Studies on Brain Hydrolytic Enzymes and their Endogenous Protein Substrates

Biochemistry Laboratory, Department of Chemistry, Kurukshetra University, Kurukshetra-132 119 

Manuscript received 26 April 1991, revised 24 October 1991, accepted 1 April 1992

The complete spectrum of proteolytic activity in the goat brain homogenate was determined by autolysis utilising only endogenous proteins as substrates. Three main peaks I, II and III of proteolytic activity were centred at pHs 3.5, 5.0 and 7.0, respectively. Inclusion of pepstatin, a specific inhibitor of cathepsin D, in assay mixtures shows that 70% of the activity in peak I is mainly due to this protease. The peak II consisted of thiol proteases like cathepsin B, L and BANA-­hydrolase. This was shown by activation of the proteases in this peak by dithioerythritol and EDTA (thiol activators). When leupeptin, a specific inhibitor of cathepsin B and L was used, only 60% of the activity in peak II was abolished indi­cating thereby the presence of leupeptin-insensitive thiol proteases in addition to the presence of cathepsin B and L. The peak III contains proteases acting at neutral and alkaline pHs. The subcellular fractionation of the brain homogenate into nuclear, mitochondrial-lysosomal and soluble fractions confirmed that most of the activity in peaks I, II and III can be attributed to lysosomal proteases.