin filaments on the basis of purely chemical data.

Oosawa and his collaborators (cf. F. Oosawa, Biophys.

Cytosolic acidification as an early transductory signal of human neutrophil chemotaxis.

The inflammatory reaction of human neutrophils consists of two successive phases. In the first, designated chemotaxis, the cells home in on a foreign intruder. In the second, the cells attempt to eliminate the intruder by secreting lysosomal enzymes and superoxide anions.

Destabilization of actin filaments as a requirement for the secretion of catecholamines from permeabilized chromaffin cells.

In the search for a functional role of cytoskeletal proteins in the mechanism(s) of stimulus-secretion coupling, we have previously demonstrated that the actomyosin system might be involved in the transport of cations across the plasma membrane of bovine adrenal chromaffin cells [(1986) J. Biol. Chem.

Control of stimulus-secretion coupling in adrenal medullary chromaffin cells by microfilament-specific macromolecules.

We have incorporated the myosin fragment heavy meromyosin (HMM), which is known to interact mechanochemically and enzymatically with actin filaments, into intact chromaffin cells of the bovine adrenal medulla, in order to study the possible involvement of actin and myosin in stimulus-secretion coupling. HMM was found to stimulate secretion of catecholamines, to cause depolarization of the plasma membrane, and to enhance 22Na+ uptake. HMM-stimulated catecholamine secretion was dependent on the presence of extracellular Na+.

Possible involvement of actin and myosin in Ca2+ transport through the plasma membrane of chromaffin cells.

The exocytosis of catecholamines by chromaffin cells following stimulation (e.g. by acetylcholine) is accompanied by a rise in the level of intracellular free Ca2+.

Active streaming against gravity in glass microcapillaries of solutions containing acto-heavy meromyosin and native tropomyosin.

Solutions containing heavy meromyosin, actin, native tropomyosin, and Mg-ATP exhibited streaming in horizontally placed glass microcapillaries. Up-hill streaming could also be observed when the capillaries were at an inclined position; this served for the clear distinction between active and passive streaming provided surface tension effects were eliminated. The presence of native tropomyosin and actin-activation of the ATPase activity of HMM were essential for the reconstitution of active streaming.

Polymerization of G-actin by hydrodynamic shear stresses.

In the absence of Ca2+ G-actin can be polymerized by the application of shear stress in low ionic strength buffer. When G-actin in low ionic strength buffer containing EGTA was sheared for predetermined times under different velocity gradients, viscosity attained a maximal value, comparable to that obtained by seeding with F-actin nuclei, at a velocity gradient of 3000 s-1 after about one hour. Such flow-polymerized actin was indistinguishable from KCl-polymerized actin.

Effect of modulators of cytoskeletal function on desensitization and recovery of PGE2-responsive ovarian adenylate cyclase.

Exposure of cultured Graafian follicles to PGE2 for 20 h resulted in a loss of the cyclic AMP response to fresh hormone. This desensitization was prevented by addition to the medium of D2O (25--50%) or Li+ (0.6--6 mM), agents believed to stabilize microtubules, as well as by phalloidin (1.