Binding of calpain fragments to calpastatin

Their cDNA-derived amino acid sequences predict that the 80-kDa subunits of the micromolar and millimolar Ca(2+)-requiring forms of the Ca(2+)-dependent proteinase (mu- and m-calpain, respectively) each consist of four domains and that the 28-kDa subunit common to both mu- and m-calpain consists of two domains. The calpains were allowed to autolyze to completion, and the autolysis products were separated and were characterized by using gel permeation chromatography, calpastatin affinity chromatography, and sequence analysis. Three major fragments were obtained after autolysis of either calpain. The largest fragment (34 kDa for mu-calpain, 35 kDa for m-calpain) contains all of domain II, the catalytic domain, plus part of domain I of the 80-kDa subunit of mu- or m-calpain. This fragment does not bind to calpastatin, a competitive inhibitor of the calpains, and has no proteolytic activity in either the absence or presence of Ca2+. The second major fragment (21 kDa for mu-calpain and 22 kDa for m-calpain) contains domain IV, the calmodulin-like domain, plus approximately 50 amino acids from domain III of the 80-kDa subunit of mu- or m-calpain. The third major fragment (18 kDa) contains domain VI, the calmodulin-like domain of the 28-kDa subunit. The second and third major fragments bind to a calpastatin affinity column in the presence of Ca2+ and are eluted with EDTA. The second and third fragments are noncovalently bound, so the 80- and 28-kDa subunits of the intact calpain molecules are noncovalently bound at domains IV and VI. After separation in 1 M NaSCN, the 28-kDa subunit binds completely to calpastatin, approximately 30-40% of the 80-kDa subunit of mu-calpain binds to calpastatin, and the 80-kDa subunit of m-calpain does not bind to calpastatin in the presence of 1 mM Ca2+.     

Effects of autolysis on properties of mu- and m-calpain

Although the biochemical changes that occur during autolysis of mu- and m-calpain are well characterized, there have been few studies on properties of the autolyzed calpain molecules themselves. The present study shows that both autolyzed mu- and m-calpain lose 50-55% of their proteolytic activity within 5 min during incubation at pH 7.5 in 300 mM or higher salt and at a slower rate in 100 mM salt. This loss of activity is not reversed by dialysis for 18 h against a low-ionic-strength buffer at pH 7.5. Proteolytic activity of the unautolyzed calpains is not affected by incubation for 45 min at ionic strengths up to 1000 mM. Size-exclusion chromatography shows that ionic strengths of 100 mM or above cause dissociation of the two subunits of autolyzed calpains and that the dissociated large subunits (76- or 78-kDa) aggregate to form dimers and trimers, which are proteolytically inactive. Hence, instability of autolyzed calpains is due to aggregation of dissociated heavy chains. Autolysis removes the N-terminal 19 (m-calpain) or 27 (mu-calpain) amino acids from the large subunit and approximately 90 amino acids from the N-terminus of the small subunit. These regions form contacts between the two subunits in unautolyzed calpains, and their removal leaves only contacts between domain IV in the large subunit and domain VI in the small subunit. Although many of these contacts are hydrophobic in nature, ionic-strength-induced dissociation of the two subunits in the autolyzed calpains indicates that salt bridges have an important, possibly indirect, role in the domain IV/domain VI interaction.     

Autolysis of mu- and m-calpain from bovine skeletal muscle

The rate of autolysis of mu- and m-calpain from bovine skeletal muscle was measured by using densitometry of SDS polyacrylamide gels and determining the rate of disappearance of the 28 and 80 kDa subunits of the native, unautolyzed calpain molecules. Rate of autolysis of both the 28 and 80 kDa subunits of mu-calpain decreased when mu-calpain concentration decreased and when beta-casein, a good substrate for the calpains, was present. Hence, autolysis of both mu-calpain subunits is an intermolecular process at pH 7.5, 0 or 25.0 degrees C, and low ionic strength. The 78 kDa subunit formed in the first step of autolysis of m-calpain was not resolved from the 80 kDa subunit of the native, unautolyzed m-calpain by our densitometer, so autolysis of m-calpain was measured by determining rate of disappearance of the 28 kDa subunit and the 78/80 kDa complex. At Ca2+ concentrations of 1000 microM or higher, neither the m-calpain concentration nor the presence of beta-casein affected the rate of autolysis of m-calpain. Hence, m-calpain autolysis is intramolecular at Ca2+ concentrations of 1000 microM or higher and pH 7.5. At Ca2+ concentrations of 350 microM or less, the rate of m-calpain autolysis decreased with decreasing m-calpain concentration and in the presence of beta-casein. Thus, m-calpain autolysis is an intermolecular process at Ca2+ concentrations of 350 microM or less. If calpain autolysis is an intermolecular process, autolysis of a membrane-bound calpain would require selective participation of a second, cytosolic calpain, making it an inefficient process. By incubating the calpains at Ca2+ concentrations below those required for half-maximal activity, it is possible to show that unautolyzed calpains degrade a beta-casein substrate, proving that unautolyzed calpains are active proteases.     

