Masanori Matsuishi

Liberation of actin from actomyosin in meats heated to 65 °C

This study investigated whether actin liberation from myofibrils occurs during the heating of various muscles, as well as squid mantle muscle at temperatures, such as 60°C, employed for vacuum cooking of meats. Actin liberation was demonstrated in scallop striated adductor muscle, but not in beef, pork, or chicken, using the detection method previously employed with squid muscle, in which liberated actin was detected with SDS-PAGE, in the supernatant obtained by centrifugation of the homogenate of heated muscle in 0.2M KCl at a neutral pH. However, actin liberation was demonstrated in beef, pork and chicken by a new detection method, in which heated muscle was homogenized in 0.6M KCl or NaCl at a slightly alkaline pH and maintained at 4°C for 16h with stirring, after which the homogenate was diluted three times with water and centrifuged to obtain the supernatant containing the liberated actin. This new method indicated that actin liberation in beef, pork, and chicken was marked by heating at 65°C, but scarcely induced at 80°C. Thus, the liberation of actin from myofibrils may contribute to the greater tenderness of vacuum-cooked meat (meat heated at a low temperature for long time), as compared with meat prepared by cooking at a higher temperature.

AMP and IMP Dissociate Actomyosin into Actin and Myosin

We investigated to determine why heating of squid muscle at 60 °C induced the liberation of actin from myofibrils. When a mixture of a myofibrillar fraction and a low-molecular sarcoplasmic fraction prepared from squid muscle was heated at 60 °C, actin liberation occurred. When a myofibrillar fraction was heated with ATP, AMP, or IMP, actin liberation occurred. Hence, AMP is perhaps one of the factors causing actin liberation in postmortem squid muscle. It was found that AMP and IMP reversibly dissociated actomyosin of chicken, bovine, and porcine skeletal muscles into actin and myosin on incubation at 0 °C at pH 7.2 in 0.2 M KCl. These results led us to conclude that AMP and IMP were the most responsible factors causing actin liberation from myofibrils in the heated muscle and causing reversible dissociation of actomyosin on storage of skeletal muscle at a low temperature. Hence, AMP and IMP are possible factors causing the resolution of rigor mortis in muscles.

Cysteine proteinase inhibitors from rabbit skeletal muscle

1. Two cysteine proteinase inhibitors, I-T (Mr = 29,000) and I-S (Mr = 10,700), were isolated from rabbit skeletal muscle by means of succesive extraction with a neutral buffer solution, precipitation at pH 3.7, acetone fractionation and gel permeation on Sephadex G-75. 2. I-T is a formed trimer of a monomeric inhibitor, I-M (Mr = 10,500), through disulfide bonds. 3. I-S is almost completely stable between pH 3 and 8, while I-M is unstable in the same pH range. 4. I-M acts most effectively towards cathepsins H and L, showing moderate activity towards cathepsin B and only weak activity towards papain. I-S acts most effectively towards cathepsin L, followed by, in decreasing order, cathepsin H, cathepsin B and papain.

Purification and properties of cysteine proteinase inhibitors from rabbit skeletal muscle

Two cysteine proteinase inhibitors, CPI-L and CPI-H, were purified from rabbit skeletal muscle by means of successive extraction with a neutral buffer solution, precipitation at pH 3.7, acetone fractionation and gel permeation on Sephadex G-75 and affinity chromatography on carboxymethyl-papain-Sepharose. The molecular mass of CPI-L was 13 kDa on gel permeation chromatography and SDS-PAGE under reducing conditions and was 15 kDa on SDS-PAGE under non-reducing conditions. The molecular mass of CPI-H was 23 kDa on gel permeation chromatography and it was converted to 13 kDa by SH-reducing agent. Although CPI-H showed single protein band with 13 kDa on SDS-PAGE under reducing conditions, it showed four protein bands with 21, 20, 15 and 13 kDa on SDS-PAGE under non-reducing conditions. Therefore, CPI-H was suggested to have a complicated subunit structure for which S-S bonds and some non-covalent bonds would be responsible. CPI-L and CPI-H were stable in the range of pH 3.0-9.5 and up to 80 degrees C. CPI-L and CPI-H were suggested to inhibit cathepsins B, H and L by a non-competitive mechanism. The inhibition constants (Ki) of CPI-L and CPI-H showed that both CPIs have much higher affinity against cathepsins H and L than against cathepsin B.

Enhancing Effect of IMP on Myosin and Actin Extraction from Porcine Meat

We examined the effects of nucleoside monophosphates on the dissociation of actomyosin into myosin and actin. GMP was effective only among GMP, CMP, dTMP, and UMP. Hence we concluded that purine-based nucleoside monophosphates such as GMP, AMP, and IMP are effective, incorporating this with our previous results (Okitani A et al., Biosci. Biotechnol. Biochem., 72, 2005-2011 (2008)). Then we examined whether IMP enhances the extraction of myosin and actin as well as pyrophosphate (KPP), using homogenates of pork with 9 volumes of 0.3, 0.4, and 0.5 M NaCl solutions containing 0-36 mM IMP or 0-9 mM KPP. Maximum extractability, about 70% for both proteins, was attained by means of NaCl solutions containing 36 mM IMP. These values were comparable to the maximum values, about 90% for myosin and 50% for actin, attained by means of solutions containing 9 mM KPP. Hence we concluded that IMP enhances the extraction of myosin and actin from porcine meat.

