Early structural changes in myosin rod upon heating of carp myofibrils

Upon heating carp myofibrils at 40 degrees C, the amount of myosin that is soluble and monomeric dropped very quickly, roughly 5 times faster than the ATPase inactivation. This rapid decrease of solubility was well explained by a rapid denaturation of the rod portion as measured by chymotryptic digestibility. Chymotryptic digestion of heated myofibrils in a low-salt medium with EDTA generated a reduced amount of rod and subfragment-1 (S-1). The decrease of S-1 produced from the heated myofibrils was consistent with the ATPase inactivation. The decrease of rod produced from the heated myofibrils was explained by the increased susceptibility of the heavy meromyosin (HMM)/light meromyosin (LMM) junction to chymotryptic. It was, therefore, concluded that the fastest event occurring in the myosin molecule upon heating of myofibrils is the irreversible exposure of the HMM/LMM junction.     

Oxidation of porcine Myosin by hypervalent myoglobin: the role of thiol groups

Oxidation of the myofibrillar muscle protein myosin from pork by hypervalent myoglobin species (MbFe(III)/H 2O2 radical generating system) was investigated in aqueous solution in the pH range of 5.0-7.8 by electron spin resonance (ESR) spectroscopy using N- tert-butyl-alpha-phenylnitrone (PBN) as spin trap and indirectly by determination of the rate of reduction of hypervalent myoglobin species by UV spectroscopy. Cross-linking of myosin was examined by SDS-PAGE. The target for oxidative modification of myosin was studied by thiol blocking by N-acetylmaleimide (NEM) and by determining oxidative modification of myosin thiols. The reaction between myosin and hypervalent myoglobin was fast and showed little dependence on pH. The myosin radicals formed were observed to be short-lived. Myosin thiols are suggested to be the main target for oxidative modification, as NEM-treated myosin did not form radicals in the presence of hypervalent myoglobin. A significant decrease in thiol content was already demonstrated 25 s after initiation of oxidation of myosin. The majority of myosin heavy chain (MHC) was demonstrated to be cross-linked through intermolecular disulfide bonding 1 h after initiation of oxidation. This demonstrates that thiols are important for radical formation and cross-linking of myosin during oxidation with hypervalent myoglobin at the pH of meat products.     

Morphological examinations of oxidatively stressed pork muscle and myofibrils upon salt marination and cooking to elucidate the water-binding potential

Pork longissimus muscle samples were subjected to the following three marination conditions: (A) oxidation (40 min) in hydroxyl radical-generating solutions (HRGS; 10 μM FeCl(3)/100 μM ascorbate with 5 or 20 mM H(2)O(2), pH 6.2) containing 0.1 M NaCl and then marination (40 min) in 0.6 M NaCl with 15 mM pyrophosphate (PP); (B) simultaneous oxidation/marination (40 min) in HRGS containing 0.6 M NaCl and 15 mM PP; or (C) the same as condition B except that PP was omitted. Protein oxidation, measured by the carbonyl and tryptophan fluorescence changes, enhanced hydration but increased cooking loss of meat. Light microscopy revealed a dense muscle structure characterized by swollen fibers and reduced intercellular spacing in intermediately oxidized muscle samples marinated with 0.6 M NaCl and 15 mM PP. However, oxidized fibers were more susceptible to transverse shrinkage upon cooking than nonoxidized fibers, which was supported by the dynamic ultrastructural changes in myofibrils observed using phase contrast microscopy. These findings provide a further understanding of the complex impact of oxidation on meat hydration and water-binding.     

Enzyme-aided modification of chicken-breast myofibril proteins: effect of laccase and transglutaminase on gelation and thermal stability

The effect of laccase and transglutaminase (TG) on cross-linking, gelation, and thermal stability of salt-soluble chicken-breast myofibril proteins was investigated at pH 6. Both enzymes modified the protein pattern detected by SDS-PAGE. Identification of proteins by peptide mass mapping showed that myosin heavy chain (MHC) and troponin T were the most affected proteins. These proteins faded or disappeared as a function of the incubation time with both enzymes on SDS-PAGE. The molecular weight of actin was not, however, affected by either enzyme. The effects that the enzymes had on the gel formation of chicken-breast myofibrils were studied in 0.35 and 0.60 M NaCl solutions at 3% protein content and a constant temperature of 40 degrees C by using a small deformation viscoelastic measurement. TG substantially increased the storage modulus (G') of 3% protein in 0.35 M NaCl. Without the enzymes, gelation was insignificant in 0.35 M NaCl. The increased solubility of the proteins at 0.60 M NaCl intensified gelation with TG. G' increased 32 and 64% at dosages of 10 and 100 nkat of TG, respectively. Also, laccase increased G' of the gel in 0.60 M salt concentration. However, a high laccase dosage decreased the magnitude of G' below the control level. Differential scanning calorimetric (DSC) measurements indicated slightly reduced myosin heat stability after TG pretreatment and increased actin heat stability with both enzymes. Maximum transition temperatures did not alter with either enzyme.     