Ca(2+)-induced structural changes in rat m-calpain revealed by partial proteolysis

Partial proteolysis by exogenous proteases in the presence and absence of Ca(2+) was used to map the protease-resistant domains in m-calpain, and to obtain evidence for the conformational changes induced in this thiol protease by Ca(2+). The complication of autoproteolysis was avoided by using the inactive Cys105Ser calpain mutant. Both trypsin and chymotrypsin produced similar cleavage patterns from the large subunit (domains I-IV), while the small subunit (domain VI) was largely unaffected. N-Terminal sequencing of the major products showed that hydrolysis occurred in the N-terminal anchor peptide, which binds domain I to domain VI, at a site close to the C terminus of domain II, and at several sites within domain III. Of particular importance to the overall Ca(2+)-induced conformational changes was the increase in mobility and accessibility of domain III. The same sites were cleaved in the presence and absence of Ca(2+), but with one exception digestion was much more rapid in the presence of Ca(2+). The exception was a site close to residue 255 located within the active site cleft. This site was accessible to cleavage in the absence of Ca(2+), when the active site is not assembled, but was protected in the presence of Ca(2+). This result supports the hypothesis that Ca(2+) induces movement of domains I and II closer together to form the functional active site of calpain.     

Crystal structure of calpain reveals the structural basis for Ca(2+)-dependent protease activity and a novel mode of enzyme activation

The combination of thiol protease activity and calmodulin-like EF-hands is a feature unique to the calpains. The regulatory mechanisms governing calpain activity are complex, and the nature of the Ca(2+)-induced switch between inactive and active forms has remained elusive in the absence of structural information. We describe here the 2.6 A crystal structure of m-calpain in the Ca(2+)-free form, which illustrates the structural basis for the inactivity of calpain in the absence of Ca(2+). It also reveals an unusual thiol protease fold, which is associated with Ca(2+)-binding domains through heterodimerization and a C(2)-like beta-sandwich domain. Strikingly, the structure shows that the catalytic triad is not assembled, indicating that Ca(2+)-binding must induce conformational changes that re-orient the protease domains to form a functional active site. The alpha-helical N-terminal anchor of the catalytic subunit does not occupy the active site but inhibits its assembly and regulates Ca(2+)-sensitivity through association with the regulatory subunit. This Ca(2+)-dependent activation mechanism is clearly distinct from those of classical proteases.     

Activation of calpain by Ca2+: roles of the large subunit N-terminal and domain III-IV linker peptides

The calpains are a family of cysteine proteases with closely related amino acid sequences, but a wide range of Ca(2+) requirements (K(d)). For m-calpain, K(d) is approximately 325microM, for mu-calpain it is approximately 50microM, and for calpain 3 it is not strictly known but may be approximately 0.1microM. On the basis of previous structure determination of m-calpain we postulated that two regions of the calpain large subunits, the N-terminal peptide (residues 1-20) and a domain III-IV linker peptide (residues 514-530 in m-calpain) were important in defining K(d). The mutations Lys10Thr in the N-terminal peptide, and Glu517Pro in the domain linker peptide, reduced K(d) of m-calpain by 30% and 42%, respectively, revealing that these two regions are functionally important. The increased Ca(2+)-sensitivity of these mutants demonstrate that the Lys10-Asp148 salt link and the short beta-sheet interaction involving Glu517 are factors contributing to the high K(d) of m-calpain. Though these two regions are physically remote from the active site and Ca(2+)-binding site, they play significant roles in regulating the response of calpain to Ca(2+). Differences in these interactions in mu-calpain and in calpain 3 are also consistent with their progressively lower K(d) values.     

