Stabilization of myosin by ionic compounds as affected by F-actin

ABSTRACT:   Suppressive effects of non-ionic (sorbitol, maltose, and trehalose) and ionic (Na-glutamate, Na-acetate, Na-sulfate, and ammonium sulfate) compounds on the thermal inactivation of myosin subframgent-1 (S-1) and myofibril Ca²⁺-ATPase were compared. All compounds suppressed S-1 denaturation. When myofibrils were used (at 0.1 M KCl), sugars and sugar alcohol (non-ionic compounds) suppressed denaturation similar to S-1, while Na-glutamate, Na-acetate, and Na-sulfate weakly suppressed them. Ammonium sulfate accelerated denaturation, but suppressed denaturation when heated in 2 M KCl, at which myosin lost protection by F-actin. It was thus concluded that ionic compounds affected the denaturation of myofibrils in two ways; suppression as established with S-1, and acceleration as a result of loss of protection by F-actin caused by increase in ionic strength.     

Suppressing effect of neocarrabiose 4-O-sulfate on thermal and freeze denaturation of myosin subfragment-1 and myofibrils

Suppressive effects of neocarrabiose 4-O-sulfate on the thermal and freeze denaturation of myosin subfragment-1 (S-1) and on myofibrils were investigated. The compound strongly suppressed the thermal denaturation of S-1. Its suppressive effect was greater than that of sorbitol and similar to that of maltose. However, it tended to accelerate the denaturation of myofibrils, suggesting a loss of protection by F-actin upon its addition. The compound suppressed the freeze denaturation of myofibrils and S-1. The effect was similar to that of sorbitol or maltose, and completely different from that of Na-sulfate. The compound solubilized myofibrils at concentrations similar to KCl. Therefore, it was concluded that neocarrabiose 4-O-sulfate behaved as an ionic salt in the thermal treatment process, whereas it behaved as a sugar in the frozen storage process.     

Thermal denaturation profiles of catfish and tilapia myofibrils as affected by pH for heating

Thermal denaturation profiles of catfish myosin when heated as myofibrils (Mf) were compared with those of tilapia. The Ca²⁺-ATPase inactivation rate of catfish myofibrils was the same as that of tilapia myofibrils. The conclusion was the same with isolated myosin. Catfish Mf was clearly distinguished from tilapia Mf in terms of subfragment-1 (S-1) and rod denaturation. Quick denaturation of rod relative to S-1 was characteristic of catfish Mf, while slower denaturation of rod relative to S-1 was the pattern for tilapia Mf. These patterns were greatly affected by the pH for heating. Rod denaturation was accelerated with increasing pH for heating and oppositely suppressed by lowering the pH, for both Mf. Tilapia Mf showed a S-1 and rod denaturation pattern similar to that for catfish Mf, but at 1 pH unit higher; for example, the pattern of catfish Mf at pH 7.5 was similar to that for tilapia Mf at pH 8.5. Less rigid filament structure of catfish Mf than tilapia Mf was demonstrated by studying chymotryptic digestion at various pH values. Accordingly, the difference in the S-1/rod denaturation patterns between the two fish species can be explained by the different rigidity of their myosin filaments.     

Effect of calcium ion on the thermal denaturation of subfragment-1 and rod regions of squid myosin upon the heating of myofibrils

Thermal inactivation of Ca²⁺ ATPase of squid myofibrils was significantly suppressed in the presence of Ca²⁺. Monomeric myosin content decreased much faster than Ca²⁺ ATPase inactivation in Ca medium, which was well explained by fast rod denaturation. In contrast, rod denaturation was slower than S-1 in EDTA medium. The decrease in monomeric myosin content was explained by faster S-1 denaturation. Comparing the S-1 and rod denaturation rates at a fixed temperature, it was concluded that S-1 denaturation was suppressed by Ca²⁺ whereas the rod denaturation was not. An unfolding experiment with isolated myosin rod confirmed that there was no stabilizing effect of Ca²⁺ on myosin rod. It was concluded that significant stabilization of the S-1 portion by Ca²⁺ generated the apparently different myosin denaturation patterns in the two media.     

