Interaction Of Heavy Meromyosin Research Articles

Protein‐Protein Interaction of Fish Myosin Fragments

ABSTRACTProtein‐protein interaction of myosin fragments from flying fish and white marlin muscles was studied by means of absorbance changes resulting from aggregation at temperatures of 20°C to 70°C. Subfragment‐1 (S‐1) exhibited a high extent of interaction with the transition temperature of 35–36°C, while the interaction of heavy meromyosin (HMM) was very weak. Though light meromyosin (LMM) gave lower interaction values throughout the heating temperature, the addition of butanol promoted markedly the interactions at the temperature above 50°C. The degree of promotion was high for flying fish and low for white marlin.

Effect of phosphorylation of myosin light chains on interaction of heavy meromyosin with regulated F-actin in ghost fibers.

The binding of phosphorylated heavy meromyosin to regulated F-actin in ghost fibers at high Ca2+ concentration increases, and at low Ca2+ concentration decreases, the anisotropy of intrinsic tryptophan fluorescence of F-actin. The effect is opposite to the effect of the binding of dephosphorylated heavy meromyosin.

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.

A Study of Cell Motility Using the Reconstituted Systems

The muscle contraction is the process in which the chemical energy of ATP is transformed to the mechanical energy. Since it is very difficult to analyze quantitatively the chemical change accompanying mechanical work in a living muscle, the mechanochemical coupling of energy conversion was studied using a reconstituted motile system, “stream cell”. In the cell circulating flow could be induced by the interaction of heavy meromyosin (HMM) with attached polarized actin, both of which were extracted from skeletal muscle of a rabbit. Improving the way of attaching F-actin, a new biological motor, which was based on microscopic energy conversion, was also developed. The motor, “actomyosin motor”, has six blades, on which F-actin-HMM assembly is formed, and can rotate in a specific directon in the HMM solution. It was shown in these reconstituted systems that the assembly, not necessarily in the fibrous form, produces a vectorial force utilizing the energy of ATP hydrolysis.The physiological significance of these reconstituted systems depends on to what extent chemical and physical properties of the systems resemble those of muscle system under contraction. The reconstituted systems worked only when the physiological condition was the same as that of muscle. In the case of the stream cell, the ATPase activity at higher streaming velocity was higher than that at lower velocity. This corresponds to the Finn's effect in the muscle system. A close analysis of the relation between chemical and mechanical properties during active streaming led to a conclusion that the extent of self-organization of macroscopic streaming, or the resulting flow velocity, controls the molecular dynamics of the elementary cycles, namely, the ATPase activity of individual myosin molecules.

Dissociation of actomyosin by vanadate plus ADP, and decomposition of the myosin-ADP-vanadate complex by actin.

In the presence of vanadate (Vi) and ADP, myosin ATPase forms a stable inactive complex (myosin.ADP.Vi) at the active site. To elucidate the nature of the inactive complex, we studied the effect of Vi plus ADP on the interaction of heavy meromyosin (HMM) with F-actin. 1) Viscosity measurements showed that the actin-HMM rigor complex was dissociated into actin and HMM by Vi and ADP (both 10(-3) M range). 2) When the HMM.ADP.Vi complex isolated by gel filtration was mixed with actin in the absence of free Vi, about 60% of the added HMM formed a complex with actin, and more than 70% of the HMM bound to actin released Vi and ADP. 3) When a mixture of the isolated HMM.ADP.Vi complex with actin was dialyzed against a buffer without free Vi and free ADP, only less than 10% of Vi and ADP, which were originally bound to the HMM, were retained in the dialysis tube after 4 days. In contrast, if actin was omitted, about 80% of Vi and ADP were retained. 4) These results indicate that the HMM.ADP-Vi complex is dissociated from actin, and that Vi and ADP originally trapped at the HMM active site can be almost completely released from the active site by actin if free (released) Vi and ADP are concomitantly removed.

The interaction of heavy meromyosin and subfragment 1 with actin. Physical measurements in the presence and absence of adenosine triphosphate.

Viscosity, turbidity, and laser-light fluctuation autocorrelations of acto-heavy merymyosin (HMM) and acto-subfragment 1 (S-1) solutions were measured under conditions where the actin-activated ATPase is close to its maximal value. The results were compared to similar data obtained in the absence of ATP where the actin and myosin fragments were completely domplexed, and in the presence of ATP but at 0.1 M KLC where the actin and HMM or S-1 were almost completely dissociated. It was found that at maximal actin activation, the viscosity, turbidity, and autocorrelation data were all much closer to the values for the completely dissociated systems than to the values for the completely complexed systems. Assuming that viscosity, turbidity, and autocorrelation measurements approximate a linear measure of binding between actin and HMM or S-1, the results suggest that at maximal actin activation less than 10% of the HMM or S-1 are bound to the actin. Therefore as was suggested previously by ultracentrifuge and kinetics studies, it appears that under conditions of maximal actin activation, most of the HMM and S-1 occur in a refractory state unable to bind to actin.

