Subcellular compartmentalization of calcium‐dependent and calcium‐independent neutral proteases in brain

In the present experiments, we studied the subcellular distribution of three types of extralysosomal, neutral proteolytic activities in rat telencephalon: (1) nonthiol proteases (NTP), (2) thiol proteases (TP), and (3) calcium‐activated thiol proteases (calpains I and II). Subcellular fractionation was performed by using conventional differential and sucrose‐gradient centrifugation techniques. The only significant proteolytic activity detected in crude homogenates could be assigned to calpain II, the high‐threshold calcium‐activated protease. Within the primary fractions prepared from the homogenates, the highest levels of calpain II were found in S 3 , or the soluble cytoplasmic fraction. Significant activity of the enzyme was also present in P 2 , the crude mitochondrial/synaptosomal fraction. In contrast, the specific activity of calpain I was greatest in P 2 with somewhat lesser enzymatic activity in P 1 and S 3 . Most of the calpain I in P 2 was recovered after differential centrifugation through sucrose gradients and lysis of the resultant subfractions. In marked contrast, only a small percentage of the calpain II activity was recovered in the gradient bands. In all, calpain II appears to be predominantly localized in the soluble cytoplasmic compartment while the greatest concentrations of calpain I are found in the soluble components of small glial and neuronal processes (pinched off during homogenization) that constitute the P 2 fraction. The highest specific activity of the calcium‐independent proteases was obtained in P 3 , a fraction essentially devoid of calpain, with a secondary peak in P 2 . Subfractionation of P 2 revealed that calcium‐independent TP in P 2 was associated with mitochondria while the calcium‐independent NTP was more uniformly distributed across myelin, synaptosomes, and mitochondria. In all cases, calcium‐independent neutral proteases proved to be associated with membranes to a far greater degree than the calpains. These results indicate that neutral proteases in brain are compartmentalized to a surprising degree and therefore are likely to be operating at different stages or aspects of extralysosomal protein degradation. The concentration of calpain I in the P 2 fraction and synaptosomal subfractions provides a plausible explanation for the occurrence of partially digested structural proteins obtained in highly purified postsynaptic densities isolated from cerebral cortex.