Species-specific thermal denaturation pattern of fish myosin when heated as myofibrils as studied by myosin subfragment-1 and rod denaturation rates

Thermal denaturation of myofibrils from various species of fish was investigated by measuring ATPase inactivation, myosin aggregation, myosin subfragment-1 (S-1) and rod denaturation rates as studied by chymotryptic digestion. Decrease in monomeric myosin (myosin aggregation) was always faster than the ATPase inactivation for all myofibrils tested. The relative denaturation rate of rod to that of S-1 differed from species to species. Preceded denaturation of rod was observed with some species, and the opposite was true with other species. The denaturation pattern was explained by the different magnitude of S-1 stabilization by F-actin in myofibrils at low salt medium. Myofibrils which receive a great stabilization by F-actin as studied by ATPase inactivation showed the preceded rod denaturation pattern, and vice versa. S-1 portion, not F-actin, determined the different stabilization of S-1 by F-actin in myofibrils.     

Comparative study on thermal denaturation modes of myosin in walleye pollack and carp myofibrils as affected by salt concentration

ABSTRACT:   The effect of salt concentration on the thermal denaturation profile of myosin in walleye pollack and carp myofibrils was compared by studying the subfragment-1 (S-1) and rod denaturation rates upon heating. Species-specific denaturation mode observed at 0.1 M KCl was no longer detected when samples were heated above 0.5 M KCl, where S-1 and rod denaturation rates were identical to each other. As the heating of the chymotryptic digest of myofibril formed practically no rod aggregates, S-1 denaturation in a form of myosin was the rate limiting step for rod aggregate formation. As the aggregate formation by rod was remarkably suppressed by lowering the temperature, the free movement of myosin tail upon heating was suggested to play an important role in the rod aggregate formation in a high salt medium.     

Denaturation mode of carp myofibrils when affected by temperature and salt concentration

ABSTRACT: Heating temperatures of 30–40°C and KCl concentrations of 0.1–0.5 M altered the denaturation mode of carp myofibrils. In 0.1 M KCl medium, heating temperature affected the denaturation of rod more significantly than of subfragment-1 (S-1), and a slow decrease in solubility at 30°C was accompanied by a slow denaturation of rod. KCl concentration at heating altered the denaturation mode differently at 30°C and 40°C. Increased KCl concentrations for heating reduced the rod denaturation rate at 40°C, but it was increased at 30°C. At concentrations above 0.3*Τ*M KCl, the denaturation rate for rod became identical to that for S-1 at both temperatures. Upon heating of chymotryptic digest of myofibrils, S-1 denaturation was similarly detected as in intact myofibrils, whereas practically no rod denaturation was detected. Thus, it was concluded that myosin structure connecting S-1 and rod has an important role in the denaturation process.     

Myosin and actin denaturation in frozen stored kuruma prawn Marsupenaeus japonicus myofibrils

Myosin and actin denaturation in kuruma prawn myofibrils stored frozen (0.1 M NaCl, pH 7.5) at ?20 °C was investigated. The inactivation profile of Ca²⁺-ATPase in the myofibrils was identical to that for myosin, indicating that myosin in myofibrils was not protected by actin. The presence of myosin detached from actin in the soluble fraction was proven by ammonium sulfate fractionation in the absence and presence of Mg-ATP. Actin denaturation in myofibrils was further confirmed by its increased susceptibility to chymotryptic degradation. In the frozen myofibrils, actin denatured more rapidly quicker than myosin: actin had completely denatured by storage day 1, followed by a gradual denaturation of myosin. Both myosin and actin in the frozen stored myofibrils retained their high salt-solubility, which decreased slowly during the frozen storage period. The presence of aggregated inactivated myosin in the salt-soluble fraction was proven by precipitation at 40 % saturation of ammonium sulfate in the presence of Mg-ATP, leaving active monomeric myosin in the soluble fraction. Almost no actin denaturation was observed with heated myofibrils.     

Effect of protein hydrolysate from Antarctic krill meat on the state of water and denaturation by dehydration of lizard fish myofibrils

ABSTRACT: To utilize Antarctic krill as functional food, protein hydrolysates were prepared by enzymatic hydrolysis. Their effects on the state of water in myofibrils of lizard fish and dehydration-induced denaturation were compared with those from two species of shrimp, glucose, and sodium glutamate. Peptides are major components in hydrolysates, occupying approximately 85–93% of the total materials. The Antarctic krill protein hydrolysates stabilized the bonding of water molecules, leading to suppressed denaturation of myofibrils during the dehydration process. Similar effects were observed for shrimp protein hydrolysates. The effect of the hydrolysate was less than that by glucose and sodium glutamate.     

