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

Abstract

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 S3, or the soluble cytoplasmic fraction. Significant activity of the enzyme was also present in P2, the crude mitochondrial/synaptosomal fraction. In contrast, the specific activity of calpain I was greatest in P2 with somewhat lesser enzymatic activity in P1 and S3. Most of the calpain I in P2 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 P2 fraction. The highest specific activity of the calcium-independent proteases was obtained in P3, a fraction essentially devoid of calpain, with a secondary peak in P2. Subfractionation of P2 revealed that calcium-independent TP in P2 was associated with mitochondria while the calcium-independent NTP was more uniformly distributed across myelin, synaptosomes, and mitochondria.(ABSTRACT TRUNCATED AT 250 WORDS)

Subcellular Distribution of Calpain and Calpastatin Immunoreactivity and Fodrin Proteolysis in Rabbit Hippocampus After Hypoxia and Glucocorticoid Treatment

“…In contrast, Chakrabarti et al (1988) found >60% of rat CALPAINS IN HYPOXIC HIPPOCAMPUS 1073 brain caseinolytic activity in the primary particulate fractions . In a similar manner, Baudry et al . (1987) found -40% of m-calpain and 90% of p-calpain activity in the primary particulate fractions, but further analysis by these investigators indicated that virtually all of the activity in the particulate fractions was actually soluble .…”

N Terminus of Calpain 1 Is a Mitochondrial Targeting Sequence

“…Ca 2ϩ -induced calpain activity in liver mitochondria was previously observed, but the calpains were not identified (20,47). In subcellular fractionation studies of rat brain, m-calpain activity was greatest in the soluble cytoplasmic fraction, while -calpain activity was greatest in the crude mitochondrial P2 fraction (48,49). We previously localized -calpain to mitochondria from rat brain and SH-SY5Y neuroblastoma cells, but the submitochondrial localization was not determined (21).…”

Calpain expression varies among different rat and bovine central nervous system regions

“…Immunohistochemical studies have observed mcalpain predominantly in glial cells, with µcalpain more prevalent in neuronal soma and processes (Siman et al, 1985;Hamakubo et al, 1986;Onizuka et al, 1995). At the subcellular level, mcalpain in the CNS is found in various cellular organelles as well as myelin membranes, synaptic plasma membranes, and cytosol (Burgoyne and Cumming, 1983;Siman et al, 1985;Baudry et al, 1987;Kolehmainen and Kaisto, 1989;Banik et al, 1992;Banay-Schwartz et al, 1994;. Further, systematic studies of the regional distribution of calpain in bovine and human brain have revealed mcalpain activity present primarily in areas rich in white matter (Chakrabarti et al, 1989;Banay-Schwartz et al, 1994).…”