Immunoaffinity purification of the calpains

A monoclonal antibody to the small subunit common to both mu- and m-calpains can be used in an immunoaffinity column to purify either mu- or m-calpain in a proteolytically active form. Extracts in 150 mM NaCl, pH 7.5, are loaded onto a column containing the anti-28-kDa antibody; the column is washed with 500 mM NaCl, pH 7.5, and the bound calpain is eluted with 150 mM NaCl, 50 mM Tris-HCl, pH 9.5, and 1 mM EDTA. These elution conditions do not affect the proteolytic activity of either mu- or m-calpain. It is most efficient to reduce the volume and to remove any proteolytic activity from crude extracts by using successive phenyl Sepharose and ion-exchange columns before loading onto the immunoaffinity column. The column purifies m-calpain more effectively than mu-calpain; m-calpain is greater than 90% pure after a single pass through this column, whereas mu-calpain can be purified to >70% purity. The epitope for the monoclonal antibody is between amino acids 92 and 104 (numbers for human calpain) in the 28-kDa subunit. Evidently, this area is shielded in the calpain molecule in a way that affects binding of the antibody to the native molecule.     

Effect of phosphatidylinositol and inside-out erythrocyte vesicles on autolysis of mu- and m-calpain from bovine skeletal muscle

The finding that phospholipid micelles lowered the Ca2+ concentration required for autolysis of the calpains led to a hypothesis suggesting that the calpains are translocated to the plasma membrane where they interact with phospholipids to initiate their autolysis. However, the effect of plasma membranes themselves on the Ca2+ concentration required for calpain autolysis has never been reported. Also, if interaction with a membrane lowers the Ca2+ required for autolysis, the membrane-bound-calpain must autolyze itself, because it would be the only calpain having the reduced Ca2+ requirement. This implies that the autolysis is an intramolecular process, although several studies have shown that autolysis of the calpains in an in vitro assay and in the absence of phospholipid is an intermolecular process. Inside-out vesicles prepared from erythrocytes had no effect on the Ca2+ concentration required for autolysis of either mu- or m-calpain, although phosphatidylinositol (PI) decreased the Ca2+ concentration required for autolysis of the same calpains. The presence of a substrate for the calpains, beta-casein, reduced the rate of autolysis of both mu- and m-calpain both in the presence and in the absence of PI, suggesting that mu- and m-calpain autolysis is an intermolecular process in the presence of PI just as it is in its absence. Because IOV have no effect on the Ca2+ concentration required for calpain autolysis, association with the plasma membrane, at least with erythrocyte plasma membranes, does not initiate calpain autolysis by reducing the Ca2+ concentration required for autolysis as suggested by the membrane-activation hypothesis. Interaction with a membrane may serve to bind calpains to their substrates rather than promoting autolysis.     

Domain II of m-calpain is a Ca(2+)-dependent cysteine protease

Calpain, a Ca(2+)-dependent cytosolic cysteine protease, proteolytically modulates specific substrates involved in Ca(2+)-mediated intracellular events, such as signal transduction, cell cycle, differentiation, and apoptosis. The 3D structure of m-calpain, in the absence of Ca(2+), revealed that the two subdomains (domains IIa and IIb) of the protease domain (II) have an 'open' conformation, probably due to interactions with other domains. Although the presence of an EF-hand structure was once predicted in the protease domain, no explicit Ca(2+)-binding structure was identified in the 3D structure. Therefore, it is predicted that if the protease domain is excised from the calpain molecule, it will have a Ca(2+)-independent protease activity. In this study, we have characterized a truncated human m-calpain that consists of only the protease domain. Unexpectedly, the proteolytic activity was Ca(2+)-dependent, very weak, and not effectively inhibited by calpastatin, a calpain inhibitor. Ca(2+)-dependent modification of the protease domain by the cysteine protease inhibitor, E-64c, was clearly observed as a SDS-PAGE migration change, indicating that the conformational changes of this domain are a result of Ca(2+) binding. These results suggest that the Ca(2+) binding to domain II, as well as to domains III, IV, and VI, is critical in the process of complete activation of calpain.     

Expression of human, mouse, and rat m-calpains in Escherichia coli and in murine fibroblasts