Effect of Cryoprotectants on Suppression of Protein Structure Deterioration Induced by Freeze-thaw Cycle in Pacific White Shrimp

This study aimed to elucidate the changes in Pacific white shrimp (Litopenaeus vannamei) myofibrillar protein as influenced by multiple freeze-thaw cycles as well as the stabilization effects of sucrose and trisodium citrate on shrimp myofibrils. Shrimp myofibrils in 0.1 M NaCl, 20 mM Tris-HCl (pH 7.5) were mixed individually with sucrose and citrate at concentrations of 0.05 M and were evaluated for Ca²⁺-ATPase activity, salt solubility, total and reactive sulfhydryl, and surface hydrophobicity during three freeze-thaw cycles. Sucrose and citrate had strong cryoprotective effects against freeze denaturation by retaining higher Ca²⁺-ATPase activity and salt-soluble myosin and actin, by slowing the reduction of reactive sulfhydryl (SH) and by exposing less hydrophobic groups at the surface of the protein compared with the no-additive sample. Results indicated that both cryoprotectants had suppressive effect against protein denaturation and helped stabilize white shrimp myofibrillar protein during the freeze-thaw process. This study suggests that sucrose and citrate stabilized the protein structure by retarding the unfolding of protein; thus, the native protein could be protected during frozen storage.     

冻结速率和冻藏温度对鲢肉蛋白质冷冻变性的影响

研究了冻结和冻藏温度对鲢肉肌原纤维Ca-ATPase活性和盐溶性蛋白溶解度的影响并作了冷冻切片观察，结果发现，冻结速率对具有一定细胞形态的鲢肌蛋白质的冷冻变性有一定的影响，对无完整细胞形态的碎鱼肉和鱼糜基本无影响，而冻藏温度对鱼肌、碎鱼肉和鱼糜蛋白质冷冻变性都有显著的影响，即温度越低，变性越小，而抗冻剂可有效防止蛋白质的冷冻变性，尤其是使鱼糜肌原纤维蛋白质的稳定性大大提高。     

Myosin denaturation in heated myofibrils of scallop adductor muscle

The thermal inactivation of Ca²⁺ ATPase of scallop myofibrils (0.1 M KCl, pH 7.5) was found to be unaffected by the presence of Ca²⁺. Monomeric myosin content and salt solubility decreased much faster than Ca²⁺ ATPase inactivation in both Ca and EDTA media, which was well explained by faster denaturation of the rod portion than subfragment-1 of myosin. In contrast, when the myofibrils were heated at 0.5 M KCl, a slow decrease in salt solubility was observed, which was also explained by slow denaturation of the rod portion of myosin. Myofibrils from scallop smooth muscle showed the same denaturation pattern as those from adductor muscle. These results show that mollusk myosin is not always stabilized by Ca²⁺.     

Effect of shrimp head protein hydrolysates on the state of water and denaturation of fish myofibrils during dehydration

ABSTRACT:   To utilize fisheries waste products as food materials with functional properties, shrimp head protein hydrolysates (SHPH) from three species of shrimp, that is, Northern pink shrimp ( Pandalus eous ), Endeavour shrimp ( Metapenaeus endeavouri ) and Black tiger shrimp ( Penaeus monodon ), were produced by enzymatic hydrolysis using endopeptidase derived from Bacillus subtilis and exopeptidase derived from Aspergillus oryzae at a level of 0.1% (w/w). SHPH were rich in protein (90–91%) and amino acids (71–84%) but little fat (0.01–0.02%). The average molecular weight of SHPH was 300–1400. The effect of 5% SHPH (dry basis) addition on the state of water and denaturation of lizard fish myofibrils (Mf) during the dehydration process was evaluated by the desorption isotherm and the Ca-ATPase activity, and compared with the effect of sodium glutamate (Na-Glu). SHPH decreased the water activity and the Ca-ATPase inactivation, and increased monolayer sorbed water and multilayer sorbed water of Mf, although these effects of SHPH were smaller than those of Na-Glu. These findings suggest that the SHPH suppressed dehydration-induced denaturation of myofibrillar protein by stabilizing the hydrated water surrounding myofibrils.     

