Polyclonal antisera specific for the proenzyme form of each calpain

Croall, Dorothy E.; Slaughter, Clive A.; Wortham, Helen S.; Skelly, Colleen M.; DeOgny, L.; Moomaw, Carolyn R.

doi:10.1016/0167-4838(92)90335-b

Cited by 27 publications

(23 citation statements)

References 21 publications

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“…Our observation does not necessarily contradict the data presented by Brown and Crawford [8] and by Croall et al [12] showing that autolysis of m-calpain large subunit proceeded very slowly in the time span 2-10 min. Because no exogenous substrates were added in the reaction mixture in which calpain was subjected to activation in their experiments, it is likely that the 'autolysis' of the large subunit was caused by intermolecular reactions between activated calpains.…”

Section: Discussioncontrasting

confidence: 56%

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Distinct kinetics of subunit autolysis in mammalian m‐calpain activation

1994

FEBS Letters

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Subunit autolysis of mammalian m-calpain upon activation was examined in kinetic terms using a set of antibodies recognizing different portions of the protease. Activation of m-calpain by calcium resulted in no apparent autolysis in the large catalytic subunit, whereas the small regulatory subtmit underwent immediate autolysis followed by substrate proteolysis. This profile of subunit autolysis is distinct from that of the other ubiquitous isozyme, g-calpain, in which autolysis of the large subunit and then of the small subunit precedes substrate proteolysis under the normal conditions. The activation state of m-calpain thus is not reflected by the large subunit autolysis. The mode and role of autolysis may vary among calpain isozymes.

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Section: Discussioncontrasting

confidence: 56%

“…Because the large subunit autolysis of m-calpain can not be resolved by electrophoresis, it has been only poorly analyzed in kinetic terms [7,8,12]. In contrast, autolysis of/t-calpain is electrophoretically detectable and thus has been much better characterized [9,13].…”

Section: Introductionmentioning

confidence: 99%

Distinct kinetics of subunit autolysis in mammalian m‐calpain activation

1994

FEBS Letters

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“…Croall et al [18] have raised antibodies specific for unautolysed m-and pcalpain. Saido et al [19] have raised antibodies against unautolysed and autolysed forms ofp-calpain.…”

Section: Discussionmentioning

confidence: 99%

Structural modifications associated with the change in Ca²⁺ sensitivity on activation of m‐calpain

Brown

Crawford

1993

FEBS Letters

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“…Immunoblot analysis of cytoplasmic extracts was performed after SDS-PAGE and transfer to 0.2 m nitrocellulose filters. Blots were blocked (5% nonfat dry milk, 50 mM Tris-HCl, 50 mM NaCl, 1 mM EDTA and 1 mM dithiothreitol) overnight and probed with primary antibodies, including monoclonal murine anti-human calpastatin PC-6 (17) (Research Diagnostics Inc., Flanders, NJ), rabbit anti-human m-calpain (D. E. Croall, University of Maine) (18), monoclonal murine anti-human ezrin-specific antibody 3C12 (Sigma), which reacted with a single 80-kDa protein in Caco 2 cell extracts, rabbit polyclonal ezrin-specific antibody 7-130 (Upstate Biotechnology, Lake Placid, NY), rabbit anti-human ␤-actin (5), and monoclonal murine anti-human villin (Chemicon International Inc., Temecula, CA). Secondary antisera were horseradish peroxidase-coupled goat anti-mouse IgG (Bio-Rad) or goat anti-rabbit IgG (Bio-Rad).…”

Section: Methodsmentioning

confidence: 99%

Calpain Regulates Enterocyte Brush Border Actin Assembly and Pathogenic Escherichia coli-mediated Effacement

Potter

Srirangam

Fiacco

et al. 2003

Journal of Biological Chemistry

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This study identifies calpain as being instrumental for brush border (BB) microvillus assembly during differentiation and effacement during bacterial pathogenesis. Calpain activity is decreased by 25-80% in Caco 2 lines stably overexpressing calpastatin, the physiological inhibitor of calpain, and the effect is proportional to the calpastatin/calpain ratio. These lines exhibit a 2.5-fold reduction in the rate of microvillus extension. Apical microvillus assembly is reduced by up to 50%, as measured by quantitative fluorometric microscopy (QFM) of ezrin, indicating that calpain recruits ezrin to BB microvilli. Calpain inhibitors ZLLYCHN 2 , MDL 28170, and PD 150606 block BB assembly and ezrin recruitment to the BB. The HIV protease inhibitor ritonavir, which inhibits calpain at clinically relevant concentrations, also blocks BB assembly, whereas cathepsin and proteasome inhibitors do not. Microvillus effacement is inhibited after exposure of calpastatin-overexpressing cells to enteropathogenic Escherichia coli. These results suggest that calpain regulates BB assembly as well as pathological effacement, and indicate that it is an important regulator involved in HIV protease inhibitor toxicity and host-microbial pathogen interactions.Although it is accepted that actin-associated cross-linking and membrane linker proteins such as villin and ezrin, found in intestinal microvilli, are Ca 2ϩ -sensitive, the role that Ca 2ϩ plays in the assembly and stability of microvilli is undetermined. Interest in Ca 2ϩ as a regulator of microvillus remodeling has been focused on its role in disrupting villin crosslinks of the microvillus actin filaments (reviewed in Ref. 1) and the activation of the actin filament severing activity of villin. It has been suggested that the Ca 2ϩ -dependent protease, calpain, cleaves the membrane linker protein ezrin, during cell motility-associated remodeling of cortical ␤-actincontaining structures (2-4). Because ␤-actin is the predominant actin isoform of microvilli (5) and because ezrin, which is abundant in microvilli, associates with ␤-actin in an isoform-specific and calpain-sensitive fashion (2), the question has arisen whether calpain regulates ␤-actin-ezrin interactions in microvilli, and microvillus assembly. Additionally, the finding that calpain levels exceed calpastatin levels in intestinal epithelial cells (6) suggests that calpain could play a role in intestinal differentiation.Calpain has been implicated in cytoskeletal remodeling, including disruption of cell-matrix interactions at the rear of the cell during crawling (7) and lamellipodial and protrusion formation during spreading (4). These examples illustrate the role of calpain in remodeling dynamic actin filament structures at the periphery of the cell. Calpain has not been implicated in assembly of filamentous actin structures in the BB apical domain of enteric epithelial cells or membrane recruitment of ezrin, where it may play an important role in assembly of actin filaments (2,8).A recently developed approach to study the r...

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Polyclonal antisera specific for the proenzyme form of each calpain

Cited by 27 publications

References 21 publications

Distinct kinetics of subunit autolysis in mammalian m‐calpain activation

Distinct kinetics of subunit autolysis in mammalian m‐calpain activation

Structural modifications associated with the change in Ca²⁺ sensitivity on activation of m‐calpain

Calpain Regulates Enterocyte Brush Border Actin Assembly and Pathogenic Escherichia coli-mediated Effacement

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Polyclonal antisera specific for the proenzyme form of each calpain

Cited by 27 publications

References 21 publications

Distinct kinetics of subunit autolysis in mammalian m‐calpain activation

Distinct kinetics of subunit autolysis in mammalian m‐calpain activation

Structural modifications associated with the change in Ca2+ sensitivity on activation of m‐calpain

Calpain Regulates Enterocyte Brush Border Actin Assembly and Pathogenic Escherichia coli-mediated Effacement

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Structural modifications associated with the change in Ca²⁺ sensitivity on activation of m‐calpain