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+.     

Autolysis of the millimolar Ca2+-requiring form of the Ca2+-dependent proteinase from chicken skeletal muscle

The millimolar Ca2+-requiring form of the Ca2+-dependent proteinase from chicken breast skeletal muscle contains two subunit polypeptides of 80 and 28 kDa, just as the analogous forms of this proteinase from other tissues do. Incubation with Ca2+ at pH 7.5 causes rapid autolysis of the 80-kDa polypeptide to 77 kDa and of the 28-kDa polypeptide to 18 kDa. Autolysis of the 28-kDa polypeptide is slightly faster than autolysis of the 80-kDa polypeptide and is 90-95% complete after 10 s at 0 degrees C. Autolysis for 15 s at 0 degrees C converts the proteinase from a form requiring 250-300 microM Ca2+ to one requiring 9-10 microM Ca2+ for half-maximal activity, without changing its specific activity. The autolyzed proteinase has a slightly lower pH optimum (7.7 vs. 8.1) than the unautolyzed proteinase. The autolyzed proteinase is not detected in tissue extracts made immediately after death; therefore, the millimolar Ca2+-requiring proteinase is largely, if not entirely, in the unautolyzed form in situ.     

Investigation of the structural basis of the interaction of calpain II with phospholipid and with carbohydrate.

Two forms of pig kidney calpain II were isolated, both of which appeared to contain an intact 80 kDa large subunit, but which showed specific proteolytic degradation at the N-terminal end of the 30 kDa small subunit. The structure of each of these molecules was investigated by amino acid sequence analysis. The forms corresponded to molecules with small subunits starting at residue 38 (degraded calpain A) and at residue 62 (degraded calpain B) of the complete sequence. These molecules were tested for their ability to interact with phosphatidylinositol and with carbohydrate (agarose gel-filtration media). Calpain and degraded calpain A, but not degraded calpain B, would interact with phosphatidylinositol. Thus the sequence (G)17TAMRILG (residues 38-61) is essential for the interaction. Neither calpain nor the degraded forms of the enzyme showed specific interaction with carbohydrate.     

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.     

Limited autolysis of calcium-activated neutral protease (CANP): reduction of the Ca2+-requirement is due to the NH2-terminal processing of the large subunit

Calcium-activated neutral protease (rabbit mCANP), composed of large and small subunits, was converted to a lower-Ca2+-requiring form (derived microCANP) by limited autolysis in the presence of Ca2+. The NH2-terminal regions of the two subunits of mCANP were cleaved by autolysis, but the COOH-termini remained intact after autolysis. When native mCANP or derived microCANP was dissociated into subunits, the proteolytic activity of the large subunit was reduced to 2-5% of that of the native dimeric enzyme. The Ca2+-sensitivity of one hybrid CANP reconstituted from the large subunit of derived microCANP and the small subunit of native mCANP was similar to that of derived microCANP. However, the other hybrid molecule composed of the large subunit of native mCANP and the small subunit of derived microCANP required a high concentration of Ca2+ for activity, like native mCANP. These results indicate that the Ca2+-sensitivity of derived microCANP is determined by the structural change of the large subunit resulting from loss of its NH2-terminal region. The autolysis of the small subunit apparently has no effect on the reduction of the Ca2+-requirement.     

Two-stage autolysis of the catalytic subunit initiates activation of calpain I

Calcium-induced autolysis of bovine erythrocyte calpain I occurs in multiple stages. Initially, a 14 amino acid segment is cleaved from the N-terminus of the native 80 kDa catalytic subunit, yielding a 78 kDa form of the subunit. Then, an additional 12 amino acid segment is cleaved from the N-terminus, forming a 76 kDa subunit. The 76 kDa enzyme is the active form of the catalytic subunit that is able to proteolyze the 30 kDa regulatory subunit as well as exogenous substrates. While the initial autolytic step requires high calcium, the 76 kDa enzyme form is active in microM calcium and can cleave the amino termini of native 80 kDa and intermediate 78 kDa enzyme forms at low calcium. Both intramolecular and intermolecular proteolysis of the catalytic subunit appear to yield the same products.     

Free calcium and calpain I activity

Activation of purified calpain I proceeds through a Ca(2+)-induced autolysis from the 80 kDa catalytic subunit to a 76 kDa form via an intermediate 78 kDa form, and from a 30 kDa form to a 18 kDa form as the result of two autocatalytic processes (intra and intermolecular). The minimum Ca2+ requirements for autolysis and proteolysis have been determined by physico-chemical and electrophoretic methods in the presence or absence of a digestible substrate. According to our results the activation process needs less free Ca2+ than the proteolysis of a digestible substrate, which means that proteolysis is really subsequent to activation. For very low Ca2+ levels, a digestible substrate does not initiate the calpain I activation process. In the presence of phospholipid vesicles, such as PI, PS or a mixture of PI (20%), PS (20%) and PC (60%), the apparent kinetic constants of activation are greatly increased without any change in the initial velocity of the substrate proteolysis. Thus, enzyme activation and substrate proteolysis are observed as independent phenomena. These results obtained from experiments using low free Ca2+ concentrations enable us to propose a hypothesis for the mechanism of regulation by which the enzyme could be activated in the living cell.     

