| Budget Amount *help |
¥3,600,000 (Direct Cost: ¥3,600,000)
Fiscal Year 1999: ¥1,100,000 (Direct Cost: ¥1,100,000)
Fiscal Year 1998: ¥2,500,000 (Direct Cost: ¥2,500,000)
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| Research Abstract |
Intracellular protein degradation in living cells is catalyzed by lysosomal and cytoplasm pathways. In the cytoplasmic pathway, there are ATP-dependent and independent mechanisms Recent studies have suggested that the ubiquitin-proteasome pathway plays an important role in the degradation of muscle proteins under various catabolic conditions. Although the ubiquitin system of cellular protein degradation has been investigated in various fields such as clinical medicine and cellular biology, this system seems to have received little attention in the field of meat science.
The purpose of this study was to demonstrate electrophoretically that the effects of ubiquitin-proteasome pathway on the sarcoplasmic proteins of postmortem skeletal and cardiac muscle during conditioning. Sarcoplasmic proteins were prepared from the quadriceps femoris muscle from 13 animals; bovine, swine, ovine and so on, and pectoral is muscle from chicken, and bovine cardiac muscle, immediately after slaughter and po
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st rigor muscle sample for SDS-PAGE, two-dimensional electrophoresis analysis, and Western blotting.
There were a little difference observed on the SDS-PAGE and 2D-PAGE profiles among the animals and between skeletal and cardiac muscle. The anti-ubiquitin antiserum reacted with the ubiquitin (pH 7, 8. 6 kDa) and some other higher-molecular-mass proteins (25-40 kDa), which were considered to be the ubiquitin-protein conjugates. The location of high-molecular-mass proteins were differed in the 2D-PAGE patterns, when compared among animals and between skeletal and cardiac muscle indicating that the molecular weight and isoelectric point were different from each other. Moreover, the ubiquitin and the ubiquitin-protein conjugates had almost decreased and/or disappeared during conditioning, suggesting their degradation by proteinase.
One of the ubiquitin-protein conjugates purified using two-dimensional electrophoresis was subjected to the amino acid sequence analysis: the sequence of six residues was R Q T A A G. This sequence suggested that this ubiquitin-protein conjugate was not poly-ubiquitin, because there was no part corresponding to the amino acid sequence of the ubiquitin.
Proteasome inhibitors had an effect on sarcoplasmic fraction from muscle immediately after slaughter. The muscle was homogenized with 0.2M Tris buffer (pH 7.4) and an inhibitor which used were the Lactacystin, clasto-lactacystin β-lactone, MG132, and MG115 was added to the buffer. Inhibitor activity of the proteasome should prevent the degradation of ubiquitin-protein conjugates : therefore the incubation with proteasome inhibitors caused an accumulation of ubiquitin-protein conjugates.
These results suggest that the possibility of ubiquitin-proteasome pathway contributes to the intracellular protein degradation in muscle cell immediately after slaughter. Less
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