The two best known calpains, micro- and m-calpain, are Ca(2+)-dependent cysteine proteases found in all mammalian tissues. They are probably involved in many Ca(2+)-linked signal pathways, although the details are not yet clear. The enzymes are heterodimers of a specific large subunit (micro-80k or m-80k) and a common small subunit (28k). Recombinant calpains have been obtained by co-expression of large and small subunits in Escherichia coli and in Sf9 cells, with variable success. Expression with the 28k subunit is very low, but is much higher with a C-terminal 21k fragment of this subunit. Rat m-calpain (m-80k/21k) is well expressed in E. coli but mouse m-calpain (m-80k/21k) is poorly expressed, even though the amino acid sequences of rat-m-80k and mouse-m-80k are 92% identical. It had also been reported that human m-calpain could be expressed in Sf9 cells but not in E. coli. To investigate these differences, hybrid rat/mouse and rat/human m-calpains were cloned and expressed in E. coli. It was shown that Ile-6 and Pro-127, which are specific to the mouse m-80k sequence, caused poor expression. High expression of human m-calpain in E. coli could be achieved by providing the correct Shine-Dalgarno ribosome binding site. The results provide a simple method to obtain approximately 10mg amounts of human m-calpain and a slightly modified mouse m-calpain. Expression of m-80k-EGFP fusions was also studied, both in E. coli and in mammalian cells, varying both the small subunit and the promoters. m-80k-EGFP alone was not active, but with 21k or 28k subunits was active in both cell types. The EGFP domain was partially cleaved during expression, releasing an active m-80k/21k calpain.     

The role of autolysis in activity of the Ca2+-dependent proteinases (mu-calpain and m-calpain)

A recent hypothesis suggests that proteolytic activity of the micromolar and millimolar Ca2+-requiring forms of the Ca2+-dependent proteinases (mu- and m-calpain, respectively) is regulated in vivo by their association with a phosphatidylinositol-containing site on the plasma membrane followed by autolysis of the proteinases. Phosphatidylinositol association lowers the Ca2+ concentration needed for autolysis, and autolysis, in turn, lowers the Ca2+ concentration needed for proteolytic activity. To test this hypothesis, we have compared the Ca2+ concentrations needed for autolysis and for proteolytic activity of the calpains both in the presence and the absence of phosphatidylinositol. Bovine skeletal muscle mu-calpain required 40-50 microM Ca2+ for half-maximal rate of proteolysis of a casein substrate, 140-150 microM Ca2+ for half-maximal autolysis in the presence of 80 microM phosphatidylinositol, and 190-210 microM Ca2+ for half-maximal autolysis in the absence of phosphatidylinositol. Consequently, mu-calpain is an active proteinase and does not require autolysis for activation. Bovine skeletal muscle m-calpain required 700-740 microM Ca2+ for half-maximal rate of proteolysis of a casein substrate, 370-400 microM Ca2+ for half-maximal autolysis in the presence of 80 microM phosphatidylinositol, and 740-780 microM Ca2+ for half-maximal autolysis in the absence of phosphatidylinositol. These results are consistent with the idea that m-calpain functions in its autolyzed form, but the results do not demonstrate that unautolyzed m-calpain is inactive. 80 microM phosphatidylinositol had no effect on the Ca2+ requirement of the autolyzed forms of either mu- or m-calpain but lowered the specific activity of mu-calpain to 20% of its activity in the absence of phosphatidylinositol. Of the four forms of the calpains, unautolyzed m-calpain, autolyzed m-calpain, and unautolyzed mu-calpain would not be proteolytically active at the free Ca2+ concentrations of 300-1200 nM present inside normal cells, and neither mu- nor m-calpain would undergo autolysis at these Ca2+ concentrations, even in the presence of phosphatidylinositol. Cells must contain a mechanism other than or in addition to membrane association and autolysis to activate the calpains.     

Efficient expression and purification of recombinant human m-calpain using an Escherichia coli expression system at low temperature

Calpain belongs to the superfamily of Ca(2+)-regulated cysteine proteases, which are indispensable to the regulation of various cellular functions. Of the 15 mammalian calpain isoforms, μ- and m-calpains are the best characterized. Both μ- and m-calpain are ubiquitously expressed and exist as heterodimers, containing a distinct 80-kDa catalytic subunit (CAPN1 and CAPN2, respectively) and the common, 30-kDa regulatory subunit (CAPNS1). To date, various expression systems have been developed for producing recombinant calpains for use in structural and physiological studies, however Escherichia coli systems have proven incompatible with large-scale preparation of calpain, with the exception of rat m-calpain. Here, we have established a highly efficient method to purify active recombinant human m-calpain using an E. coli expression system at low temperature (22°C). This was achieved by co-expressing CAPN2 with a C-terminal histidine-tag, and CAPNS1, lacking the first Gly-repeated region at the N-terminal. After three sequential passes through a chromatographic column, ~5 mg of human m-calpain was homogenously purified from 1 l of E. coli culture. Proteins were stable for several months. This is the first report of efficient, large-scale purification of recombinant human m-calpain using an E. coli expression system.     

