Myosin Gel Research Articles

Effect of Actomyosin on Heat-induced Gelation of Myosin

Effect of Actomyosin on Heat-induced Gelation of Myosin

Effect of actomyosin on heat-induced gelation of myosin.

Myosin and reconstituted actomyosin free of regulatory proteins were mixed in 0.6M KCl and 20 HIM phosphate buffer at pH 6.0 and were tested quantitatively for thermally induced gelation properties by measuring the rigidity of the system at 65°C. Full enhancement of gelation was attained when the weight ratio of myosin-to-actomyosin was about 4. The addition of regulatory proteins to actomyosin could restore calcium sensitivity of the contractile system, but did not affect the heat-induced gelation of myosin in the presence of actomyosin, suggesting that regulatory proteins play no role in the heat-induced gelation of the system. Neither the single and double headed subfragments, both capable of interacting with F-actin, nor the helical tail subfragments, devoid of the intracting site with F-actin, exhibited changes in thermogelling properties when mixed with F-actin. However, upon addition to F-actomyosin, the tail subfragments revealed a significant effect on the gelation of actomyosin, whereas the headed subfragments exerted no influence over gelation of the system. These results indicate that the enhancing effect of F-actin on the heat-induced gelation of myosin was brought about solely by the limited amount of F-actomyosin formed in the system, which acts as a cross-linker between the tail portion of bound and free myosin molecules.

Heat induced gelling properties of actomyosin: Effect of tropomyosin and troponin.

The effects of tropomyosin and troponin on the heat-induced gelation of myosin were investigated by SDS-polyacrylamide gel electrophoresis, scanning electron microscopy and gel rigidity assay, in comparisons with natural and desensitized actomyosin. SDS-polyacrylamide gel electrophoretograms revealed that tropomyosin was almost completely removed from each desensitized actomyosin samples while it was retained in natural actomyosin samples. In spite of this, no significant differences were found in rigidity between natural and desensitized actomyosin gels. No differences could be observed in the microstructure of either actomyosin gel. It may, therefore, be concluded that tropomyosin does not affect the gel texture of the actomyosin system.

Relative Roles of the Head and Tail Portions of the Molecule in Heat‐Induced Gelation of Myosin

ABSTRACTMyosin molecules are cleaved by chymotrypsin digestion into two fragments: subfragment 1, which originates from the globular heads of myosin, and the myosin rod, which originates from the helical tail of the myosin molecule. The heat‐induced gelation of these subfragments was compared to that of intact myosin by measuring rigidity, turbidity, and other physico‐chemical characteristics of each system. Two features of the heat‐induced gelation of myosin, aggregation and three‐dimensional network formation were found to be imparted by the subfragment 1 and the rod, respectively. The former involves disulfide exchange and the latter relates to conformational changes arising from a partially irreversible helix‐coil transition during heating. Possible relationships are suggested between these physicochemical changes of the myosin head and tail regions upon heating and the heat‐induced gelation of myosin.

HEAT-INDUCED GELATION OF MYOSIN IN THE PRESENCE OF ACTIN

ABSTRACT The rabbit muscle contractile proteins, myosin, actin and reconstituted actomyosin were mixed in 0.1–1.0 M KCl, 20 mM buffers, pH 5.0–8.0, and were tested quantitatively for thermally induced gelation properties by measuring the rigidity (shear modulus) of the system at 20–70°. Scanning electronmicroscopy (SEM) was also used to study the structure of the gels formed by gelation of myosin in the presence of F-actin. Under the standard condition, i.e. at 0.6 M KCl, pH 6.0 and 65°, decrease of the myosin/actin mole ratio to about 1.5–2.0 in the reconstituted acto-myosin system resulted in substantial augmentation of the rigidity of the gel formed. Further decreases in the myosin ratio relative to F-actin reduced the rigidity value of the gel to close to the level of myosin alone. Gel-formability of the reconstituted actomyosin was maximal at pH 5.5–6.0 and between 0.5 and 0.8 M KCl and decreased considerably at other pH values and KCl concentrations. The SEM studies revealed progressive changes in three dimensional ordering as actin concentration in the actomyosin varied. These were in concordance with the results of gel strength.

Effect of blocking the myosin-actin interaction in heat-induced gelation of myosin in the presence of actin.

