Partial purification and characterization of two thiol proteases from hog thyroid lysosomes

Area of Science:

• Biochemistry
• Enzymology
• Protease research

Background:

• Lysosomes are key cellular organelles involved in degradation.
• Proteases within lysosomes play crucial roles in cellular turnover and processing.
• Understanding specific lysosomal proteases aids in elucidating cellular functions.

Purpose of the Study:

• To purify and characterize thiol proteases from hog thyroid lysosomes.
• To differentiate the properties and substrate specificities of the isolated proteases.
• To compare the identified proteases with known lysosomal cathepsins.

Main Methods:

• Purification using diethylaminoethyl cellulose chromatography, gel filtration (Sephacryl S-200), and carboxymethyl cellulose chromatography.
• Enzyme activity assays using benzoyl-L-arginine-2-naphthylamide (BANA) and casein as substrates.
• Inhibition studies with leupeptin and L-trans-epoxysuccinyl-leucylamido(4-amino)butane.
• Characterization by electrophoresis, chromatofocusing, and lectin binding (concanavalin A Sepharose).

Main Results:

• Two thiol proteases, TP-1 and TP-2, were successfully purified.
• TP-1 showed high BANA hydrolytic activity, while TP-2 hydrolyzed both BANA and casein.
• TP-1 and TP-2 exhibited differential sensitivity to leupeptin and L-trans-epoxysuccinyl-leucylamido(4-amino)butane.
• Electrophoresis and chromatofocusing revealed distinct isoelectric points for TP-1 and TP-2.
• TP-1 bound to concanavalin A Sepharose and hydrolyzed L-arginine-2-naphthylamide, whereas TP-2 did not bind and hydrolyzed carbobenzoxy-L-arginyl-L-arginine methylcoumarylamide.

Conclusions:

• Hog thyroid lysosomes contain at least two distinct thiol proteases.
• TP-1 and TP-2 possess unique substrate preferences and inhibitor profiles.
• The properties of TP-1 and TP-2 suggest similarities to liver lysosomal cathepsins H and B, respectively.