Enzymatic mechanochemistry. I. The interaction of heavy meromyosin with “immobilized adenosine triphosphate”

ATP was covalently bound to an agarose gel. The insolubilized ATP was found to be capable of specifically binding heavy meromyosin. The adsorbed heavy meromyosin could be eluted by ATP in solution. Both binding and elution by ATP of heavy meromyosin were not much effected by Ca 2+, Mg 2+ or EDTA. While the water-soluble polyalanine-myosin was also found to be adsorbed, myosin in 0.5 M KCl did not seem to be adsorbed by agarose-ATP. Both Mg 2+ and Ca 2+ appear to activate the splitting of bound ATP by heavy meromyosin to practically the same extent. We prepared water-soluble derivatives of ATP in which ATP underwent the same chemical modification required for its coupling to agarose but in which the agarose component was absent. Their splitting by heavy meromyosin was also activated by Mg 2+ though to a lesser extent but actin did not influence this reaction. Possible relations between our findings and the various stages of the reaction between myosin and ATP, as well as the potential use of columns filled with insolubilized NTPs for the separation and purification of myosin and of its subfragments, are discussed.

Interaction of heavy meromyosin with substrate. VI. The difference absorption spectra induced by ATP analogs and 2-hydroxy-5-nitrobenzyl bromide.

Interaction of heavy meromyosin with substrate. VI. The difference absorption spectra induced by ATP analogs and 2-hydroxy-5-nitrobenzyl bromide.

A two-filament system and interaction of heavy meromyosin (HMM) with thin filaments in smooth muscle.

The structure of glycerinated smooth muscle from small intestine of adult rat was investigated by electron microscopy. In the central parts of the tissue blocks a two-filament system was found, consisting of parallel thick and thin filaments with regularly spaced interconnections, closely resembling that of striated muscle. In the peripheral parts of the blocks only thin filaments were found. The thin filaments were identified as actin by the formation of arrowhead complexes after incubation with heavy meromyosin.

Interaction of heavy meromyosin with substrate III. Difference spectrum of ultraviolet absorption in subfragment 1 induced by ATP or ADP

1. 1. The ultraviolet absorption difference spectrum of Subfragment 1 induced by ATP was measured in the presence of MgCl 2. The shape of the difference spectrum was the same as that of heavy meromyosin. The difference molar extinction coefficient at the major peak, ΔE 289 m μ , was 2200 ± 400 M −1·cm −1, which was 44% of that of heavy meromyosin. It decreased with time especially near 290 mμ and reached an identical shape as that induced by ADP. 2. 2. The difference spectra of Subfragment 1 induced by the addition of ADP and of heavy meromyosin had identical forms. ΔE 288 m μ was 1300 ± 400 M −1·cm −1, which was 52% of the value of heavy meromyosin. 3. 3. The dependence on the concentration of the difference absorbance induced by ADP was measured at 288 mμ. A strong binding of ADP to Subfragment 1 was suggested. The minimum ADP concentration needed to give the maximum change in the difference absorbance was 1.2 times the concentration of Subfragment 1. 4. 4. Both difference spectra due to ATP and ADP were not dependent on the KCl concentration. 5. 5. Decay of the difference spectrum of Subfragment 1 induced by ATP was measured. The value of k s obtained from Chance's equation agreed with the maximum velocity of steady-state ATPase.

Interaction of heavy meromyosin with substrate II. Rate of the formation of ATP-induced ultraviolet difference spectrum of heavy meromyosin measured by stopped-flow method

1. 1. The rate of formation of the ultraviolet difference spectrum of heavy meromyosin, induced by ATP, was measured by the stopped-flow method. The initial velocity of formation was proportional to the initial concentration of ATP, indicating a second-order reaction. The second-order rate constant decreased with decrease of MgCl 2 and with increase of KCl concentrations, but did not depend on the kind of divalent cation (Mg 2+ or Ca 2+) in the medium. 2. 2. The maximum difference absorbance Δ; A max attained after addition of ATP was obtained as a function of initial concentration of ATP. A strong binding of ATP to heavy meromyosin was suggested in the presence of MgCl 2. The minimum ATP concentration giving the maximum Δ; A max was 2-fold the protein concentration. In the presence of CaCl 2, the binding was not as strong. 3. 3. Decay of the difference spectrum was measured. In the presence of MgCl 2, k s calculated using Chance's equation agreed well with the maximum velocity of the steady-state ATPase. In the presence of CaCl 2, k s was 3 times larger than the maximum velocity. 4. 4. It is inferred that the difference spectrum accompanies only the Michaelis-Menten complex formed at one of the two sites in heavy meromyosin.