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A Oplatka

Publications and source records attributed to A Oplatka.

At least 19 recordsLinked to original sources
PubMed2005

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. Our discovery has been ignored for a long time until the so-called "in vitro motility essays" appeared. By using this artifact--laden technique the mechanochemical reactivity of the active myosin fragments was re-discovered without giving us any credit. The essays gave continuously changing values for fundamental parameters of muscle contraction; the values were appreciably different in different laboratories and decreased in a continuous fashion in the hands of one scientist. By analyzing recent experiments which derived the rate of ATP hydrolysis of active muscles as function of the applied load I calculated the value of the sliding distance resulting from the breakdown of one ATP molecule by each of the myosin heads in contracting muscle. According to the contemporary theory this should be the same for all muscles under any environmental conditions and determined by length of the myosin head's neck. My examination led to the conclusion that the sliding distance varies from one muscle to another and with different temperatures for the same muscle. This again, contradicts the current theory and should give the final blow to both this theory and the "essays". Furthermore: it can be explained by a hydrodynamic mechanism such as that proposed by us more than 30 years ago.

Actin Cytoskeleton↗
PubMed2000

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%. It is concluded that ATP hydrolysis is actually three different reactions. The analysis leads to Hill's force-velocity relationship. Its empirical constants a and b are expressed by thermodynamic and molecular parameters. The constant a was found to be inversely proportional to the sliding distance. The same considerations and conclusions should apply also to other muscles and to the movement under load of microtubules interacting with, e.g. kinesin.

Adenosine Triphosphate↗
PubMed1998

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., rotation is the outcome of water mini-jets impinging tangentially on the rotors. The vectorial jets are formed when the cations lose part or all of their hydration water upon interacting with a properly positioned site on a protein component which is part of the rotor or is located on a peripheral protein.

Adenosine Triphosphate↗
PubMed1998

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. Myosin, kinesin and dynein are considered to be such motors, F-actin and microtubules serving as passive "tracks". However, data is presented which suggests that the actin filaments rotate in shortening muscle. Microtubules also have been reported to rotate upon interacting with kinesin and dynein. Axial protein rotation thus appears to be a common fundamental characteristic of actin- and of microtubule-based motility systems, in addition to F0-F1 and the bacterial motor. An analysis is carried out of the way ATP hydrolysis and randomly moving protons can induce rotation. It is concluded that all four engines are driven by water jets, thus operating like water turbines.

Actins↗
PubMed1997

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. Analysis of various experimental data suggests that water may play an active role in muscular contraction. Examination of both the experiments that do not fulfill the expectations of the SCBT and the measurements of water liberation during the "contractile" process suggests a new outlook according to which tension development and movement are not due to major conformational changes but rather to restructuring of the hydration shells of actin and myosin.

Actin Cytoskeleton↗
PubMed1996

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. The stoichiometry of glyceraldhehyde-3-phosphate dehydrogenase phosphorylation is not affected by varying the level of phosphatidylserine. However, actin phosphorylation is dependent on phosphatidylserine level, peaking at high phosphatidylserine concentration. Moreover, if actin and glyceraldhehyde-3-phosphate dehydrogenase are cophosphorylated at high phosphatidylserine concentration, actin phosphorylation is favored, despite lower affinity for protein kinase C. Hence, phosphatidylserine directs differential phosphorylation of these key proteins of glycolysis and cellular motility and might be capable of recruiting protein kinase C for preferential actin phosphorylation. The sedimentation of phosphorylated actin is increased 3.8 fold and total actin 1.7 fold, suggesting that phosphorylation promotes actin polymerization.

Actins↗
PubMed1996

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. Glyceraldehyde-3-phosphate dehydrogenase, creatine kinase and glutamine synthase, key enzymes of energy metabolism, are also phosphorylated at comparable stoichiometry. The data suggest that protein kinase C might be directly involved in the regulation of cellular motility and energy metabolism.

Actins↗
PubMed1994

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. Data is presented indicating that water is liberated during tension development and the extent to which it is released appears to affect the degree of tension. Since water can move (because of acquired extra energy, involvement in hydration forces etc.), it might cause protein movement.

Actins↗
PubMed1991

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.), whereas phi is due to an isomerization reaction of a.m., following the hydrolysis of ATP. Both forces are intimately coupled with appreciable changes in the structure of the hydration shell of a.m., mainly at the interface between the two proteins, which involve the release of stored energy. When an active muscle is allowed to shorten freely, psi gives rise to a sliding distance (s.d.) delta l1 which differs in character and in magnitude from the s.d. (delta l2) observed when a muscle which had developed rigor tension isometrically is released. The maximal values of the two forces (psi 0 and phi 0) as well as delta l2 are calculated on the basis of experimental data. The forces and their corresponding s.d.'s are related through the standard free energies of the chemical reactions which are responsible for them. It is claimed that the same mechanochemical (m.c.) mechanisms operate also in all microtube-based locomotion and force-generation systems and, furthermore, that practically the same values of psi 0, phi 0, delta l1, and delta l2 are shared by the two types of biological m.c. convertors.