Oxidation-induced unfolding facilitates Myosin cross-linking in myofibrillar protein by microbial transglutaminase

Myofibrillar protein from pork Longissimus muscle was oxidatively stressed for 2 and 24 h at 4 °C with mixed 10 μM FeCl(3)/100 μM ascorbic acid/1, 5, or 10 mM H(2)O(2) (which produces hydroxyl radicals) and then treated with microbial transglutaminase (MTG) (E:S = 1:20) for 2 h at 4 °C. Oxidation induced significant protein structural changes (P < 0.05) as evidenced by suppressed K-ATPase activity, elevated Ca-ATPase activity, increased carbonyl and disulfide contents, and reduced conformational stability, all in a H(2)O(2) dose-dependent manner. The structural alterations, notably with mild oxidation, led to stronger MTG catalysis. More substantial amine reductions (19.8-27.6%) at 1 mM H(2)O(2) occurred as compared to 11.6% in nonoxidized samples (P < 0.05) after MTG treatment. This coincided with more pronounced losses of myosin in oxidized samples (up to 33.2%) as compared to 21.1% in nonoxidized (P < 0.05), which was attributed to glutamine-lysine cross-linking as suggested by sodium dodecyl sulfate-polyacrylamide gel electrophoresis.     

Biochemical changes in myofibrillar protein isolates exposed to three oxidizing systems

The objective of the study was to compare three different oxidizing systems commonly present in muscle foods for their influence on the biochemical properties of muscle proteins. Myofibrillar protein isolate (MPI) prepared from pork serratus ventralis muscle was suspended (30 mg protein/mL) in 15 mM piperazine-N,N-bis(2-ethane sulfonic acid) buffer (pH 6.0). Oxidation was induced by incubating the protein suspension at 4 degrees C for 24 h with (i) a hydroxyl radical-generating system (HRGS: 10 microM FeCl3, 0.1 mM ascorbic acid, and 0.05-5.0 mM H2O2), (ii) a lipid-oxidizing system (LOS: 0.05-5.0 mM linoleic acid and 3750 units of lipoxidase/mL), or (iii) a metmyoglobin-oxidizing system (MOS: 0.05-0.5 mM metmyoglobin). Changes in oxidized MPI were measured as Ca- and K-ATPase activities, formation of protein carbonyls and 2-thiobarbituric acid-reactive substances (TBARS), loss of protein thermal stability, and protein aggregation. The three oxidizing matrixes induced complex MPI changes; for example, the Ca- and K-ATPase activities were altered mainly by low-concentration oxidants, but the changes were unique for each oxidizing system. The carbonyl content in MOS-treated MPI was the highest, while the TBARS production, changes in thermal properties, and loss of the myosin heavy chain were the greatest in HRGS-treated MPIs. Overall, the hydroxyl radical-producing medium appeared to be the most oxidative to myofibrillar proteins under the experimental conditions employed in the study.     

Effect of sodium chloride stress on the plasma membrane ATPase of barley roots: Probable cause for decrease in ATPase activity

The regulation of plasma membrane ATPase activity by salt stress was investigated in barley roots. The plasma membrane fractions were prepared from the roots treated with or without 200 mM sodium chloride (NaCl) for one day. After salt treatment, ATPase activity reduced by 20 to 30% as compared with that of control roots. No significant changes in the content of total phospholipid and sterol were detected in the plasma membrane fraction by salt stress. After extraction of most of the phospholipids in the plasma membrane vesicles with a solution containing 1% (W/V) octylglucoside and 1% (W/V) Triton X‐100, the ATPase activity in salt‐stressed roots was lower than that of control roots. After reconstitution of detergent‐extracted protein into liposome, the reduction of ATPase activity by salt stress did not recover. Based on immunoblott analysis, the relative amount of H⁺‐ATPase in plasma membrane fraction prepared, from NaCl‐stressed roots was smaller than that of control roots. These results indicate that the reduction of H⁺‐ATPase activity by salt stress was caused by the decrease in the amount of H⁺‐ATPase rather than the modification of ATPase.     

