Calpains belong to a family of calcium-dependent thiol-proteases that proteolyze a wide variety of cytoskeletal, membrane-associated, and regulatory proteins. Fifteen gene products of the calpain family are reported in mammals, which are classified as nine typical and six atypical calpains. Typical calpains are characterized by a C-terminal Ca2+-binding domain that includes EF-hand motifs, while atypical calpains lack this region, but often contain additional domains. Calpains do not generally function as destructive proteases, but act as calcium-dependent modulators that remove limited portions of protein substrates. Calpains respond to Ca2+ signals by cleaving specific proteins, frequently components of signaling cascades, thereby irreversibly modifying their function. Two major isoforms of calpain are reported in mammals, calpain 1 (m-form) and calpain 2 (m-form). They are constitutively expressed in all tissues and differ in their calcium requirement for activation (~50 mM for calpain 1 and ~500 mM for calpain 2) and contain several calcium-binding sites, which allosterically affect the enzyme activity.



Calpain 1 and 2 exhibit about 55 to 65% sequence homology and are composed of an 80 kDa and a 30 kDa subunit. The 80 kDa subunit has the catalytic site and is unique to each isozyme, whereas the 30 kDa unit is the regulatory subunit and is common to both m- and m-isozymes. The 80 kDa subunit consists of four domains (I - IV) and the 30 kDa unit has 2 domains (V and VI). Domain I is partially removed during autolysis. Domain II is the protease domain, which is structurally similar to the catalytic domain of other cysteine proteases. It contains a Cys, His, Asn triad characteristic of cysteine proteases. Domain III exhibits a homology with typical calmodulin binding proteins and interacts with calcium binding domains (IV and VI) and frees the domain II for protease activity. Domain IV is a Ca2+-binding domain structurally similar to calmodulin. Domain V contains a hydrophobic region and is essential for calpain interaction with membranes. Domain VI has five EF-hands. There are two EF-hand pairs, one pair (EF1-EF2) displays an 'open' conformation and the other (EF3-EF4) a 'closed' conformation. The fifth EF-hand (EF5) is in a 'closed' conformation. Both, calpain 1 and 2 associate non-covalently with domain V and domain VI.

Several putative substrates of calpain 1 and 2 are known that are cleaved by both isoforms, but with different efficiencies. Recently, determinants for calpain 1 and 2 have been analyzed and it is shown that amino acid preferences extend over 11 residues around the scissile bond. Calpains prefer Leu, Thr, Val in P2 position and Lys, Tyr, Arg in P1 position and proline in the region flanking the P2 - P’1 segment.

Calpain 3, another typical calpain was first described as a skeletal muscle-specific calpain isoform. However, subsequent studies have shown its presence in several other tissues. It is a 94 kDa enzyme that contains 821 amino acids. Structurally calpain 3 is similar to calpains 1 and 2, however, it has an additional N-terminal sequence of 20 -30 amino acids (NS). Also, its domain I exhibits less than 20% homology to calpains 1 and 2. Calpain 3 includes two characteristic amino acid stretches: one between the catalytic cysteine and histidine (IS1) and the other (IS2) upstream of the first EF hand motif of calcium-binding domain IV. A characteristic feature of calpain 3 is that it is not inhibited by calpastatin. While conventional calpains localize mainly in the cytosol or at the inner face of the plasma membrane, calpain 3 is detected within the nuclei of skeletal muscle cells and in the N2 region of myofibrils. More recently, attention has been focused on the pathological significance of calcium accumulation in the central nervous system following cerebral ischemia and traumatic brain injury. Over-activation of NMDA, kainate and AMPA receptors in the brain leads to sustained influx of Ca2+ through the voltage-gated calcium channels. Disturbances in calcium homeostasis result in the activation of several calcium-dependent enzymes including calpains. Over-expression of calpains has been positively linked to both acute and chronic neurodegenerative processes including ischemia, trauma, and Alzheimer’s disease. In Alzheimer’s disease the ratio of active (76 kDa) to inactive (80 kDa) calpain 1 is reported to be much higher. Calpain proteolysis is usually the late-stage common pathway towards cell death induced by excitotoxic compounds; hence, a selective inhibition of calpains to limit neuronal damage appears to be a viable therapeutic measure. However, most of the inhibitors reported are active-site targeted peptides and their limited cell permeability poses problems. Calbiochem’s PD 150606 (Cat. No. 513022), and Calpain Inhibitor XII (Cat. No. 208744) a new cell-permeable inhibitor for calpains may serve as useful tools for these studies.

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Nakajima, T., et al. 2001. Biochim. Biophys. Acta 1519, 55.
Kinbara, K., et al. 1998. Biochem. Pharmacol. 56, 415.
Kampfl, A., et al. 1997. J. Neurotrauma 14, 121.
Kinbara, K., et al. 1997. Arch. Biochem. Biophys. 342, 99
Johnson, G.V.W., and Guttmann, R.P. 1997. BioEssays 19, 1011.
Sorimachi, H., et al., 1997. Biochem. J. 328, 721.
Bartus, R.T., et al. 1995. Neurol. Res. 17, 249.
Wang, K.K.W., and Yuen, P-W. 1994. Trends Pharmacol. Sci. 15, 412.
Saito, K., et al. 1993. Proc. Natl. Acad. Sci. USA 90, 2628.
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Melloni, E., and Pontremoli, S. 1989. Trends Neurosci. 12, 438.