Scanning electron microscopy and rigidity characterization were used in this study of myosin gelation in the presence of actin. The heat-induced gel formation of myosin was aided by the addition of actin to myosin at a molar ratio of 1: 2.7. However, this actin effect was not observed when the actin-binding site(s) of myosin was blocked by p-chloromercuri-benzoate treatment or denaturation, or when the myosin-binding site(s) of actin was blocked by trinitrophenylation.The effect also disappeared with an aging myosin-actin mixture, with ATP or pyrophosphate as dissociating agent prior to thermal treatment of the system.The present findings indicate the indispensability of myosin binding to actin for enhancing the thermal gelation of myosin. The gelation process appears to involve a large electrostatic contribution, as well as such chemical reactions as oxidation of SH groups.

IONIC, pH, AND TEMPERATURE EFFECTS ON THE BINDING ABILITY OF MYOSIN

ABSTRACTThe peak force required to separate pieces of meat at a binding junction was measured with the Instron Universal Testing Machine. These measurements were used to study the effects of salt, phosphate, pH, cooking temperature and. protein concentration on the ability of crude myosin to bind meat pieces. Results showed salt and phosphate to linearly increase the ability of myosin to bind meat pieces primarily by solubilizing the protein. Temperature and the level of protein had increasing effects on binding ability in the range 45‐80°C and 0‐8%, respectively. Scanning electron micrographs of the ultrastructure of gels formed by myosin showed a three dimensional network of overlapping fibers in the presence of salt and phosphate and a sponge‐like network in their absence. Since the ultrastructure of the gel formed by myosin heavy chains was comparable to that of myosin, it was concluded that the heavy chain core of the myosin molecule plays an important role in the heat‐induced gelation of myosin. The relationship between the formation of a three dimensional network of fibers by the heavy chains of myosin and the mechanism of binding meat pieces was discussed.

HEAT‐INDUCED GELATION OF MYOSIN: FACTORS OF pH AND SALT CONCENTRATIONS

ABSTRACTThe heat‐induced gelation of myosin was optimally developed at temperatures between 60° and 70°C and at pH 6.0 as studied quantitatively by measuring shear modulus of myosin in 0.6M KCl. No difference was observed in the gelation profile, when KCl was replaced by NaCl. The shear modulus of the gel formed at 65°C and pH 6.0 increased proportionally to the 1.8 power of myosin concentration. Although the heat gelling ability of myosin as measured by the shear modulus did not increase with increasing salt concentration from 0.4–1.0M, irrespective of storage time at 0°C of the stock myosin solution (0.6M KCl) or suspension (0.1M KCl), it varied drastically with time of storage at salt concentrations between 0.1 and 0.3. This variation in shear modulus at O.1–0.3M KCl was apparent due to the storage conditions. These changes in the salt concentration dependence of the heat gelation of myosin corresponded well with the changes in solubility of myosin during storage. Studies on selected physico‐chemical properties of the original stock myosin during storage indicated gradual increase in viscosity with little inactivation of ATPase as well as very slight decrease in sulfhydryl content, suggesting the spontaneous transformation (aggregation) of myosin molecules to a less soluble state.

MlCROSTRUCTURE OF ISOLATED SOY PROTEIN IN COMBINATION HAM

ABSTRACTCombination hams were prepared by injecting excised porcine muscles with a brine containing solubilized isolated soy protein. The microstructure of the muscle in the combination ham was compared to that of a water added ham to determine the location of the isolated soy protein. The effect of massaging on the location of the isolated soy protein was also studied. The results showed that the isolated soy protein occupied primarily perimysial spaces and the massaging acts to incorporate these proteins into the endomysial spaces and mix them with extracted myofibrillar proteins. The isolated soy protein appeared to enhance myofibrillar protein extraction by binding water, thus increasing the effective concentration of salt and phosphate. Scanning electron microscopy showed that the gel formed by the isolated soy protein possessed a denser structure than the gel formed by a crude myosin preparation. The gel formed by a mixture of myosin and isolated soy protein possessed a structure more comparable to the structure of the crude myosin gel.

CHANGES IN SHEAR MODULUS, ULTRASTRUCTURE AND SPIN‐SPIN RELAXATION TIMES OF WATER ASSOCIATED WITH HEAT‐INDUCED GELATION OF MYOSIN

ABSTRACTGelation of myosin in 0.6M KC1 at pH 7.0 and 6.0 during heating at various temperatures between 25–70°C was quantitatively measured by a simple shear modulus tester devised in our laboratory. Shear modulus data indicated that gelation occurs with a stepwise elevation of temperature from 30–60°C. Heat‐treatment of the protein solution remarkably influenced the structure and water mobility in the system. Myosin gel formed by heating had a micro‐network structure, which was not‐present in the original myosin solution. The myosin gel had a slower value of spin‐spin relaxation time compared to that of the original untreated sample, suggesting that the water mobility in the gel system was somehow restricted. The heat‐induced gelation of myosin may be the result of the development of a three dimensional network structure which holds water in a less mobilized state.

