Purification of a calcium-activated neutral proteinase from bovine brain

Abstract

A calcium-activated neutral proteinase (CANP) resolved into three components has been partially purified from bovine brain. The method of isolation has resulted in 22,000, 7,100, and 8,000-fold purification for CANP I, II and III respectively. All three fractions require Ca2+ for activation. The characterization of the purified CANP I has shown that it is activated by 250 microM Ca2+ and the enzyme loses its activity when incubated in the presence of Ca2+ without substrate. Mg2+ is ineffective. The enzyme degrades neurofilament triplet proteins, tubulin and casein efficiently. The myelin basic protein is hydrolyzed after longer incubation. Bovine serum albumin and histones are unaffected. The enzyme is active at pH 5.5 to 9.0 with optimum between pH 7.5 and 8.5. It has a Km of 1.8 X 10(-7) M for the 69,000 dalton neurofilament protein. The enzyme is inhibited by sulphydryl blocking reagents and also by EGTA, leupeptin and E-64c. The SDS-PAGE analysis of the enzyme fractions has shown a major band at 66-68,000 daltons and two minor bands at 60,000 and 48-50,000 daltons for CANP I; a major band at 48-50,000 daltons and a minor band at 30-32,000 daltons for CANP II and a predominant doublet at 30-32,000 daltons with a minor band at 48-50,000 daltons for CANP III. The degradation of neurofilament proteins suggests that the CANP(s) may be involved in the turnover of these proteins.

Chronic intermittent ethanol induced axon and myelin degeneration is attenuated by calpain inhibition

“…Protein samples were resolved in 4–20 or 7.5 % (for SBDP) precast sodium dodecyl sulfate–polyacrylamide gel (Bio-Rad Laboratories, Hercules, CA) at 100 V for 1 h or 1 and 1/2 h respectively; transferred to the Immobilon™-polyvinylidene fluoride microporous membranes (Millipore). Membranes were incubated with 5 % non-fat milk in Tris-HCl buffer (20 mM Tris-HCl, pH 7.6, 0.1 % Tween-20), then incubated overnight at 4 °C with following primary IgG antibodies: rabbit polyclonal MBP and PLP, (1:500; raised and characterized in the lab; (Li and Banik, 1995), mouse monoclonal CNPase (1:500, Sigma), neurofilaments light and heavy (NFL and NFH; both 1:1000; Sigma), active calpain (1:500; raised and characterized in the lab; (Banik et al, 1983), calpastatin (1:250, Santa Cruz Biotechnology), α Fodrin (1:10,000, Enzo, Biomol). Next day, the membranes were washed 3 × 5 min in Tris-HCl buffer, and then incubated with horseradish peroxidase-conjugated corresponding secondary IgG antibodies (1:2000; MP Biomedicals) for 1 h. Immunoblots were developed with chemiluminescent reagent (ECL, Amersham) and imaged on Alpha-Innotech, equipped with FluorChem FC2 Imaging System (Cell Biosciences).…”

“…Tissue sections (5-10 μm) were fixed in 95% EtOH, rinsed 3 × 5 min in phosphate-buffered saline (PBS, containing 137 mM NaCl, 2.7 mM KCl, 11.9 mM phosphates, pH 7.4). For double staining, sections were blocked in PBS containing 2 % horse and goat serum for 1 h, next incubated with respective mouse monoclonal NFL and rabbit polyclonal NFH primary IgG antibodies (1:1000; Sigma) or mouse monoclonal NeuN and rabbit polyclonal active calpain (Banik et al, 1983; Samantaray et al, 2007) overnight at 4 °C. Next day, slices were rinsed in PBS and incubated with secondary horse anti-mouse IgG, DyLight™ 594 (red) or goat anti-rabbit, DyLight™ 488 (green) conjugated antibodies (Thermo Scientific) for 1 h. For single staining, mouse monoclonal SMI 311 (1:1000; Covance) antibody was used for detection of dephosphorylated neurofilament protein (deNFP).…”

Combination of all-trans retinoic acid and taxol regressed glioblastoma T98G xenografts in nude mice

“…The antibodies were purchased from either BD Biosciences (San Jose, CA, USA) or Santa Cruz Biotechnology (Santa Cruz, CA, USA). The rabbit polyclonal primary IgG antibody against calpain was raised in our laboratory [22]. The blots were incubated with a primary IgG antibody followed by incubation with an alkaline horseradish peroxidase (HRP)-conjuaged secondary IgG antibody (ICN Biomedicals, Aurora, OH, USA).…”

Administration of low dose estrogen attenuates gliosis and protects neurons in acute spinal cord injury in rats

“…Western blotting was carried out according to Samantaray et al (Samantaray et al 2007). Calpain was detected with rabbit polyclonal antibody [1: 500 dilution; (Banik et al 1983, Samantaray et al 2007)]. Spectrin breakdown products (SBDP), caspase-3, Bax, Bcl 2 , ER-α, ER-β and VEGF were detected (antibody specifications in Table S1).…”