Thermal denaturation and aggregation properties of Atlantic salmon myofibrils and myosin from white and red muscles

Thermal denaturation and aggregation abilities of salmon myofibrils and myosin were studied measuring turbidity, intrinsic fluorescence, 8-anilino-1-naphthalene sulfonic acid binding, and 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide cross-linking. The thermal behaviors of protein preparation from white and red muscles were compared, and the relationship with thermal gelation properties is discussed. The low gelation ability of salmon muscle proteins was related to a limited extent of protein denaturation and aggregation upon heating. These properties seemed to be carried by myosin molecules as a similar behavior was observed for both myofibrils and myosin preparations. The higher thermal stability observed for red muscle proteins with higher transition temperatures in rheological profiles was related to a shift to higher temperature in denaturation and aggregation processes. The extent of denaturation and aggregation was very similar for both muscle types as was the final rigidity of the gels formed.     

自由基引起的氧化对牛乳清蛋白凝胶特性的影响

为了深入了解蛋白氧化对凝胶特性的影响,以此探讨乳清蛋白氧化对其功能性质的影响机制,该文主要研究了氧化对乳清蛋白凝胶质地、流变学特性和微观结构变化的影响。试验采用羟基自由基氧化体系,在不同H2O2浓度(1～20mmol/L)及不同FeCl3浓度(0.1～1mmol/L)对乳清蛋白分别氧化3h,通过质构仪、流变仪和扫描电镜对凝胶特性和微观结构进行研究。结果显示:同未氧化乳清蛋白相比,在所有氧化条件下,凝胶硬度降低了90%以上,贮藏模量(G')值降低了17%以上,复合模量(G*)值降低了20%以上;高浓度氧化条件下,弹性降低了20%以上。氧化明显改变了凝胶的微观结构,随着氧化剂的加入,导致了疏松多孔且不规则凝胶的形成。上述结果表明,氧化对蛋白凝胶质地和凝胶形成能力起着很大的破坏作用,并影响着其微观结构。     

Does oxidation affect the water functionality of myofibrillar proteins?

Water-binding properties of myofibrils extracted from porcine muscle, and added hemoglobin with and without exposure to H2O2, were characterized using low-field proton NMR T2 relaxometry. The effects of pH and ionic strength in the samples were investigated as pH was adjusted to 5.4, 6.2, and 7.0 and ionic strength was adjusted to 0.29, 0.46, and 0.71 M, respectively. The formation of dityrosine as a measure of oxidative protein cross-linking revealed a significant increase in dityrosine concentrations upon H2O2 activation. The formation of dityrosine was strongly pH-dependent and increased with decreasing pH. In addition, increased levels of thiobarbituric acid reactive substances were observed upon addition of H2O2, implying that lipid oxidation was enhanced, however, with a different oxidation pattern as compared to the myofibrillar proteins. Low-field NMR relaxation measurements revealed reduced T2 relaxation times upon H2O2 activation, which corresponds to reduced water-holding capacity upon oxidation. However, a direct relationship between degree of oxidation and T2 relaxation time was not observed with various pH values and ionic strengths, and further studies are needed for a complete understanding of the effect of oxidation on myofibrillar functionality.     

Kinetics of heat-induced polymerization of gliadin

The kinetics of heat-induced polymerization of gliadin, that is, a mixture of monomeric wheat storage proteins, was studied using a model system. Samples were heated at pH 6.0 and 8.0 at 110, 120, and 130 °C for up to 240 min, and their extractabilities were compared under nonreducing and reducing (with 1% dithiothreitol) conditions. Extraction media were sodium dodecyl sulfate (SDS) containing buffer (pH 6.8, SDS buffer) and/or 70% ethanol. Gliadin cross-linking mainly resulted from intermolecular disulfide (SS) bond formation. At higher temperatures and, preferably, alkaline pH, intramolecular SS bonds in gliadin underwent β-elimination reactions, leading to the formation of dehydroalanine (DHA) and free sulfhydryl (SH) groups. The latter interchanged rapidly with SS bonds, leading to intermolecular SS bonds and gliadin extractability loss. When free SH groups had been formed, gliadin extractability in SDS buffer decreased following first-order reaction kinetics, the reaction rate constant of which increased with temperature and pH. Furthermore, the extractabilities of α- and γ-gliadin in 70% ethanol decreased according to first-order reaction kinetics. ω-Gliadin extractability was much less affected. Under the experimental conditions, gliadin polymerization through SH-SS interchange occurred much more rapidly than β-elimination of cystine.     