Actomyosin↗
PubMed1990

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. Analysis of the data in terms of Doppler broadening led to an approximate evaluation of the average velocity of translation of the mobile filaments. This velocity was found to increase with increasing HMM concentration up to a maximum attained at a molar ratio HMM/actin of 1:2, and then decreased. Total intensity measurements indicate that the mobile scatterer is actually a complex of HMM with an isolated actin filament. HMM subfragment-1 was found to be ineffective. These results suggest that cooperation between the two myosin heads is necessary for efficient induction of active streaming along isolated actin filaments.

Actins↗
PubMed1988

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. Chem. 11 (1980) 443), employing various optical techniques, have shown that the flexibility of actin filaments increases upon interacting with the enzymatically active myosin fragments, particularly heavy meromyosin (HMM). It has been reported (S. Hitchock, L. Carlsson and U. Lindberg, Cell 7 (1976) 53) that HMM can accelerate the DNase 1-induced depolymerization of F-actin, provided MgATP is also present. Since, as we have demonstrated (cf. J. Borejdo myosin, is endowed with mechanochemical capability, we made an attempt to correlate the enhanced rate of depolymerization with the decrease in rigidity of the G-G bonds in F-actin. On the basis of the chemical kinetic data of Hitchcock et al. we could derive the approximate value of the HMM-MgATP-induced change in rigidity which is a mechanical molecular parameter. Since interaction between HMM or HMM subfragment-1 and F-actin in the presence of MgATP leads to the movement of the myosin heads along the actin filaments, it is argued that the enzymic behavior of this system should not be analyzed on the basis of simple, equilibrium, complex formation.

Actins↗
PubMed1987

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. The initiation of chemotaxis involves prompt morphological changes that are manifested by a sharp biphasic drop in light scattering, accompanied by a transient cytosolic acidification. In a search for a causal relation between these two events, the neutrophil cytoplasm was abruptly acidified by the application of sodium propionate. This evoked a pulse of decreasing light-scattering, the time course and amplitude of which were practically identical to the rapid response induced by chemoattractants such as N-formyl-L-methionyl-L-leucyl-L-phenylalanine (fMLP). Both fMLP- and sodium propionate-induced responses were unaffected by amiloride, but were inhibited with a similar dose-dependence by a series of proton uncouplers. The initial phase of the cytosolic acidification seems, therefore, to fulfill the criteria for a second messenger for the initiation of chemotaxis.

Amiloride↗
PubMed1986

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. 261, 5745-5750]. To establish whether actin and myosin might also be involved in later stages of the cellular response, we have examined the possible effects of various actin-specific reagents on the calcium-mediated secretion of catecholamines from digitonin-permeabilized cells. F-Actin-destabilizing agents, such as cytochalasin D or DNase 1, were found to promote Ca2+-stimulated (as well as basal) secretion. By contrast, stabilizers, like phalloidin, produced the opposite effect. It is concluded that stimulus-secretion coupling in chromaffin cells might require the reorganization of actin for modulating both ion transport across the plasma membrane and exocytotic secretion per se.

Actin Cytoskeleton↗
PubMed1986

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+. The Na+ uptake caused by HMM was inhibited by 10 microM amiloride. Acetylcholine-stimulated catecholamine secretion and 22Na+ uptake were both enhanced by HMM incorporation. A Na+/H+ antiporter, activated by the interaction of HMM with the cells' microfilaments, seems to be involved in HMM action and could possibly also be a component of stimulus-secretion coupling in chromaffin cells, induced by regular agonists.

3-Pyridinecarboxylic acid, 1,4-dihydro-2,6-dimethy↗
PubMed1984

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+. Actually, secretion can be induced merely by making the cells leaky to Ca2+ from the external medium. We have recently demonstrated that secretion can be increased by the introduction of DNase-I, the F-actin depolymerizing agent, or of heavy meromyosin, the enzymatically active fragment of myosin. Suspecting that these changes might be associated with a higher intracellular level of Ca2+, we now have measured the influx of 45Ca2+ into chromaffin cells which have undergone fusion with DNase-I- or with heavy meromyosin-loaded liposomes. In both cases, a marked increase in Ca2+ uptake has been observed, which could be abolished by Co2+ ions (a Ca2+ channel blocker), suggesting an intimate involvement of the cellular actomyosin system in the process of Ca2+ ions transport through the Ca2+ channels of the plasma membrane.

Actins↗
PubMed1982

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. The significance of the results for cytoplasmic streaming and muscle contraction is discussed.

Animals↗