Biochemical Properties of Actomyosin and Expressible Juice of Cold Stored Striated Adductor Muscles of Aulacomya ater ater (Molina):

The mechanism that lead to actomyosin denaturation in striated adductor muscles of Aulacomya ater ater (Molina) during cold storage was investigated. By intrinsic viscosity and surface hydrophobicity determinations evidences for a conformational change of the protein were obtained. The effect of muscle immersions in dip solutions: 3% NaCl, 3% Na glutamate and 10% Na-polyphosphates on expressible juice and biochemical properties of actomyosin of muscles stored at 2-4¦C were investigated. The 3% NaCl and 3% Na-glutamate treatments were not effective inhibitors of the increase of expressible juice or the actomyosin denaturation. Polyphosphates reduced the amount of expressible juice of the muscles and delayed the decrease in Mg2+ ATPase activity of actomyosin. Treatment with polyphosphates did not affect the viscosity of actomyosin.     

Permeation of carboxylates as ionic compounds into fish meats by soaking and its osmotic dehydrating effect

Permeability of Na-gluconate, Na-malate and Na-acetate into fish-meat strips by soaking, and their osmotic dehydrating effects were investigated in comparison with those of sodium chloride (NaCl) and sorbitol. Carboxylic acids, anion species of carboxylates, were less permeable into soaked meats than chlorine of NaCl, and their permeability was similar to that of sorbitol. However, the permeation of sodium, cation species of the carboxylates and NaCl, was promoted with the concentration of free sodium ion dissociated in soaking solutions. The ratio of sodium to the anion species in soaked meats was varied, depending on the numbers of the sodium atom in the molecules as well as the dissociation degree of the ionic compounds in the soaking solution. Furthermore, the osmotic dehydrating effects of Na-gluconate and Na-malate were analogous with that of sorbitol, and higher than those of NaCl and Na-acetate. These results indicated that the permeation characteristics and the osmotic dehydrating effects of the carboxylates were associated with their ionization in soaking solution as well as their molecular weights.     

Rheological properties and structural changes in steamed and boiled abalone meat

ABSTRACT: Changes in tissue structures, rheological properties, and water content of abalone meat were studied in relation to boiling and steaming time. The adductor muscle of abalone Haliotis discus, which was removed directly from the shell, was boiled or steamed for 1 h, 2 h, and 3 h. When observed under a light microscope and by scanning electron microscopy, structural changes in the myofibrils were greatest in the boiled abalone meat compared with the steamed meat. When heating time was increased from 1 h to 3 h, the instantaneous modulus E ₀ of boiled abalone meat decreased gradually with increased heating time, whereas the E ₀ of steamed abalone meat was reduced when heated for 2 h. When heated for 1 h, the relaxation time of steamed abalone meat was much longer than that of boiled meat. There were no significant changes in the relaxation time of abalone meat among the different boiling times, but the relaxation time of steamed abalone meat was reduced gradually with increasing heating times. The study's results confirmed that the difference in rheological properties between the boiled and steamed meats was due mainly to the denaturation level of myofibrils when heated for 1 h, as well as due to the changes in water and solid content and the manner in which the inner water was exchanged after heating time was increased from 1 h to 3 h.     

Myosin denaturation in “Burnt” Bluefin tuna meat

A quantitative conversion of tuna meat into muscle homogenate made it possible to study myosin denaturation in Bluefin tuna meat. Myosin denaturation was accessed by measuring Ca²⁺-ATPase activity, salt-solubility with and without Mg-ATP, monomeric myosin content, and amount of subfragment-1 (S-1) and rod produced by chymotryptic digestion. Commercially available tuna used in this study showed a pH around 5.4–5.7. Myosin in the meat lost the salt-solubility measured in the absence of Mg-ATP; however, such myosin showed full salt-solubility when released from actin in the presence of Mg-ATP. Incubation of tuna meat at 30 °C for up to 90 min caused obvious myosin denaturation. However, the tuna meat dialyzed against neutral pH buffer showed practically no myosin denaturation by the same heating. It was suggested that exposure to lowered pH to around 5.5 and increased temperature of 30 °C led myosin denaturation. Myosin denaturation in the “Burnt” Bluefin tuna sample was analyzed. A significant myosin denaturation was observed with the part showing the “Burning” symptom, the inner part of the tuna meat near the spine. Myosin in that part showed almost no Ca²⁺-ATPase activity, no salt-solubility even with Mg-ATP, no recovery of monomeric myosin, and almost no production of S-1 by chymotryptic digestion. However, myosin denaturation was not detectable for the meat taken from outer parts of the same tuna near the skin with normal appearance. It was demonstrated that “Burning” of tuna meat occurring in the deep part of the body is accompanied by myosin denaturation. The above results suggested that insufficient cooling of the deep part of body would be the reason for “Burning” of tuna meat.     

