A high molecular weight protease in liver cytosol

Area of Science:

• Biochemistry
• Molecular Biology
• Enzymology

Background:

• Proteases play crucial roles in cellular protein turnover.
• Liver cytosol is generally considered inefficient in breaking down endogenous proteins.
• Existing cathepsins do not fully explain all cellular proteolytic activities.

Purpose of the Study:

• To identify and characterize novel proteases in mouse tissues.
• To investigate the unexpected presence and activity of a protease in mouse liver.
• To differentiate this novel protease from known cathepsin enzymes.

Main Methods:

• Fractionation of mouse tissues (kidney, brain, heart, spleen, tumor, liver) to isolate postmicrosomal fractions.
• Assay of protease activity using [3H]leucine-labeled globin as a substrate.
• Characterization of enzyme properties including molecular weight, optimal pH, thermal stability, and cofactor requirements.
• Comparison of enzyme characteristics with known cathepsins and lysosomal enzymes.

Main Results:

• A high molecular weight ( > 400,000 Da) protease was identified in the postmicrosomal fraction of multiple mouse tissues, with highest activity in the liver.
• The enzyme exhibits optimal activity at pH ~7.5.
• Purified enzyme is unstable above 20°C but stabilized by metal chelating agents (citrate, creatine-P, glycerate-3-P).
• It is an -SH protease, but its thermal instability is not mitigated by dithiothreitol.
• The protease is distinct from known cathepsins and is not lysosomal.

Conclusions:

• A novel, high molecular weight, neutral-acting protease exists in mouse tissues, notably in the liver.
• This enzyme possesses unique biochemical properties differentiating it from cathepsins.
• Its non-lysosomal nature and distinct characteristics suggest a unique role in cellular protein degradation pathways.