Mammalian Calpain Family

Calpain Pseudonyms Tissue Type Species
Calpain 1
CAPN1, m-calpain
Human, Mouse, Rat
Calpain 2
CAPN2, m-calpain
Human, Mouse, Rat
Calpain 3
CAPN3, nCL-1, p94
Skeletal muscle, lens, retina
Human, Mouse, Rat
Calpain 5
htra3, nCL-3
Ubiquitous (high in colon, small intestine and testis)
Human, Mouse, Rat
Calpain 6
CAPNX, Calpamodulin
Human, Mouse, Rat
Calpain 7
Human, Mouse, Rat
Calpain 8
Stomach mucosa
Human, Mouse, Rat
Calpain 9
Digestive track
Human, Mouse, Rat
Calpain 10
Human, Mouse, Rat
Calpain 11
Human, Mouse, Rat
Calpain 12
Ubiquitous (High in hair follicle)
Human, Mouse, Rat
Calpain 13
Calpain 14
Calpain 15
Sol H
Human, Mouse, Rat
Calpain Small Subunit 1
Human, Mouse, Rat
Calpain Small Subunit 2
Human, Mouse, Rat


Product Application Species Reactivity Cat. No.
Anti-Calpain-1/2, Small Subunit Mouse mAb (28F3) ELISA (1:2000)
Immunoblotting (1:2000)
Immunoprecipitation (1:500) 
bovine, human, not mouse, not rat 208730
Anti-Calpain-1, C-Terminal Rabbit pAb Immunoblotting (1:1000, colorimetric; 1:5000, chemiluminescence)  human, mouse, rat 208753
Anti-Calpain-1, Domain III Mouse mAb (9A4H8D3) Immunoblotting (1:2000)
Immunocytochemistry (1:100) 
bovine, human, porcine, rat 208728
Anti-Calpain-1, Domain II Mouse mAb (2H2A7C2) Immunoblotting (1:1000)
Immunocytochemistry (1:25) 
bovine, human, rat 208727
Anti-Calpain-1, Domain I Rabbit pAb Immunoblotting (1:1000, colorimetric; 1:5000, chemiluminescence; see application references)  human, mouse, rat 208751
Anti-Calpain-1, Large Subunit Rabbit pAb Immunoblotting (1:5000, colorimetric)  human 208732
Anti-Calpain-2 Domain III Rabbit pAb Immunoblotting (1:5000, chemiluminescence; 1:1000, colorimetric) Immunoprecipitation (see comments) ELISA (see comments) Immunohistochemistry (see comments)  rat 208737
Anti-Calpain-2, Domain III Rabbit pAb Immunoblotting (1:1000, colorimetric; 1:5000, chemiluminescence)  human, mouse, rat 208755
Anti-Calpain-2, Domain I Rabbit pAb Immunoblotting (1:1000, colorimetric; 1:5000, chemiluminescence)  human, mouse, rat 208754
Anti-Calpain-2, Domain IV Rabbit pAb Immunoblotting (1:1000, colorimetric; 1:5000, chemiluminescence)  human, mouse, rat 208756
Anti-Calpain-2, Domains III/IV Mouse mAb (107-82) Immunoblotting (1:500)
Immunocytochemistry (1:50) 
bovine, human, porcine, rat 208729
Anti-Calpain-3, Domain III (Insert #2) Rabbit pAb Immunoblotting (1:1000, colorimetric; 1:5000, chemiluminescence)  human 208759
Anti-Calpain-3, Domain I Rabbit pAb Immunoblotting (1:1000, colorimetric; 1:5000, chemiluminescence)  human, rat 208757


Product Cat. No.
ALLM - CAS 110115-07-6 - Calbiochem 208721
ALLN - CAS 110044-82-1 - Calbiochem 208719
Calpain Inhibitor III - CAS 88191-84-8 - Calbiochem 208722
Calpain Inhibitor IV - Calbiochem 208724
Calpain Inhibitor Set - Calbiochem 208733
Calpain Inhibitor V - Calbiochem 208726
Calpain Inhibitor VI - CAS 190274-53-4 - Calbiochem 208745
Calpain Inhibitor X 208742
Calpain Inhibitor XI - CAS 145731-49-3 - Calbiochem 208743
Calpain Inhibitor XII 208744
Calpastatin, Human Erythrocytes 208901
Calpastatin, Human, Recombinant, Domain I - Calbiochem 208900
Calpastatin Peptide - Calbiochem 208902
Calpastatin Peptide, Negative Control - Calbiochem 208904
Calpeptin 03-34-0051
EST - CAS 88321-09-9 - Calbiochem 330005
InSolution™ ALLN - CAS 110044-82-1 - Calbiochem 208750
PD 145305 513021
PD 150606 513022
PD 151746 513024

Kits and Tools

Product Format Form Sensitivity Assay Range Assay Time Sample Type Cat. No.
Calpain Activity Assay Kit, Fluorogenic 96-well plate 96 Tests     1.5 h Cell lysates, plasma, serum QIA120
InnoZyme™ Calpain 1/2 Activity Assay Kit 96-well plate 96 Tests   63-1000 ng/ml as measured with purified human calpain 1.   Cell lysates, tissues extracts CBA054


Product Cat. No.
Calpain-1, Human Erythrocytes 208713
Calpain-1, Porcine Erythrocytes 208712
Calpain-2, Porcine Kidney 208715
Calpain-2, Rat, Recombinant, High Purity, E. coli 208718


Product Cat. No.
Calpain-1 Substrate, Fluorogenic - Calbiochem 208748
Calpain-1 Substrate II, Fluorogenic - Calbiochem 208772
Calpain Substrate II, Fluorogenic - CAS 94367-20-1 - Calbiochem 208731
Calpain Substrate III, Fluorogenic - Calbiochem 208771

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