Tensile strength of heat-coagulated myosin fibres

Myosin fibres, formed by heat-coagulation under conditions of pH and ionic strength similar to those prevailing in post-rigor meat, exhibited tensile strengths which decreased with fibre diameter. Tensile strengths of the smaller diameter fibres (10–100 μm) ranged from 30 to 3 kg/cm 2. These values resemble breaking strengths reported for cooked meat (e.g. Davey & Gilbert, 1977) but are an order of magnitude higher than strengths reported for heat-set myosin gels (Nakayama & Sato, 1971) and myosin-bonded meat pieces (Macfarlane et al., 1977).

Electrophoretic analysis of multiple forms of rat cardiac myosin: Effects of hypophysectomy and thyroxine replacement

Electrophoretic analysis in pyrophosphate gels of intact myosin of adult rat myocardium revealed the presence of five distinct components, two in atrial myosin (A 1, A 2) and three in ventricular myosin (V 1, V 2, V 3). Analysis of Ca 2+-activated myosin ATPase activity in the gels revealed that A 1, A 2 and V 1 had about the same specific activity; V 3 had the lowest activity, while that of V 2 was intermediate. At 3 weeks of age, ventricular myosin was exclusively V 1, there being a slow age-dependent shift in myosin distribution toward the adult pattern. Hypophysectomy of juvenile rats caused a shift towards V 3, which by 45 days after operation accounted for 90% of total myosin. This shift was prevented by daily administration of 5 μg of thyroxine. When chronically hypophysectomized rats were similarly treated, there was a rapid shift of myosin components towards V 1. These changes in distribution of myosin components were associated with appropriate changes in Ca 2+-activated ATPase activity of electrophoretically purified ventricular myosin as measured in the gel. Sodium dodecyl sulphate gel analysis of purified myosin showed little or no contaminants. For both V 1 and V 3 rich myosins, the ratio of the two ventricular light chains (V-LC 1, V-LC 2) and the ratio of light chains to heavy chains are consistent with the model that each intact molecule contains two each of V-LC 1 and V-LC 2. Sodium dodecyl sulphate electrophoresis of purified atrial myosin revealed two types of light chains: A-LC 1 (mol. wt 27 000, not resolved from V-LC 1) and A-LC 2 (mol. wt 22 000), the latter being distinct from V-LC 2.

Heat Gelling Properties of Myosin, Actin, Actomyosin and Myosin‐subunits in a Saline Model System

SUMMARY—Myosin and actin were isolated from rabbit skeletal muscle. The gelation of myosin, actin and actomyosin, as well as of heavy and light meromyosins derived from myosin by trypsin treatment, by heat was studied in various systems. The data indicate that the heat gelling properties of these protein solutions do not run parallel with those of saline model systems composed of these proteins and stroma. Actin does not exert any influence on the binding properties of the system, but when F‐actin and myosin A were both present the resulting binding properties were considerably improved. Since heavy and light meromyosins have little influence on binding properties, it may be concluded that an intact molecule of myosin is required for development of binding properties upon heating.

Effects of cations, polyanions, and sulfhydryl reagents on muscle proteins

Myosin gel, in dilute salt solution, can hydrate or dehydrate at pH 7.4; Ca ++ and polyanions are required for dehydration, and Mg ++ and polyanions are required for hydration. Addition of sulfhydryl reagents which add negative charges to three of the 42 sulfhydryl groups in myosin mimics the effect of ATP on hydration of myosin but not on actomysin, although ATPase of both is inhibited by the sulfhydryl reagents. Sulfhydryl reagents which do not add charge to the protein cause no change in hydration but do block the effect of ATP and PCMS on hydration. The binding of actin to myosin results in (a) greater specificity of the hydration reaction for ATP, rather than other polyphosphoric nucleotides; (b) greater insolubility (super precipitation) at comparable concentration of Ca ++ and Mg ++; (c) inability to hydrate on addition of sulfhydryl reagents. Conditions which cause dehydration of myosin gels increase ATPase activity, whereas hydration of the protein decreases ATPase activity. A role for the hydration and dehydration of myosin in muscle contraction is hypothesized.

