Decrease in platelet, erythrocyte and lymphocyte acetylcholinesterase activities due to the presence of protease...

Area of Science:

• Biochemistry
• Enzymology
• Membrane protein stability

Background:

• Acetylcholinesterase (AChE) is crucial for neurotransmission.
• Understanding AChE stability during sample preparation and storage is vital for accurate research.
• Protease activity can potentially affect enzyme integrity.

Purpose of the Study:

• To investigate the impact of protease inhibitors on AChE activity in membrane preparations.
• To determine optimal conditions for preserving AChE activity during storage.

Main Methods:

• Crude membrane preparations from sheep platelets were used.
• AChE activity was measured before and after Triton X-100 solubilization.
• Storage buffer composition, including various protease inhibitors (PMSF, EGTA, EDTA, trypsin inhibitor), was varied.
• Enzyme activity was assessed after different storage durations at 4°C.

Main Results:

• AChE activity remained stable for 6 days without inhibitors at 4°C.
• PMSF (10⁻⁵ M) reduced AChE activity by 50% within 24 hours.
• Combinations of PMSF with EGTA, EDTA, and trypsin inhibitor protected AChE from PMSF-induced inactivation but not from longer-term degradation.
• EDTA, EGTA, and trypsin inhibitor alone also decreased AChE activity after 24 hours.
• Complex inhibitor cocktails reduced AChE activity only when PMSF or EDTA was present.

Conclusions:

• Certain protease inhibitors, particularly PMSF and EDTA, can negatively impact AChE activity in membrane preparations.
• While some inhibitor combinations offer short-term protection, long-term storage of AChE requires careful buffer optimization.
• Findings are consistent across different cell types, including sheep and bovine erythrocytes and lymphocytes.