Purification and characterization of calpain and calpastatin from rainbow trout, Oncorhynchus mykiss

Although the calpain system has been studied extensively in mammalian animals, much less is known about the properties of μ-calpain, m-calpain, and calpastatin in lower vertebrates such as fish. These three proteins were isolated and partly characterized from rainbow trout, Oncorhynchus mykiss, muscle. Trout m-calpain contains an 80-kDa large subunit, but the  26-kDa small subunit from trout m-calpain is smaller than the 28-kDa small subunit from mammalian calpains. Trout μ-calpain and calpastatin were only partly purified; identity of trout μ-calpain was confirmed by labeling with antibodies to bovine skeletal muscle μ-calpain, and identity of trout calpastatin was confirmed by specific inhibition of bovine skeletal muscle μ- and m-calpain. Trout μ-calpain requires 4.4 ± 2.8 μM and trout m-calpain requires 585 ± 51 μM Ca²⁺ for half-maximal activity, similar to the Ca²⁺ requirements of μ- and m-calpain from mammalian tissues. Sequencing tryptic peptides indicated that the amino acid sequence of trout calpastatin shares little homology with the amino acid sequences of mammalian calpastatins. Screening a rainbow trout cDNA library identified three cDNAs encoding for the large subunit of a putative m-calpain. The amino acid sequence predicted by trout m-calpain cDNA was 65% identical to the human 80-kDa m-calpain sequence. Gene duplication and polyploidy occur in fish, and the amino acid sequence of the trout m-calpain 80-kDa subunit identified in this study was 83% identical to the sequence of a trout m-calpain 80-kDa subunit described earlier. This is the first report of two isoforms of m-calpain in a single species.     

In vitro Proteolytic Degradation of Bovine Brain Calcineurin by m-Calpain

A major cause of neuronal dysfunction is due to altered Ca2+ regulation. An increase in Ca2+ influx can activate Ca2+-dependent enzymes including calpains, causing the proteolysis of its specific substrates. In the present study, calcineurin (CaN) was found to be proteolysed by a Ca2+-dependent cysteine protease, m-calpain. In the presence of Ca2+, the 60 kDa subunit (CaN A) was degraded to a 46 kDa immunoreactive fragment, whereas in the presence of Ca2+ /calmodulin (CaM) immunoreactive fragments of 48 and 54 kDa were observed. The beta-subunit (CaN B) was not proteolysed in either condition. The proteolysis of CaN A increased its phosphatase activity and rendered it totally CaM-independent after 10 min of proteolysis. The molecular weight of the proteolytic fragments suggested that the m-calpain cleaved CaN A in the CaN B binding domain. A CaM-overlay experiment revealed that the CaM-binding site was present only in the 54 kDa fragment produced by CaN A proteolysis in the presence of Ca2+ /CaM. Thus, the increase in CaN A phosphatase activity observed in many neuronal disorders, may be due to the action of calpain.     

Domain III of calpain is a ca2+-regulated phospholipid-binding domain

The X-ray structure of m-calpain shows that domain III of the large subunit is structurally related to C2 domains, Ca2+-regulated lipid binding modules in many enzymes. To address whether this structural similarity entails functional analogy, we have characterized recombinant domain III from rat micro- and m-calpain and Drosophila CALPB. In a Ca2+ overlay assay domain III displays a large capacity for Ca2+ binding, commensurable with that of domain IV, the principal Ca2+-binding domain of calpains. The amount of Ca2+ bound to domain III increases 2- to 10-fold upon the addition of liposomes containing 20-40% di- and triphosphoinositides. Conversely, phospholipid-binding in spin-column size-exclusion chromatography is significantly promoted by Ca2+, in a manner similar to known C2 domains. These results suggest that domain III might be the primary lipid binding site of calpain and may play a decisive role in orchestrating Ca2+- and lipid activation of the enzyme.     

Ca²+-induced release of mitochondrial m-calpain from outer membrane with binding of calpain small subunit and Grp75

Although mitochondrial μ- and m-calpains play significant roles in apoptotic cell death, their activating mechanisms have not been determined. The purpose of this study was to determine the core factors that are involved in activating mitochondrial outer membrane (OM)-bound calpains. To accomplish this, we solubilized OM-bound calpains and separated them by DEAE-Sepharose column chromatography, and identified them by immunoblots. We also determined the core factors that activated the OM-bound calpains and release them from the OM by calpain assays, immunoprecipitations, and immunoblots. The OM-bound m-calpain large subunit was not associated with the small subunit or with Grp75 chaperone. Free calpain small subunit was located in the IMS and caused the release of the OM-bound m-calpain large subunit from the OM together with Grp75, ATP, and Ca2+. Our results showed that the activating mechanism of mitochondrial OM-bound m-calpain and the release of mitochondrial m-calpain from the OM have important implications in facilitating apoptotic cell death.     