Stabilizing effect of Ca<Superscript>2+</Superscript> on myosin and myofibrils of squid mantle muscle as affected by heating conditions

The suppressive effects of Ca²⁺ on the thermal denaturation of myosin and myofibrils of squid mantle muscle were compared. The stabilization effect on myosin was smaller than that on myofibrils and was not affected by KCl concentration. The stabilizing effect of Ca²⁺ on myosin decreased as the heating temperature dropped, showing no stabilization at 20°C, while the effect on myofibrils was the same at all temperatures tested. The stabilizing effect of Ca²⁺ on myosin disappeared even at 30°C in the presence of sorbitol, where a small inactivation rate was found, while the effect of Ca²⁺ on myofibrils was equally detected irrespective of the reduction in inactivation rate in the presence of sorbitol. Stabilization of myosin by Ca²⁺ again appeared even in the presence of sorbitol when the heating temperature was raised to 38°C. It was suggested that Ca²⁺ confers stabilization on myosin only when myosin is under unstable conditions. The stabilization effect of Ca²⁺ on myosin was enhanced upon F-actin binding: Ca²⁺-bound myosin was more significantly stabilized by F-actin binding, and the effect was no longer affected by the conditions for heating.     

Preventive effects of amino acids and glutathione on the formaldehyde-induced denaturation of myofibrillar proteins

ABSTRACT:     Formaldehyde (FA)-induced denaturation of myofibrillar proteins and its prevention were investigated by means of measuring the solubility, adenosine triphosphatase (ATPase) activity, and thermal gel formability of myofibrils and surimi proteins in the presence and absence of free amino acids and glutathione, reduced form. The addition of FA decreased the solubility of myofibrils in 0.5 M NaCl at pH 7.0 and 0°C depending on its concentration and incubation time. The solubility decrease was completely inhibited by the presence of equal, twofold, and threefold amounts of cysteine (Cys), glutathione, and histidine (His) to the amount of FA, respectively. Myofibrillar Ca-ATPase was markedly activated at the initial phase and then decreased later by the addition of FA. The K-ATPase was inactivated with an increase in the amount of FA. The FA-induced changes in both ATPase activities were inhibited in the presence of Cys and His. Thermal gel formability of surimi paste increased only in a short period after the addition of a low concentration of FA. Practically, FA inhibited the thermal gelation and setting effect through the inactivation of transglutaminase. In the presence of Cys, His or glutathione, a strong elastic surimi gel was produced because FA-induced detrimental effects were inhibited.     

Effects of urea and trimethylamine-N-oxide on ATPase of requiem shark myofibril and its constituents

ABSTRACT: The effects of trimethylamine- N -oxide (TMAO) on the urea-resistibility of requiem shark myofibrils were investigated, using Ca²⁺- and Mg²⁺-ATPase activities as a parameter. Both activities were hardly changed or activated up to 0.6 M urea. In contrast, the two activities both decreased to less than 50% in the presence of TMAO up to 0.5 M. When measured at a 2 : 1 molar ratio of urea and TMAO, Ca²⁺- and Mg²⁺-ATPase activities were similar to those in the presence of TMAO alone, indicating that TMAO reduced the urea-resistibility of myofibrils. Myosin, the most abundant protein in myofibrils, from requiem shark exhibited the effects of urea and TMAO on its Ca²⁺-ATPase activity, which was primarily similar to those of myofibrils. However, Ca²⁺-ATPase activities in the coexistence of urea and TMAO for actomyosin reconstituted from requiem shark myosin and chicken F-actin were approximately average of those measured independently in the presence of either urea or TMAO alone. Carp myofibrils, reconstituted actomyosin and myosin, which were used as teleost references, all showed a tendency in the effects of urea and TMAO on Ca²⁺-ATPase activities that was similar to those of requiem shark counterparts.