Immunoassay and Activity of Calcium‐Activated Neutral Proteinase (mCANP): Distribution in Soluble and Membrane‐Associated Fractions in Human and Mouse Brain

Abstract

The millimolar form of calcium-activated neutral proteinase (mCANP) is generally regarded as a cytosolic enzyme in nonneuronal systems, although its subcellular localization in brain is less well established. To resolve conflicting reports on the localization of mCANP based on activity measurements, we developed an immunoassay for CANP and compared the content and activity of the molecule in soluble and membrane fractions of mouse and human brain. Western blot immunoassays, using two different antibodies specific for mCANP, demonstrated that mCANP content is 4.5 ng/g in human or mouse brain, about 0.0005% of the total protein. More than 95% of the total immunoreactive mCANP remained in the soluble fraction after 15,000 g centrifugation of the whole homogenate. mCANP activity was determined with [14C]azocasein as substrate after removing endogenous CANP inhibitor(s) by ion-exchange chromatography on DEAE-cellulose. Caseinolytic activity was detected only in fractions derived from the supernatant extract. The distribution of mCANP content and enzyme activity were unchanged when tissues were extracted with different concentrations of Triton X-100. These findings establish the usefulness and validity of the CANP immunoassay and demonstrate that mCANP in mouse and human brain is localized predominantly within the cytosol.

Specificity of calcium-activated neutral proteinase (CANP) inhibitors for human ?CANP and mCANP

“…In the case of mCANP, halfmaximal and maximal activities were observed at calcium concentrations of 100 IxM and 1 mM (Fig. 1), consistent with previous results (23), Enzyme activities were assayed using [14C]azocasein as substrate (21,23). The test compounds were preincubated with 12.5 units of CANP at 0~ for 30 min.…”

Widespread activation of calcium-activated neutral proteinase (calpain) in the brain in Alzheimer disease: a potential molecular basis for neuronal degeneration.

“…Human erythrocyte ,pCANP was purified from blood of normal adults by anion-exchange and phenyl-Sepharose chromatographies (31,32). Enzyme activity was determined by using [14C]azocasein as substrate at a final free calcium concentration of 125 AM (33). Human spectrin was isolated from fresh human erythrocyte ghost membranes by the low ionic strength buffer extraction method (34) and purified by chromatography on Sepharose 4B (35).…”

“…The 15,000 x g supernatant was applied to a column (1.0 x 15 cm) of DEAEcellulose (DE-52; Whatman) that was developed with stepwise increments of KCI from 0 to 3 mM. ,uCANP, eluted at 0.15 M KCI, was subjected to SDS/PAGE (7.5% acrylamide) and immunoblot analysis using a mouse monoclonal antibody to human ,uCANP (25,26) as described (33). In some experiments, homogenates of prefrontal cortex were centrifuged at 15,000 x g for 30 min at 4°C to obtain cytosolic (supernatant) fractions and membrane (pellet) fractions before immunoblot analysis.…”

“…The weight of ,lCANP on the immunoblot was calculated from standard curves prepared by measuring the immunostaining intensity of various known amounts of human erythrocyte ,gCANP on immunoblots (33). ,uCANP standards were analyzed on every immunoblot that contained brain samples to control for possible minor immunostaining variations.…”

Differential PARP Cleavage: An Indication of Heterogeneous Forms of Cell Death and Involvement of Multiple Proteases in the Infarct of Focal Cerebral Ischemia in Rat

“…Calpain activity was measured using azocasein (SigmaAldrich) as substrate (Takeuchi et al 1992;Shukla et al 2006) at a final concentration 0.6% in 1.0 ml of reaction mixture containing 0.02% b-mercaptoethanol, 100 mM Tris-acetate buffer (pH 7.5), 10 mM KCl, 5 mM CaCl 2 , and 50 lg of enzyme solution. The control tubes were treated the same way except CaCl 2 was replaced by EGTA (5 mM).…”