PROTEIN CATABOLISM: III. Proteolytic Enzymes of Guinea Pig Cerebral Cortex and Synaptosomal Localization

Two proteinases of guinea pig cerebral cortex, one of pH optimum 3.4, the other of pH optimum 7.6, were purified 130- and 900-fold by centrifugation and Sephadex G-100 and G-200 gel chromatography. Both proteinases were assayed for utilizing ³H]acetyl hemoglobin as substrate. The purified pH 3.4 proteinase had two bands on sodium dodecyl sulfate polyacrylamide gel electrophoresis while the purified pH 7.6 proteinase gave three bands when stained for protein; none of the purified material stained for glycoprotein. The purified pH 3.4 proteinase had pI of 4.0 to 5.0 and a K_m of 1 ± 10^-4 M hemoglobin. This acid proteinase showed no cofactor requirements, was inhibited 51% by 0.1 M HgCl₂, and was relatively inactive against low molecular weight synthetic substrates. The purified pH 7.6 proteinase had pI of 6.7 to 7.4 and a K_m of 1 ± 10^-5 M. This neutral proteinase showed no cofactor requirements, was severely inhibited by HgCl₂ and diisopropylphosphofluoridate, and was also relatively inactive against low molecular weight synthetic substrates. On fractionation of the guinea pig cerebral cortex into ‘myelin’, ‘purified synaptosomes’ and ‘mitochondria’ fractions proteinase activity was found in each fraction. Highest activity of both pH 3.4 and pH 7.6 enzymes was in the ‘mitochondrial’ fraction. Fractionation of synaptosomes indicated highest pH 7.6 and pH 3.4 proteolytic activity in the fraction D ‘synaptic vesicle’ fraction. This activity was further purified on continuous sucrose gradients and separation of the pH 3.4 and pH 7.6 enzymes achieved. The purified pH 7.6 proteinase was found to be capable of releasing peptides and glycopeptides from isolated synaptosome surface electrokinetically in a manner similar to trypsin.