Tyrosinase-aided protein cross-linking: effects on gel formation of chicken breast myofibrils and texture and water-holding of chicken breast meat homogenate gels

The effects of Trichoderma reesei tyrosinase-catalyzed cross-linking of isolated chicken breast myofibril proteins as a simplified model system were studied with special emphasis on the thermal stability and gel formation of myofibrillar proteins. In addition, tyrosinase-catalyzed cross-linking was utilized to modify the firmness, water-holding capacity (WHC), and microstructure of cooked chicken breast meat homogenate gels. According to SDS-PAGE, the myosin heavy chain (MHC) and troponin T were the most sensitive proteins to the action of tyrosinase, whereas actin was not affected to the same extent. Calorimetric enthalpy (DeltaH) of the major thermal transition associated with myosin denaturation was reduced and with actin denaturation increased in the presence of tyrosinase. Low-amplitude viscoelastic measurements at constant temperatures of 25 degrees C and 40 degrees C showed that tyrosinase substantially increased the storage modulus (G') of the 4% myofibrillar protein suspension in the 0.35 M NaCl concentration. The effect was the most pronounced with high-enzyme dosages and at 40 degrees C. Without tyrosinase, the G' increase was low. Tyrosinase increased the firmness of the cooked phosphate-free and low-meat chicken breast meat homogenate gels compared to the corresponding controls. Tyrosinase maintained gel firmness at the control level of the low-salt homogenate gel and weakened it when both salt and phosphate levels were low. Tyrosinase improved the WHC of the low-meat and low-salt homogenate gels and maintained it at the level of the corresponding controls of phosphate-free and low-salt/low-phosphate homogenate gels. Microstructural characterization showed that a collagen network was formed in the presence of tyrosinase. Keywords: Chicken myofibrillar proteins; protein modification; cross-linking; tyrosinase; gelation; thermal stability; texture; water-holding capacity; microstructure.     

Maillard reaction and protein cross-linking in relation to the solubility of milk powders

Protein changes in relation to solubility, Maillard reaction (MR), and protein cross-linking in whole milk powder (WMP), skim milk powder (SMP), and whey protein concentrate (WPC) stored at different relative humidities (RHs) were investigated by chemical and electrophoretic methods. WMP and SMP reached minimum solubility rapidly, while WPC showed no change in solubility. The loss of solubility corresponded with development of high-molecular-weight protein complexes observed by two-dimensional electrophoresis. The maximal MR rate occurred at 66% RH for WMP and SMP (high lactose/protein ratios) and 84% RH for WPC (low lactose/protein ratios) based on the furosine and hydroxymethylfurfural contents. However, browning was greatest at 84% RH in all powders. The minimum solubility corresponded with the casein and fat contents. The retention of solubility and minimal protein cross-linking of WPC compared to casein-containing powders suggest that the casein content and cross-linking strongly influence the decrease in the solubility of milk powder.     

Solubility of the labile forms of soil carbon and nitrogen in K2SO4 of different concentrations

The general pattern of the changes in the solubility of the labile carbon and nitrogen compounds with the changes in the concentration of the salt extractant (0.05 and 0.5 M K₂SO₄) has been determined for soils differing in their acidity and in their contents of organic matter and nitrogen. Different forms of extracted compounds react differently to changes in the salt concentration. The solubility of inorganic nitrogen compounds (NH ₄ ⁺ and NO ₃ ^? ) does not depend on the concentration of K₂SO₄. In most cases, the carbon and nitrogen of the microbial biomass manifest a tendency for increasing extractability with an increase in the concentration of the K₂SO₄ solution. A fundamental difference is characteristic of the organic carbon and nitrogen compounds, the solubility of which in 0.5 M K₂SO₄ increases in different soils by 1.5–3.9 times in comparison with their solubility in 0.05 M K₂SO₄.     