Purification and some properties of cathepsin H from rabbit skeletal muscle.

Rabbit muscle cathepsin H classified as an aminoendopeptidase was purified and its properties were investigated to clarify its contribution to the proteolysis of postmortem muscle. The purification was performed by ammonium sulfate fractionation and successive chromatographies on Sephadex G-75, phosphocelluose, DEAE-Sephadex A-50 and Sephadex G-100. The purified enzyme gave a single protein band on SDS-PAGE. Its molecular mass was found to be 28 kDa by gel permeation and 30 kDa by SDS-PAGE. The optimum pHs for alpha-N-benzoyl-DL-arginine-beta-naphthylamide (BANA)- and L-leucine-beta-naphthylamide (Leu-NA)-hydrolyzing activities were 6.6 and 7.0, respectively. This enzyme was almost stable in the range of pH 4-5 and up to 50 degrees C at pH 5.0. The Km values of BANA- and Leu-NA-hydrolyzing activities were 0.367 and 0.203 mM, respectively. The enzyme was inhibited by monoiodoacetic acid, antipain, leupeptin, TLCK and TPCK, but was not affected by pepstatin, bestatin, puromycin, PMSF or trypsin inhibitor. This enzyme strongly acted on Arg-, Lys-, Met-, Ala-, Ser- and Leu-NAs, weakly acted on Val- and Glu-NAs, and hardly acted on Pro- and Gly-NAs. The amount of cathepsin H in muscle was estimated to be less than one-fourth of the sum of the amount of aminopeptidases C and H by the Leu-NA-hydrolyzing activity on the chromatography. This enzyme degraded myosin heavy chain, actin, tropomyosin and troponin I clearly at pH 4.0, while it slightly degraded troponin I at pH 5.0-5.6. Therefore, the contribution of cathepsin H to the proteolysis of postmortem muscle is presumed to be relatively small.

Elucidation of a basic protein, glyceraldehyde-3-phosphate dehydrogenase, as a contributing factor to raise Mg-ATPase activity of myofibrils during meat conditioning

We investigated the factor which increased the maximum value of the Mg-ATPase activity of myofibrils existing at low KCl concentrations during meat conditioning. On the treatment of myofibrils with the solution extracted with the buffer of pH 7.2 from muscle, the Mg-ATPase activity in the presence of 0-0.15 M KCl increased time-dependently. This change was most remarkable in the range of pH 5.6-7.0. Trypsin treatment of the extract abolished such effect, suggesting that the responsible factors were proteins. The fractionation of the extract with isoelectric focusing demonstrated that the factors were basic proteins (pI 8.3-9.6). The treatment of myofibrils with those basic proteins under various conditions suggested that the time-dependent adhesion of those basic proteins, through a denaturation at around pH 5.5, to myofibrils was assumed to raise the Mg-ATPase activity. Analysis of myofibrils prepared from rabbit muscles stored at 0°C for 12 days postmortem showed the appearance of the 35,000 Da protein, accompanied the increase in the Mg-ATPase activity. Therefore, the adhesion of this protein to myofibrils in situ probably caused the increase in the Mg-ATPase activity. Successive treatment with the basic protein and the crude cathepsin increased the dependency of the Mg-ATPase activity on KCl concentrations and the maximum value of the Mg-ATPase activity. Therefore, the coordinate action of a basic 35,000 Da protein and cathepsins was presumed to induce the changes in the Mg-ATPase activity of myofibrils during meat conditioning. The basic protein was concluded to be glyceraldehyde-3-phosphate dehydrogenase (its subunit molecular mass: 35,000 Da), since the incubation of myofibrils with its commercial preparation raised the Mg-ATPase activity of myofibrils.

Mode of IMP and Pyrophosphate Enhancement of Myosin and Actin Extraction from Porcine Meat

We examined the mode of IMP and pyrophosphate enhancement of myosin and actin extraction from porcine meat. Extractabilities were determined after homogenates, prepared by adding 9 volumes of 0.3, 0.4, or 0.5 M NaCl solutions containing 0 to 36 mM IMP and 0 to 9 mM tetrapotassium pyrophosphate (KPP) to minced pork, were incubated at 4 °C for 0 or 12 h. Irrespective of the NaCl concentrations, IMP-induced extraction of both proteins increased with increasing extraction time. In contrast, that of KPP did not. When 0.3 M NaCl solutions containing both IMP and KPP were used, the solutions with 1.5 mM KPP showed marked enhancement of IMP-induced myosin and actin extraction. Incorporating these results with our previously published data (Nakamura et al., Biosci. Biotechnol. Biochem., 76, 1611–1615 (2012)), we hypothesized that IMP and KPP have the ability to release thick and thin filaments from restraints in myofibrils, in addition to the ability to dissociate actomyosin into myosin and actin, and that the restraint-releasing ability of IMP is dependent on reaction time and NaCl concentration while that of KPP is not.