The Importance of Cell Lysis Methods in Measuring Proteasome Activity

Abstract

Proteasome dysfunction has been implicated in a wide range of diseases, including neurodegenerative disorders such as Alzheimer's or Parkinson's diseases, as well as cardiomyopathies and diabetes. The role of the proteasome is critical to the normal functioning of the body's degradation pathways. Numerous methods of isolating and measuring proteasome activity in cell lysates are currently being used by different groups. In particular, the homogenization and lysis buffers vary considerably between different laboratories. In this study, we investigated how different cell lysis procedures and buffers affect both 20S and 26S proteasome proteolytic activity. Our results suggest that the inclusion of glycerol in the lysis buffer is important for optimal 20S proteasome activity. We also found that in comparison to cell lysis buffers containing 1% Triton X-100, buffers containing NP-40 as the detergent resulted in up to a 50% decrease in beta5 20S proteasome activity. For 26S proteasome measurements, many labs utilize varying freeze-thawing methods for cell lysis. We found that brief cycles of freeze-thawing at −20°C resulted in higher beta5 26S proteasome activity compared to freezing cells overnight at −80°C or shaking cells vigorously at 4°C for 1 hour. Optimization of cell lysis techniques is important for helping future studies investigate smaller changes in proteasome activity by allowing the same amount of protein to show significantly higher activity. It also allows proteasome activity to be measured using less protein sample. Our results indicate that the type of cell lysis buffer used as well as the procedure used to disrupt cells is important for optimal proteasome activity measurements. This research is partially supported by NIH grant HL096819.