Masayuki Mikami

Degradation of myofibrils from rabbit, chicken and beef by cathepsin l and lysosomal lysates.

The degradation of rabbit, chicken and beef myofibrils by cathepsin L or lysosomal lysates was studied by SDS-polyacrylamide-gel electrophoresis and electron microscopy (EM). Similar degradation patterns were observed for each myofibrillar preparation incubated with cathepsin L, except that myosin heavy chain and tropomyosin of beef were more susceptible than those of rabbit and chicken. Otherwise, troponin T, troponin in I and C-protein were rapidly degraded with slower degradation of titin, nebulin, myosin heavy chain, α-actinin, α-tropomyosin, actin and myosin light chains, LC1 and LC2. However, the component of 30 000 Mr was found to be further degraded to smaller peptides. Degradation at pH 5·5 (approximate post-mortem limit value) was faster than at pH 6·0 but slower than at pH 5·0. A number of new protein bands were identified (130 000, 120 000, 90 000, 85 000, 80 000, 31 000 and 30 000 Mr). The degradation patterns of rabbit myofibrils by rabbit muscle lysosomal lysates were similar to that of myofibrils incubated with purified cathepsin L except for the retention of the 30 000 Mr component and reduced degradation of actin, due presumably to the reduced amount or stability of cathepsin L in the crude enzyme preparations. Electron micrographs revealed that myofibrillar degradation by cathepsin L occurred preferentially at the Z-lines leading to removal of the Z-line proteins and fracturing of the myofibrils at these sites. Catheptic damage was seen to be most rapid in chicken myofibrils and least rapid in beef myofibrils consistent with the more rapid conditioning process in chicken.

Species and muscle differences in L-carnitine levels in skeletal muscles based on a new simple assay

We have adapted the enzymatic method [Biochemical and Biophysical Research Communications 176 (3) (1991) 1617] for the safe and rapid assay of L-carnitine (L-CA) in skeletal muscle using a microplate reader. The concentration of L-CA in fresh semitendinosus muscle from broiler chicken, pig, beef cattle, deer, horse and goat muscle were 0.69, 1.09, 1.86-3.57, 4.57, 4.95 and 11.36 μmol/g wet weight, respectively. The animals which had higher concentration of L-CA, also had the highest amounts of myoglobin as an index to the redness of the muscle. Furthermore, we investigated this relationship between white muscle, M. pectoralis profundus, and red muscle, M. soleus, in laying hens. The L-CA and myoglobin concentration in red muscle were significantly higher than those in white muscle (p<0.01). These findings suggest that L-CA concentration in muscle is related to oxygen metabolism and to myofiber types.

Degradation of ubiquitin in beef during storage.

Sarcoplasmic proteins were prepared from the quadriceps femoris muscle immediately after slaughter (2.5 hr) and from stored muscle samples at 10 days post mortem for SDS-PAGE analysis and Western blotting. Characterization with ubiquitin antiserum (Sigma, St. Louis, MO, USA) showed clear and strong recognition of ubiquitin (8.6 kDa) and another minor band (17 kDa) in purified ubiquitin (Sigma, St. Louis, MO, USA). Among the sarcoplasmic proteins prepared, this antiserum also reacted with the bands corresponding to purified ubiquitin (8.6 kDa and 17 kDa) and a small amount of some other higher-molecularmass proteins which were considered to be ubiquitin-protein conjugates. However, at 10 days post mortem, both ubiquitin and the ubiquitin-protein conjugates had almost disappeared, suggesting their degradation by proteinases.

Presence of ubiquitin in bovine post-mortem cardiac muscle

Abstract We prepared sarcoplasmic proteins from bovine cardiac muscle immediately after slaughter (1.0 h; 0 days) and from stored muscle at 1, 2 and 7 days post-mortem for SDS-PAGE and Western blotting analysis. Characterization of the ubiquitin antiserum (Sigma, St Louis) showed clear and strong recognition of the ubiquitin band (8.6 kDa) and another minor band (17 kDa) in the purified ubiquitin sample (Sigma, St Louis). Among the sarcoplasmic proteins prepared from stored muscle at 0 and 7 days, this antiserum also reacted with bands corresponding to purified ubiquitin and small amounts of some other, higher-molecular-mass proteins (about 25 and 30 kDa) which were considered to be ubiquitin–protein conjugates. However, the 25 kDa band was faint in the 7 days sample, suggesting that it had degraded. We compared these results with those from our previous study of bovine skeletal muscles, in which both ubiquitin and the ubiquitin-protein conjugates had almost disappeared in the samples tested at 10 days post-mortem.

Effect of proteasome inhibitor on sarcoplasmic protein of bovine skeletal muscle during storage

Bovine skeletal muscle, immediately after slaughter (1.5 h; 0 d), was buffered, homogenized treated with proteasome inhibitor, and stored (2 d) for SDS-PAGE and Western blotting analysis. Ubiquitin antiserum (Sigma, St Louis) reacted with bands corresponding to purified ubiquitin and small amounts of other higher-molecular-mass proteins (about 30 and 40 kDa) which were considered to be ubiquitin-protein conjugates. These bands were faint in the control 2 d sample, suggesting that they had degraded. However, these tendencies of the ubiquitin positive bands to decrease were not clearly observed in the sample treated with proteasome inhibitors (MG132 and Lactacystin). These results suggest that both ubiquitin and the ubiquitin-protein conjugates were present in the skeletal muscle immediately after slaughter and they were then degraded during storage. This degradation was partially due to the action of proteasome.

Bacteriological changes of colostrum naturally fermented, and preserved with propionic acid or hydrogen peroxide.

余剰初乳を仔牛に給与する試みが最近行なわれているが,本実験では分娩後5日間の初乳を集め,過酸化水素またはプロピオン酸を添加して,実験室規模で保存試験を行なった.1. 初乳の微生物は,分娩直後に比較約多く,大腸菌群,ブドウ球菌も含まれているものもあった.2. 混合初乳の保存中の一般細菌数は,20°Cで3～5日目にピーク(1×109/ml)があり,その後わずかに減少するが,大腸菌群は21日目で1×103/mlまで減少した.3. 過酸化水素を添加すると大腸菌群は1日目で0となり,一般細菌数においても0.5%添加では5日目で,1.0および1.5%添加では1日目で0となった.4. 過酸化水素の残存量は0.5%添加では1日後に0になったが,二次汚染に対する注意が必要である.1.0%添加では約4.000ppm,1.5%では約10.000ppm残存した.5. プロピオン酸を添加すると一般細菌数は0.5%添加では3日目まで,1.0%添加では5日目まで,1.5%添加では21日目まで増殖が抑制された.一方大腸菌群は1.0および1.5%添加では1～3日目でほぼ0となった.6. NPNは細菌数の増加と関係があって,過酸化水素は0.5%,プロピオン酸は1.0%添加することによって増加がほぼ抑えられた.