Conformational and rheological changes in catfish myosin as affected by different acids during acid-induced unfolding and refolding

Changes in the conformation of catfish (Ictalurus punctatus) myosin due to (i) anions, (ii) acid pH, and (iii) salt addition were determined using tryptophan fluorescence, hydrophobicity measurements, differential scanning calorimetry, and circular dichroism. The relationship between conformation and storage modulus (G') of acid-treated myosin was studied. Three acids, HCl, H2SO4, and H3PO4, were used for unfolding myosin at three acidic pH conditions, 1.5, 2.0, and 2.5. Unfolded myosin was refolded to pH 7.3. Denaturation and unfolding of myosin was significantly (p < 0.05) lower when salt (0.6 M NaCl) was present during acid unfolding than in the absence of salt. When salt was added before unfolding, the alpha-helix content of myosin treated at pH 1.5 was significantly lower than that treated at pH 2.5. When salt was added after refolding, the alpha-helix content of myosin was unaffected by different pH treatments. The G' of myosin increased with an increase in myosin denaturation. The G' of myosin was significantly (p < 0.05) higher when salt was added to myosin after refolding than before acid unfolding. Among the different anion treatments, the G' of acid-treated myosin decreased in the order Cl- approximately SO42- > PO43-. Among the different pH treatments, the G' of myosin treated at pH 1.5 was significantly (p < 0.05) higher than myosin treated at pH 2.5. The conditions that would result in maximum myosin denaturation and maximum G' were unfolding of myosin at pH 1.5 using Cl- (from HCl) followed by refolding at pH 7.3 and subsequent addition of 0.6 M NaCl.     

Benzothiadiazole-induced resistance modulates ozone tolerance

The effects of ozone on bean plants pretreated with the SAR activator benzothiadiazole (BTH) have been investigated after fumigations with an acute dose of the pollutant (200 nL x L(-1) for 4 h), carried out at different times from BTH application. BTH pretreatment induced opposite effects on bean susceptibility to O(3), depending on the time elapsed before fumigation. When this time was only 1-2 days, bean plants were more susceptible to O(3) than untreated controls, showing rapid and extensive cell death in both palisade and spongy mesophyll. These damages appeared to be closely correlated with the amount and localization of H(2)O(2) in the leaf tissues. In BTH-pretreated, but not fumigated, plants, H(2)O(2) accumulation occurred in the cell walls and no dead cells were detected, whereas O(3) fumigation of untreated plants produced H(2)O(2) accumulation also inside some palisade mesophyll cells, causing their death. When BTH pretreatments were carried out 5-7 days before fumigation, plants appeared to be more tolerant to O(3) compared to untreated controls. Under these conditions, no visible symptoms of phytotoxicity were observed for at least 2 weeks after fumigation and no H(2)O(2) accumulation was detected. Biochemical assays showed a significant increase in the ascorbate (AA) level, taking place from the fifth to the seventh day after BTH treatment and unaffected by O(3) when given at these times. Ascorbate peroxidase (APX) activity appeared to decrease during the first 2 days after BTH treatment, and the decrease was somewhat enhanced by fumigation. On the contrary, guaiacol peroxidase (GuPX) activity was found to steadily increase up to the fifth day after BTH treatment but showed a bimodal trend upon fumigation. These results suggest that, during the first 1-2 days after BTH application, the H(2)O(2) level is enhanced by O(3) over a critical threshold for cell viability. However, in the absence of the pollutant, H(2)O(2) decreases in the following days under the effect of AA accumulation and increased GuPX activity. As GuPX is known to promote cell wall lignification and protein cross-linking, these effects would protect plasmalemma from O(3) irreversible damage, provided the priming by BTH has been fully developed.     

Effect of Clay Minerals on Immobilization of Heavy Metals and Microbial Activity in a Sewage Sludge-Contaminated Soil (8 pp)