Ubiquitous calpains promote caspase-12 and JNK activation during endoplasmic reticulum stress-induced apoptosis

Ubiquitously expressed mu- and m-calpain proteases are implicated in development and apoptosis. They consist of 80-kDa catalytic subunits encoded by the capn1 and capn2 genes, respectively, and a common 28-kDa regulatory subunit encoded by the capn4 gene. The regulatory subunit is required to maintain the stability and activity of mu- and m-calpains. Accordingly, genetic disruption of capn4 in the mouse eliminated both ubiquitous calpain activities. In embryonic fibroblasts derived from these mice, calpain deficiency correlated with resistance to endoplasmic reticulum (ER) stress-induced apoptosis, and this was directly related to a calpain requirement for activation of both caspase-12 and the ASK1-JNK cascade. This study provides compelling genetic evidence for calpain's role in caspase-12 activation at the ER, and reveals a novel role for the ubiquitous calpains in ER-stress induced apoptosis and JNK activation.     

Dissociation of m-calpain subunits occurs after autolysis of the N-terminus of the catalytic subunit, and is not required for activation.

Calpain is a heterodimeric, intracellular Ca(2+)-dependent, "bio-modulator" that alters the properties of substrates through site-specific proteolysis. It has been proposed that calpains are activated by autolysis of the N-terminus of the large subunit and/or its dissociation into the subunits. It is, however, unclear whether the dissociation into subunits is required for the expression of protease activity and/or for in vivo function. Recently, the crystal structure of m-calpain in the absence of Ca(2+) has been resolved. The 3D structure clearly shows that the N-terminus of the m-calpain large subunit (mCL) makes contact with the 30K subunit, suggesting that autolysis of the N-terminus of mCL changes the interaction of both subunits. To examine the relationship between autolysis, dissociation, and activation, we made and analysed a series of N-terminal mutants of mCL that mimic the autolysed forms or have substituted amino acid residue(s) interacting with 30K. As a result, the mutant m-calpains, which are incapable of autolysis, did not dissociate into subunits, whereas those lacking the N-terminal 19 residues (Delta 19), but not those lacking only nine residues (Delta 9), dissociated into subunits even in the absence of Ca(2+). Moreover, both Delta 9 and Delta 19 mutants showed an equivalent reduced Ca(2+) requirement for protease activity. These results indicate that autolysis is necessary for the dissociation of the m-calpain subunits, and that the dissociation occurs after, but is not necessary for, activation.     

Effect of Ca2+ on binding of the calpains to calpastatin

Autolyzed mu-calpain, unautolyzed mu-calpain, autolyzed m-calpain, and unautolyzed m-calpain (mu-calpain is the micromolar Ca2+-requiring proteinase, m-calpain is the millimolar Ca2+-requiring proteinase) were passed through a calpastatin-affinity column at different free Ca2+ concentrations, and binding of the calpains to calpastatin was compared with proteolytic activity of that calpain at each Ca2+ concentration. Unautolyzed m-calpain, autolyzed m-calpain, and autolyzed mu-calpain required less Ca2+ for half-maximal binding to calpastatin than for half-maximal activity. Unautolyzed mu-calpain, however, required slightly more Ca2+ for half-maximal binding to calpastatin than for half-maximal activity. Half-maximal binding of oxidatively inactivated mu- or m-calpain to calpastatin required approximately the same Ca2+ concentrations as half-maximal binding of unautolyzed mu- or m-calpain, respectively, to calpastatin. Binding of unautolyzed m-calpain and autolyzed mu-calpain to calpastatin occurred over a wide range of Ca2+ concentrations, and it seems likely that two or more Ca2+-binding sites with different Ca2+-binding constants are involved in binding of the calpains to calpastatin. Proteolytic activity occurs at different Ca2+ concentrations than calpastatin binding, suggesting a second set of Ca2+-binding sites associated with proteolytic activity. Third and fourth sets of Ca2+-binding sites may be involved in autolysis and in binding to phosphatidylinositol or cell membranes; these four Ca2+-dependent properties of the calpains may require the eight potential Ca2+-binding sites that amino acid sequences predict are present in the calpain molecules.