Proteasomal Activity in Skeletal Muscle: A Matter of Assay Design, Muscle Type and Age...
Proteasomal Activity in Skeletal Muscle: A Matter of Assay Design, Muscle Type and Age Scientific Poster
The ubiquitin proteosome system (UPS) is a major non-lysosomal protein degradation machinery of the cell, which cleaves regulatory, misfolded and damaged proteins into small peptides. The ATP-dependent UPS represents a cascade of enzymatic factors, ubiquitin-activating enzymes (E1), ubiquitin-conjugating enzymes (E2), and ubiquitin protein ligases (E3), which are associated with the 26S proteasome and give rise to a large macromolecular complex. The barrel-shaped conventional 26S proteasomal structure is composed of one or two regulatory cap domains (19S subunits) and a proteolytically active central core domain (20S subunit), which harbors the three main catalytic activities, the chymotrypsin-like, trypsin-like and peptidyl-glytamyl-like (caspase-like) activities.
In the present study we established and validated a luminescence-based proteasomal activity assay applicable to milligram quantities (typically 5 mg) of skeletal muscle tissue. Special emphasis of our work was put on the influence of tissue preparation and storage, the amount of total protein extract yielding in a stable luminescent signal with optimal signal-to-noise ratio, the usage of different substrates in conjunction with a specific and irreversible proteasomal inhibitor, the difference in the use of individual muscle groups, and the effect of ageing.
- Part# PS118
- Printed in USA.