Carp Myofibrils Research Articles

Effect of MTGase on silver carp myofibrillar protein gelation behavior after peroxidation induced by peroxyl radicals

The objective of this study was to explore the oxidative modification induced by AAPH (2,2′-azobis (2-amidinopropane) dihydrochloride) on the microbial transglutaminase (MTGase) cross-linking reaction and gelling properties of silver carp myofibril protein (SCMP). Compared to AAPH treatment, MTGase addition resulted further changes of protein properties as evidenced by the decreasing free amino and thiol group content, the decreased secondary and tertiary structure, and the increasing storage modulus (G′) and gel strength (P < 0.05). The proper oxidation induced by AAPH (5 mM) promoted the glutamine-lysine and disulfide cross-linking due to MTGase (10 U/g). Therefore, the quality of the SCMP gel was improved with a good water-holding capacity (WHC), gel strength and G′. This results could lay a theoretical foundation for the development of silver carp surimi products with good quality.Chemical compounds: (2,2′-azobis(2-amidinopropane)dihydrochloride (PubChem CID:76344); O-Phthalaldehyde (PubChem CID:4807); 5,5′-Dithiobis(2-Nitrobenzoic Acid) (PubChem CID:6254); 8-Anilino-1-naphthalenesulfonic acid (PubChem CID:1369); Acrylamide (PubChem CID: 6579); β-Mercaptoethanol (PubChem CID: 1567); Sodium dodecyl sulfate (PubChem CID:3423265)

Effect of high hydrostatic pressure (HHP) on myofibril-bound serine proteinases and myofibrillar protein in silver carp (Hypophthalmichthys molitrix)

Silver carp (Hypophthalmichthys molitrix) is a popular cultured freshwater fish in China, and becomes a potential raw material for surimi product. However, silver carp surimi exhibits considerable gel softening (modori) and myofibril-bound serine proteinases (MBSP) involved in the degradation of myofibrillar proteins have been assumed to be crucial to the modori phenomenon. High-pressure technology has demonstrated its potential application to control enzyme-related seafood texture deterioration. Herefrom, it could be further applied to the freshwater fish processing and the effect of high pressure treatments on the activity of MBSP implicated in texture deterioration in myofibrils and crude enzyme extracts was evaluated. The inactivation kinetics of MBSP pressured from 200MPa to 500MPa at room temperature (20°C) were fitted first-order kinetics, The extent of enzyme inactivation was lower in silver carp myofibrils in comparison with the crude enzyme extracts from myofibrils showing a protective effect against the high pressure treatment. The effect of HHP on MBSP myofibrillar degradation shown by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) was associated with a corresponding decrease coincided with the elevated high pressure levels in intensity of myosin heavy chain (MHC). Dynamic rheological measurements indicated that high-pressure treatment exerted considerable influence on MBSP activities as well as proteins (mainly myofibrils), resulting in structural modifications and texture changes. In this study, 300MPa (≥10min) was the process condition (pressure and time) that caused apparent MBSP inactivation derived from the first order kinetic model and had the efficacy in controlling silver carp texture deterioration both verified by SDS-PAGE and dynamic rheological measurements.

Acid-induced Gel Formation of Silver Carp (Hypophthalmichthys molitrix) Myofibrils as Affected by Salt Concentration

Acid-induced Gel Formation of Silver Carp (Hypophthalmichthys molitrix) Myofibrils as Affected by Salt Concentration

Three types of proteinases in Japanese common squid Todarodes pacificus hepatopancreas as studied by using carp myofibrils as substrate

Three types of proteinases, namely cysteine, metallo, and serine proteinases, were found in squid hepatopancreas by studying the inhibition spectra using carp myofibril as substrate. The cysteine, metallo, and serine types showed the highest activities at 50, 35, and 40°C, respectively. The optimal pHs were 5, 7, and 9 for the cysteine, metallo, and serine types, respectively. When assayed at 20°C and pH 7.5, the metallo type showed the highest activity. The metallo type was characterized by a high selectivity in the digestion of myosin. Among the three enzymes, the cysteine type was found to be the most stable against thermal and acid treatments. Heat-treated myofibrils were more susceptible to the cysteine and serine types, but less susceptible to the metallo type. Acid treatment of myofibrils also enhanced the digestibility by the cysteine type. The results indicated that the cysteine type seemed to be the most suitable enzyme to produce peptides from denatured myofibrils by their random digestion.

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

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 de naturation 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.

Degradation of myofibrillar proteins by a myofibril-bound serine proteinase in the skeletal muscle of crucian carp ( Carasius auratus)

A myofibril-bound serine proteinase (MBSP) in the skeletal muscle of crucian carp ( Carasius auratus) was identified. Hydrolysis of myofibrillar proteins by the endogenous MBSP was studied. Myosin heavy chain (MHC) was degraded markedly when crucian carp myofibril was incubated at 55 °C, as shown by SDS-PAGE. Prolonged incubation of myofibril at 55 °C also caused the obvious degradation of tropomyosin, while the decomposition of other myofibrillar proteins, such as α-actinin and actin, was slight, as detected by Western blotting. The results suggest the existence of an endogenous myofibril-associated proteinase in crucian carp myofibril, which efficiently cleaves MHC and tropomyosin. Serine proteinase inhibitors (Lima bean trypsin inhibitor, PMSF and benzamidine) greatly suppressed the degradation of MHC, caused by the enzyme, while inhibitors for cysteine, metallo-, and aspartic proteinases showed only partial or incomplete inhibitory effects, indicating that the endogenous proteinase is a serine proteinase. Substrate specificity analysis, using partially purified crucian carp MBSP, suggested that the enzyme is a trypsin-like serine proteinase.