Role of anions in mitochondrial swelling and contraction

Summary 1. The effects of large anions on mitochondria, as compared with chloride, are: (a) Increase in rates of swelling, (b) Increase in rates of oxygen uptake, (c) Inhibition of metabolic contraction, (d) Acceleration of the loss of inorganic phosphate from mitochondria. 2. Addition of agents, which cause contraction and restoration of intramito-chondrial concentration of ATP (EDTA or ATP) in chloride buffers, causes further swelling and loss of intramitochondrial ATP (EDTA only) in buffers, containing large anions. 3. Large anions do not alter the semipermeable properties of mitochondrial membranes, i.e ., osmotic contraction of sodium acetate loaded mitochondria by addition of sucrose can be obtained. 4. Swelling by large anions is independent of respiration. 5. Addition of sulfhydryl reagents which add negative charge to mitochondria ( p -chloromercuriphenylsulfonate) markedly increases swelling under all conditions. However, inactivation of mitochondrial sulfhydryl groups by sulfhydryl reagents which do not add negative charge also does not cause swelling. Both reagents completely inhibit respiration in mitochondria. 6. The above data are interpreted to indicate that mitochondrial shrinking and swelling is very similar to gel—sol transformations of myosin gels, which phenomena is accomplished by alterations in surface charge of the gels. These surface charges are specifically altered by the binding of such substances as Ca 2+ , Mg 2+ , ATP, and p -chloromercuriphenylsulfonate to the gels.

致死条件が魚肉蛋白におよぼす影響-II

Effect of freezing and cold storage upon the extractability and salting-out behavior of muscle protein was examined, using carp killed in two different ways; one was instantly put to death by beheading and the other was beheaded after allowed to struggle in air for several hours. Fillets taken from these two groups of carp were frozen at -15°C and stored at this temperature for a period varying from 2 to 14 days. Before freezing and at each different time of cold storage, myosins were extracted from these fillets with 0.6M KCI (pH 6.6), precipitated by dilution. and the amount was measured. With isolated myosins salting-out behavior toward ammonium sulfate was determined at pH 6.9 and at the temperature of 0°C. Extractability Myosins were always extracted greater in amount from death-after-struggle group than from instant death group throughout the period of the present experiment. No significant difference, however, in the ratio of amount of myosins (M) to salt extractable protein (L), was noticed between the two groups. After frozen storage of 10 days, a slight diminution in the values of L and M was observed in the muscle of instantly killed fish, but it was not appreciable for death-after-struggle group within the limited duration of the present test. Meanwhile, the ratio M : L seemed to decrease somewhat in both groups on prolonged storage (Table 1). Salting-out Curves Around the range of 30-40% (v/v) saturation of ammonium sulfate in the salting-out curves for either group, there was found a dominant peak, which presumably pepresented myosin and actomyosin. With fresh fillet before freezing, this peak was more prominent for the instant death group than for the other. However, by freezing and subsequent frozen storage, the pattern of the curves for instant death group turned out close to those of unfrozen fillet of death-after-struggle group, and inversely the pattern of the latter group became kin to those of the former group that was not frozen. This will be obviously perceived from the mode of slope and fall in the s-c curves in the range of 30-40% saturation of ammonium sulfate (Figs. 1 and 2). In another series of experiments similarly conducted for isolated myosin gel, the results given were not quite the same as obtained for the fillets. The salting-out curve of the gel from fish killed after struggle became analogous to that of fresh, unfrozen gel from instant death group, just as seen in the case of the fillets. But the curves of frozen myosin gel from instant death group was not altered by freezing process and cold storage (compare Figs. 1. 2 and Figs. 3 and 4). It is notable in Figs. 1-4 that there was found concentration range of ammonium sulfate in which components salted-out decreased in amount with increasing concentration as the result of freezing and subsequent storage. This means that a part of components which were saltedout at concentration c1 remained in solution in the range of higher concentrations between c1 and c2. This effect will be valid for either fillet or isolated myosins gel. Actin fraction in the salting-out pattern In the previous paper ??, it was shown with fresh fillets that actin fraction appeared in the salting-out pattern of myosin fraction from carp killed after struggle, but the corresponding fraction was absent in the case of carp put to an instant death. In addition, it was mentioned that this actin fraction would not appear, if the extraction of myosins from carp killed after struggle was carried out in the presence of ATP. In the present work, it was found that when the fillets were frozen and kept for 2 days or 6 days, a prominent peak presumed as actin fraction, appeared also in the case of carp put to an instant death.