Calcium-activated neutral protease (CANP) in brain and other tissues

Zimmerman, Un‐Jin P.; Schlaepfer, William W.

doi:10.1016/0301-0082(84)90012-1

Cited by 88 publications

(32 citation statements)

References 95 publications

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“…The calcium-activated neutral cysteine proteinase calpain (CANP) 1 is a heterodimer of a catalytic 80-kDa subunit and a regulatory 30-kDa subunit (1)(2)(3). The physiological role of CANP is not fully established, but its participation in events such as cell division, signal transduction, and long term potentiation has been suggested (4 -7).…”

Section: ؉mentioning

confidence: 99%

Functional Properties of Recombinant Calpain I and of Mutants Lacking Domains III and IV of the Catalytic Subunit

Vilei

Calderara

Anagli

et al. 1997

Journal of Biological Chemistry

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The catalytic subunit (L-CANP) of human calpain I (CANP, the high Ca 2؉ affinity form) and two of its mutants were expressed in Escherichia coli or using the baculovirus Sf9 system. The mutants lacked domain III (L-CANP⌬3) and the calmodulin-like domain IV (L-CANP⌬4), respectively. The bacterially expressed proteins were solubilized from the inclusion bodies and refolded with polyethylene glycol. In Sf9 cells, co-expression of the inhibitor calpastatin was necessary to prevent autolysis of L-CANP, whereas co-expression of the regulatory subunit enhanced it. Only very low levels of mRNA of the truncated form L-CANP⌬4 were found in bacmid-transfected Sf9 cells, and it proved impossible to isolate this mutant using the baculovirus expression system. While the apparent K m (Ca 2؉ ) of freshly isolated human erythrocyte CANP was about 60 M, the recombinant monomeric forms L-CANP and L-CANP⌬3 required 65-215 and 400 -530 M Ca 2؉ , respectively. Bacterially expressed L-CANP⌬4 was Ca 2؉ -independent; the presence of inhibitors during its renaturation was necessary to prevent its autolysis. A chimeric form (LmCANP) composed by domains I-III of CANP and domain IV of calpain II (mCANP, the low Ca 2؉ affinity form) was also expressed in Sf9 cells. This mutant required less Ca 2؉ (about 50 M) than native erythrocyte calpain for half-maximal activity and had the highest specific activity of all calpains tested. Domain III proved unnecessary for the activity of the recombinant catalytic subunit, but its absence raised the K m (Ca 2؉) and removed its inactivation at high Ca 2؉ concentrations. All recombinant proteins were active as monomers in polyethylene glycol-containing buffers; the in vitro association with the regulatory subunit enhanced only slightly the V max and the Ca 2؉ dependence of the expressed proteins. Activation by Ca 2؉ promoted the separation of the two subunits of the expressed recombinant proteins.

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Section: ؉mentioning

confidence: 99%

Functional Properties of Recombinant Calpain I and of Mutants Lacking Domains III and IV of the Catalytic Subunit

Vilei

Calderara

Anagli

et al. 1997

Journal of Biological Chemistry

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“…One consequence of increasing intracellular Ca2~levels is the sustained activation of many Ca2-dependent enzymes and proteins, including members of the calpain family (calpain I and calpain II) of neutral proteases (Zimmerman and Schlaepfer, 1984;Suzuki et al, 1987;Saido et al, 1994). The substrates of the calpains include the cytoskeletal proteins microtubule-associated protein 2 (MAP2), spectrin, and neurofilaments (Schlaepfer and Zimmerman, 1985;Siman and Noszek, 1988;Johnson et al, 1991).…”

mentioning

confidence: 99%

Rapid Calpain I Activation and Cytoskeletal Protein Degradation Following Traumatic Spinal Cord Injury: Attenuation with Riluzole Pretreatment