Uniquely Stable 40 kDa Subfragment-2 in Carp Myosin

Digestion of carp myofibrils at 30 degrees C in 0.5 M KCl medium with calcium ion generated unique 135 kDa heavy meromyosin (HMM). The HMM was not produced when digested at 10 degrees C. A further digestion of the 135 kD HMM isolated in the absence of calcium ion generated uniquely short subfragment-2 (S-2) with a size of 40 kDa (40 kDa S-2) together with subfragment-1 (S-1). The 40 kDa S-2 was identified by N- and C-end sequencing, and demonstrated to locate the amino end of the rod portion. The unfolding temperature for the 40 kDa S-2 was around 52 degrees C as studied by circular dichroism measurement. The same unfolding peak was also detected with the intact rod together with a large unfolding peak at around 36 degrees C coming from the rest of the rod portion, light meromyosin. The unfolding peak for the 40 kDa S-2 in myosin was a little lower (48 degrees C) than that in free form, suggesting the involvement of the head portion in the stability of the 40 kDa S-2 in the structure.

IDENTIFICATION OF A MYOFIBRIL-BOUND SERINE PROTEINASE IN THE SKELETAL MUSCLE OF SILVER CARP

Myofibril-bound serine proteinase (MBSP) in the skeletal muscle of silver carp was characterized. Myosin heavy chain (MHC) degraded markedly when silver carp myofibril was incubated at 55-60C as shown by SDS-PAGE. Prolonged incubation of myofibrils also caused the degradation of other myofibrillar proteins such as α-actinin, actin and tropomyosin to some degree. The results suggest the existence of an endogenous myofibril associated proteinase. Serine proteinase inhibitors (Pefabloc SC and Lima bean trypsin inhibitor) greatly suppressed the degradation of myosin heavy chain, while inhibitors for cysteine, metallo, and aspartic proteinases did not show any effect, indicating that the endogeneous proteinase is a myofibril-bound serine proteinase.

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

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 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.

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

: 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.

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.

Proteolytic degradation of myofibrillar components by carp cathepsin L

Carp cathepsin L, which is the best candidate to produce textural change in the arai-treated carp fillet, exhibited maximum hydrolytic activity for Z-Phe-Arg-MCA and soluble casein at pH 5·0–5·5. The proteolytic action of the enzyme was evaluated by complete degradation of various carp myofibrils at pH 5·0 over 30 min and by potent degradation of the same proteins at pH 5·5–6·0 over 20 h. All myofibrillar components were partially degraded by the enzyme at pH 6·5–7·0, but varying amounts of them remained undegraded after 20 h. These findings indicate that carp cathepsin L degrades not only carp myofibrillar components but also their resultant products between pH 5·0 and 7·0 and that it markedly acts on myosin heavy chain, α-actinin and troponin-T and -I. Carp cathepsin L likely contributes to postmortem muscle tenderisation of carp fillet over an extensive pH range during storage. © 1998 SCI.

Freeze Denaturation of Carp Myofibrils Compared with Thermal Denaturation

Freeze Denaturation of Carp Myofibrils Compared with Thermal Denaturation

Aggregation of Actin and Myosin in Carp Myofibrils during Frozen Storage under Phosphate Buffer Conditions

Aggregation of Actin and Myosin in Carp Myofibrils during Frozen Storage under Phosphate Buffer Conditions

Changes in Solubility and ATPase Activity of Carp Myofibrils during Frozen Storage under Phosphate Buffer Conditions

Changes in Solubility and ATPase Activity of Carp Myofibrils during Frozen Storage under Phosphate Buffer Conditions

2. Estimation for Storage Stability of Freeze-Dried Carp Myofibrils by DSC Method(Papers presented at the 41st Annual Meeting)

2. Estimation for Storage Stability of Freeze-Dried Carp Myofibrils by DSC Method(Papers presented at the 41st Annual Meeting)

Influence of KCl on the Decrease of Ca&lt;sup&gt;2+&lt;/sup&gt;-ATPase Activity and Slubility of Carp Myofibrils during Frozen Storage

Influence of KCl on the Decrease of Ca&lt;sup&gt;2+&lt;/sup&gt;-ATPase Activity and Slubility of Carp Myofibrils during Frozen Storage

Subunit Components in Salt-soluble and Insoluble Fractions of Carp Myofibrils during Frozen Storage

Subunit Components in Salt-soluble and Insoluble Fractions of Carp Myofibrils during Frozen Storage

Changes of the Solubility and ATPase Activity of Carp Myofibrils during Frozen Storage at Different Temperatures

Changes of the Solubility and ATPase Activity of Carp Myofibrils during Frozen Storage at Different Temperatures

筋原繊維タンパク質の凍結乾燥変性に及ぼすタンパク質とソルビトール濃度の影響

筋原繊維タンパク質の凍結乾燥変性に及ぼすタンパク質とソルビトール濃度の影響