Background   Aims, and Scope. Reducing heavy metal solubility and bioavailability in contaminated area without removing them from the soil is one of the common practices in decreasing the negative impacts on the environment and improving the soil quality. Therefore, our aim was to study the effect of clay minerals: Na-bentonite, Ca-bentonite, and zeolite applied to a contaminated soil on immobilization of heavy metals, as well as on some soil parameters related with microbial activity. Methods   A soil derived from sewage sludge was incubated with clay minerals of either Na-bentonite, Ca-bentonite, or zeolite for 111 days (d). During the incubation experiment, concentrations of water soluble Zn, Cd, Cu, and Ni were measured after extraction of 2 g air-dry soil with 50 ml of H2O for 2 h. After the water extraction, the soil sediment was extracted with 50 ml of 1 M NH4NO3 for 2 h to estimate the exchangeable amounts of heavy metals. Furthermore, soil microbial respiration, microbial biomass C, Corg mineralization, metabolic quotient (qCO2), and inorganic N were also investigated. Results and Discussion   Water extractable and exchangeable forms of heavy metals were changed by incubation and addition of clay minerals. Incubation of soil without addition of clay minerals (control) increased water extractable Cu by 12, 24 and 3.8% of initial content after 21, 62, and 111 d of incubation, respectively. The water extractable Zn decreased by 9% during 62 d of incubation and it tended to increase by 14% at the end of the incubation, as compared with the initial soil. Water extractable Cd decreased by 71, 66 and 33% of initial content, and Ni decreased by 54, 70, and 58%, after 21, 62, and 111 d of incubation, respectively. During the incubation experiment, the exchangeable form of all tested metals was decreased by incubation. The addition of clay minerals led to a significant decrease in water soluble and exchangeable forms of heavy metals during the incubation experiment, resulting in low metal extractability. The reduction in metal extractability was greater due to the addition of Na-bentonite or Ca-bentonite than that due to the addition of zeolite. During the first 3 weeks after addition of clay minerals, the studied biological parameters were not affected. However, as incubation progressed, the addition of Na- or Ca- bentonite led to a significant increase in soil respiration, microbial biomass C, Corg mineralization, and inorganic N; and a significant decrease in qCO2. This result is explained by sorption of heavy metals on Na-bentonite and Ca-bentonite and strong reduction of their toxicity. Conclusions   Our results clearly show that the addition of clay minerals, especially of Na-bentonite and Ca-bentonite, decreased the extractability of four metals during incubation. The decreased metal extractability was accompanied by an increase of soil respiration, Corg mineralization, microbial biomass C, and inorganic N and a decrease of metabolic quotient (qCO2), showing positive effect of clay mineral addition on soil biological parameters. Recommendations and Outlook   The use of Na-bentonite and Ca-bentonite is promising tool for reduction the extractability and possible toxicity of heavy metals in sewage sludge-contaminated soil. Therefore, the soils polluted with heavy metals may be ameliorated by addition of clay minerals, especially Na-bentonite and Ca-bentonite.     

Electrophoretic pattern, thermal denaturation, and in vitro digestibility of oxidized myosin

Physicochemical changes and in vitro digestibility of chicken breast myosin oxidized with a nonenzymic free-radical-generating system (FeCl(3)/H(2)O(2)/ascorbate) were studied by SDS-PAGE, differential scanning calorimetry, and o-phthaldialdehyde assay. Oxidation caused fragmentation and polymerization of myosin. Myosin polymers were cross-linked mainly through disulfide bonds. Hydroxyl radicals destabilized myosin, lowering its denaturation temperature by up to 4 degrees C. Oxidized myosin also produced a new thermal transition in the 60-80 degrees C temperature range, which could be attributed to the formation of disulfide-stabilized polymers. The proteolytic susceptibility of myosin to pepsin, trypsin, and chymotrypsin was increased by oxidation. Under nonreducing conditions, however, oxidized myosin showed decreased digestibility. The results may help explain variations in the functionality and nutritional quality of muscle foods in meat processing in which oxidation is involved.     

Interactive effects of microbial transglutaminase and recombinant cystatin on the mackerel and hairtail muscle protein

Interactive effects of microbial transglutaminase (MTGase) and recombinant cystatin on the mackerel and hairtail water soluble protein (WSP), salt soluble protein (SSP), and muscle protein (MP) were investigated. According to sodium dodecyl sulfate-polyacrylamide gel electrophoresis and enzymic activity analyses, cross-linking of mackerel and hairtail myosin heavy chain and low molecular mass compounds and formation of epsilon-(gamma-glutamyl)lysine cross-links were observed on samples with MTGase, while the recombinant cystatin could effectively inhibit the cathepsins and subsequently prevent degradation of proteins during setting. The cathepsins and MTGase activities in WSP, SSP, and MP solutions decreased, but the recombinant cystatin activity increased during setting at 45 degrees C.     

Role of neck region in the thermal aggregation of myosin.

Carp dorsal myosin formed oligomers that retained ATPase activity upon heating. Cleavage of the oligomeric myosin at subfragment-1 (S-1)/rod junction released monomeric S-1 and rod, indicating that ATPase retaining myosin associated near the S-1/rod junction. The digest also contained rod oligomers. Heating a mixture of S-1 and rod generated neither ATPase retaining S-1 oligomers nor rod oligomers. Electron microscopic observation of the heated myosin revealed that some oligomers were formed by associating at the S-1/rod joining region, exhibiting a recognized double head, probably ATPase retaining oligomers. No myosin oligomers associated at the tail region were observed, thus, rod aggregation would be formed at its very restricted region near the S-1/rod junction. Based on the findings, we proposed that the neck structure is important in the thermal oligomerization process of myosin.