Preparation and characterization of frog muscle myosin subfragment 1 and actin

Ferenczi, Michael A.; Homsher, Earl; Trentham, David R.; Weeds, Alan G.

doi:10.1042/bj1710155

Cited by 47 publications

(20 citation statements)

References 30 publications

(31 reference statements)

Supporting

Mentioning

Contrasting

Order By: Relevance

“…The in vitro state, with its lack of mechanical and geometric constraints on protein interactions, probably corresponds most closely to the rapidly shortening state in intact muscle. The rate-limiting step in this case is 5 0 sec1-(kR) in the two-state model and this is similar to the in vitro actin-activated myosin subfragment-1 ATPase rate, estimated as 4-4 sec' for frog proteins at 0-2 0C (Ferenezi, Homsher, Trentham & Weeds, 1978). The rate constant of reaction A -+ B is 30 sec1during shortening (ks) and this is similar to the rate constant for tension fall during the isovelocity release, which can be calculated from the fit in Fig.…”

Section: Ahr Adp+pisupporting

confidence: 76%

The dependence on extent of shortening of the extra energy liberated by rapidly shortening frog skeletal muscle.

Irving¹,

Woledge²

1981

The Journal of Physiology

View full text Add to dashboard Cite

SUMMARY1. Pairs of frog sartorius muscles were stimulated for 2 sec at 0 IC and, after 1 see of isometric contraction, were released at a constant velocity. The total excess heat (shortening heat) and work associated with the release were determined by comparison with isometric control tetani.2. Shortening heat and work production were non-linearly related to the distance shortened. There was proportionally more energy liberation for smaller releases.3. The dependence of shortening heat on muscle length was investigated within the sarcomere length range 2-1-2-6 ,sm (as measured in resting muscle) and was found to be similar to that of isometric tension. 4. A simple model in which heat and work are produced in a two-state cycle can describe these and previous results concerning the energetics of rapidly shortening muscle.

show abstract

Section: Ahr Adp+pisupporting

confidence: 76%

The dependence on extent of shortening of the extra energy liberated by rapidly shortening frog skeletal muscle.

Irving¹,

Woledge²

1981

The Journal of Physiology

View full text Add to dashboard Cite

show abstract

“…5). A previous investigation showed that frog's myosin heavy chain and actin are of the same relative molecular weights as rabbit's myosin heavy chain and actin when estimated by PAGE (Ferenczi et al, 1978). This indicates that the prominent 200-and 42-kD bands seen by PAGE in this study are of myosin and actin, respectively.…”

Section: Evaluation Of Enzymatic Treatmentsupporting

confidence: 75%

Energy stored and dissipated in skeletal muscle basement membranes during sinusoidal oscillations

Tidball¹

1986

Biophysical Journal

View full text Add to dashboard Cite

We subjected single skeletal muscle cells from frog semitendinosus to sinusoidal oscillations that simulated the strain experienced as the cells near the end of passive extension and begin active contraction in slow swimming. Other cells from which the basement membrane was removed by enzymatic and mechanical procedures were tested identically. Effectiveness of the basement membrane removal technique was evaluated by electron microscopy, by an electrophoretic and lectin-binding assay for depletion of cell surface glycoproteins, and by confirmation by means of electrophoretic and immunologic analyses that major intracellular, cytoskeletal proteins were not disrupted. Measurements of maximum stress, maximum strain, and phase lag between these maxima enabled the complex modulus (dynamic stiffness) and loss tangent (relative viscous losses to elastic energy storage) to be calculated for each mechanically tested preparation. We also calculated the amounts of energy stored and dissipated in each preparation. These calculations indicate that cells with intact basement membranes have complex moduli significantly greater than those of cells without basement membranes, and that cells with basement membrane store significantly more elastic energy than basement membrane depleted cells. However, when subjected to identical sinusoidal strains, energy dissipation in cells with intact basement membranes is over three times greater than dissipation in cells without basement membrane. The relative magnitudes of energy losses to energy storage, called the specific loss, is nearly three times greater for intact cells than for basement membrane depleted cells. Basement membranes may thereby serve as a brake for slowing passive extension of muscle before contraction begins.