Springer

Azbill

Kennedy

et al. 1997

Journal of Neurochemistry

129

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Abstract:Immunocytochemical and immunoblotting techniques were used to investigate calpain I activation and the stability of the calpain-sensitive cytoskeletal proteins microtubule-associated protein 2 (MAP2) and spectrin at 1, 4, and 24 h after contusion injury to the spinal cord. Spinal cord injury resulted in the activation of calpain I at all time points examined, with the highest level of activation occurring at 1 h. At the same early time point, there was a loss of dendritic MAP2 staining in spinal cord sections, accompanied by pronounced perikaryal accumulation. The loss in MAP2 staining in the injured spinal cord progressed over the 24-h survival period to affect regions 3 mm distant to the site of injury. The presence of calpain I-specific spectrin degradation was apparent in neuronal cell bodies and fibers as early as 1 h after injury, with the most intense staining occurring within and juxtaposed to the injury site. Spectrin breakdown products in neuronal cell bodies declined rapidly at 4 h and were nearly undetectable at 24 h after injury. Immunoblot studies confirmed the immunocytochemical results by demonstrating a significant increase in calpain I activation, a significant decrease in MAP2 levels, and a significant increase in spectrin breakdown. Finally, treatment of animals with riluzole, an inhibitor of glutamate release, before surgery reduced significantly the loss of MAP2 levels observed at 24 h after injury. These results demonstrate that Ca 2~-dependentprotease activation and degradation of critical cytoskeletal proteins are early events after spinal cord injury and that treatments that minimize the actions of glutamate may limit their breakdown. Key Words: Microtubule-associated protein 2-Spectrin -Cytoskeleton -Glutamate-Calcium -Calpain l-RiluzoIe-CNS injury.

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“…of the sealing process. Calpain and other proteases degrade cytoskeletal proteins such as neurofilaments (4,6,7,8), which help maintain axonal diameter and shape (9). Such degradation might produce a complete or partial collapse of the cut end (10, 11), thereby inducing sealing by enhancing the interactions between vesicles that form a seal by themselves and͞or that form a continuous membrane at the cut end.…”

mentioning

confidence: 99%

Calpain activity promotes the sealing of severed giant axons

Godell

Smyers

Eddleman

et al. 1997

Proc. Natl. Acad. Sci. U.S.A.

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A barrier (seal) must form at the cut ends of a severed axon if a neuron is to survive and eventually regenerate. Following severance of crayfish medial giant axons in physiological saline, vesicles accumulate at the cut end and form a barrier (seal) to ion and dye diffusion. In contrast, squid giant axons do not seal, even though injuryinduced vesicles form after axonal transection and accumulate at cut axonal ends. Neither axon seals in Ca 2؉ -free salines. The addition of calpain to the bath saline induces the sealing of squid giant axons, whereas the addition of inhibitors of calpain activity inhibits the sealing of crayfish medial giant axons. These complementary effects involving calpain in two different axons suggest that endogenous calpain activity promotes plasmalemmal repair by vesicles or other membranes which form a plug or a continuous membrane barrier to seal cut axonal ends.The rapid (within 60 min) repair of unmyelinated crayfish medial giant axons (MGAs) (1) and myelinated earthworm MGAs (2) is associated with the accumulation of vesicles and other membranous material at the cut axonal ends. In contrast, squid giant axons (GAs) do not seal, even though injuryinduced vesicles form after transection and accumulate at cut axonal ends (3). Calpain (4) is a calcium-activated neutral protease endogenous to squid GAs (5), other axons (4), and other cell types (4, 6) that could enhance or inhibit one or more stages (vesicle formation, movement, aggregation, membrane fusion, etc.) of the sealing process. Calpain and other proteases degrade cytoskeletal proteins such as neurofilaments (4,6,7,8), which help maintain axonal diameter and shape (9). Such degradation might produce a complete or partial collapse of the cut end (10, 11), thereby inducing sealing by enhancing the interactions between vesicles that form a seal by themselves and͞or that form a continuous membrane at the cut end. (In this paper, the term ''complete constriction or complete collapse'' is used to describe 100% closure of a cut axonal end; the term ''partial constriction or partial collapse'' is used to describe any constriction that leaves an opening at the cut end.) Alternatively, calpain or other proteases might inhibit sealing by repressing the formation of vesicles or a continuous membrane or by degrading cytoskeletal proteins (microtubules, actin, molecular motors, etc.), which might affect the movement of injury-induced or other vesicles to the cut axonal end.We examined the effects of calpain and other proteases on the sealing of two giant axons: the crayfish MGA, which normally seals after severance in physiological saline (see preceding paper, ref. 1), and the squid GA, which normally does not seal after severance in physiological saline (2). [GAs (2) and MGAs (1) transected in Ca 2ϩ -free salines do not seal.] According to our electrophysiological measures (decay of injury current, I i , and resting membrane potential, V m ) and optical measures (dye exclusion), we find that exogenous calpain, but not chymotrypsin, i...

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Calcium-activated neutral protease (CANP) in brain and other tissues

Cited by 88 publications

References 95 publications

Functional Properties of Recombinant Calpain I and of Mutants Lacking Domains III and IV of the Catalytic Subunit

Functional Properties of Recombinant Calpain I and of Mutants Lacking Domains III and IV of the Catalytic Subunit

Rapid Calpain I Activation and Cytoskeletal Protein Degradation Following Traumatic Spinal Cord Injury: Attenuation with Riluzole Pretreatment

Calpain activity promotes the sealing of severed giant axons

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