show abstract

“…This is a compelling reason for the application of in vitro studies to the contractile proteins from frog skeletal muscle. Up to now a serious impediment for these studies has been caused by problems encountered in preserving the enzymatic activity of frog myosin during and after the purification process (Ferenczi et al 1978; Pliszka et al 1978; Focant & Huriaux, 1980). Inactivation was likely to have been favoured by dissolving purified myosins in high ionic strength solution, since these molecules in situ exist only in a polymerized form.…”

Section: Introductionmentioning

confidence: 99%

“…Even after lowering the ionic strength of the extraction solution by 15 times, the rate of filament formation was minimum due to the inhibitory effect of pyrophosphate on filament formation (Davis, 1988). However, lowering pH from 6.4 to 6.1 increased the rate of filament formation (Ferenczi et al 1978). The pH of the solution was therefore adjusted to 6.1–6.2 using acetic acid and the myosin was incubated in this solution to polymerize on ice for 30 min.…”

mentioning

confidence: 99%

An integrated in vitro and in situ study of kinetics of myosin II from frog skeletal muscle

Elangovan

Capitanio²,

Melli

et al. 2012

The Journal of Physiology

View full text Add to dashboard Cite

Key points• Force and shortening in muscle are due to the ATP-powered motor protein myosin II, polymerized in two bipolar arrays of motors that pull the two overlapping actin filaments toward the centre of the sarcomere.• The parameters of the myosin motor in situ have been best characterized for the skeletal muscle of the frog, from which single intact cells can be isolated allowing fast sarcomere level mechanics to be applied.• Up to now no reliable methods have been developed for the study of frog myosin with single molecule techniques.• In this work a new protocol for extraction and conservation of frog muscle myosin allows us to estimate the sliding velocity of actin on myosin (V F ) and its modulation by pH, myosin density, temperature and substrate concentration.• By integrating in vitro and in situ parameters of frog muscle myosin we can relate kinetic and mechanical steps of the acto-myosin ATPase.Abstract A new efficient protocol for extraction and conservation of myosin II from frog skeletal muscle made it possible to preserve the myosin functionality for a week and apply single molecule techniques to the molecular motor that has been best characterized for its mechanical, structural and energetic parameters in situ. With the in vitro motility assay, we estimated the sliding velocity of actin on frog myosin II (V F ) and its modulation by pH, myosin density, temperature (range 4-30• C) and substrate concentration. V F was 8.88 ± 0.26 μm s −1 at 30.6• C and decreased to 1.60 ± 0.09 μm s −1 at 4.5• C. The in vitro mechanical and kinetic parameters were integrated with the in situ parameters of frog muscle myosin working in arrays in each half-sarcomere. By comparing V F with the shortening velocities determined in intact frog muscle fibres under different loads and their dependence on temperature, we found that V F is 40-50% less than the fibre unloaded shortening velocity (V 0 ) at the same temperature and we determined the load that explains the reduced value of V F . With this integrated approach we could define fundamental kinetic steps of the acto-myosin ATPase cycle in situ and their relation with mechanical steps. In particular we found that at 5• C the rate of ADP release calculated using the step size estimated from in situ experiments accounts for the rate of detachment of motors during steady shortening under low loads.

show abstract

Preparation and characterization of frog muscle myosin subfragment 1 and actin

Cited by 47 publications

References 30 publications

The dependence on extent of shortening of the extra energy liberated by rapidly shortening frog skeletal muscle.

The dependence on extent of shortening of the extra energy liberated by rapidly shortening frog skeletal muscle.

Energy stored and dissipated in skeletal muscle basement membranes during sinusoidal oscillations

An integrated in vitro and in situ study of kinetics of myosin II from frog skeletal muscle

Contact Info

Product

Resources

About