Dissociation and aggregation of calpain in the presence of calcium

Calpain is a heterodimeric Ca(2+)-dependent cysteine protease consisting of a large (80 kDa) catalytic subunit and a small (28 kDa) regulatory subunit. The effects of Ca(2+) on the enzyme include activation, aggregation, and autolysis. They may also include subunit dissociation, which has been the subject of some debate. Using the inactive C105S-80k/21k form of calpain to eliminate autolysis, we have studied its disassociation and aggregation in the presence of Ca(2+) and the inhibition of its aggregation by means of crystallization, light scattering, and sedimentation. Aggregation, as assessed by light scattering, depended on the ionic strength and pH of the buffer, on the Ca(2+) concentration, and on the presence or absence of calpastatin. At low ionic strength, calpain aggregated rapidly in the presence of Ca(2+), but this was fully reversible by EDTA. With Ca(2+) in 0.2 m NaCl, no aggregation was visible but ultracentrifugation showed that a mixture of soluble high molecular weight complexes was present. Calpastatin prevented aggregation, leading instead to the formation of a calpastatin-calpain complex. Crystallization in the presence of Ca(2+) gave rise to crystals mixed with an amorphous precipitate. The crystals contained only the small subunit, thereby demonstrating subunit dissociation, and the precipitate was highly enriched in the large subunit. Reversible dissociation in the presence of Ca(2+) was also unequivocally demonstrated by the exchange of slightly different small subunits between mu-calpain and m-calpain. We conclude that subunit dissociation is a dynamic process and is not complete in most buffer conditions unless driven by factors such as crystal formation or autolysis of active enzymes. Exposure of the hydrophobic dimerization surface following subunit dissociation may be the main factor responsible for Ca(2+)-induced aggregation of calpain. It is likely that dissociation serves as an early step in calpain activation by releasing the constraints upon protease domain I.     

Isolation and sequence analysis of cDNA clones for the small subunit of rabbit calcium-dependent protease

We have isolated and sequenced cDNA clones for the small subunit (30-kDa subunit) of rabbit calcium-dependent protease (Ca2+-protease) using synthesized oligodeoxynucleotide probes based on the partial amino acid sequence of the protein. A nearly full-length cDNA clone containing the total amino acid coding sequence was obtained. From the deduced sequence, the following conclusions about possible functions of the protein are presented. The kDa subunit comprises 266 residues (Mr = 28,238). The N-terminal region (64 residues) is mainly composed of glycine (37 residues) and hydrophobic amino acids and may interact with the cell membrane or an organelle. The sequence of the C-terminal 168 residues is highly homologous to the corresponding C-terminal region of the large subunit (80-kDa subunit) which has been identified as the calcium-binding domain. This region of the 30-kDa subunit contains four E-F hand structures and presumably binds Ca2+, as in the case of the 80-kDa subunit. Thus, the 30-kDa subunit may play important roles in regulating enzyme activity and/or possibly in determining the location of the Ca2+-protease. The marked sequence homology of the C-terminal regions of the two subunits may indicate that the calcium-binding domains have evolved from the same ancestral gene.     

The mitochondrial ribosomes of Neurospora crassa. II. Comparison of the proteins from Neurospora crassa mitochondrial ribosomes with ribosomal proteins from Neurospora cytoplasm, from rat liver mitochondria and from bacteria.

1. It has been shown by Datema et al. (Datema, R., Agsteribbe, E. and Kroon, A.M. (1974) Biochim. Biophys. Acta 335, 386--395) that Neurospora mitochondria isolated in a Mg2+-containing medium (or after homogenization of the mycelium in this medium and subsequent washing of the mitochondria in EDTA-containing medium) possess 80-S ribosomes; mitochondria homogenized and isolated in EDTA medium yield 73-S ribosomes. The ribosomal proteins of the subunits of 80-S and 73-S ribosomes were compared by two-dimensional electrophoresis. The protein patterns of the large, as well as of the small subunits are very similar but not completely identical; the most conspicuous difference is that the large subunit of 80 S contains about eight more proteins than the large subunit of 73 S. 2. The contamination by Neurospora cytoplasmic 77-S ribosomes in the 80-S preparations, if present, is only minor. 3. Neurospora cytoplasmic ribosomes contain 31 proteins in the large, and 21 proteins in the small subunit. 4. Neurospora 80- mitochondrial ribosomes contain 39 proteins in the large, and 30 proteins in the small subunit 30 proteins. 5. Rat liver mitochondrial ribosomes contain 40 proteins in the large and at least 30 proteins in the small subunit. About 50% of these proteins has an isoelectric point below pH 8.6. 6. The pattern of Paracoccus denitrificans is very similar to that of other bacterial ribosomes, the large subunit contains 29, the small subunit 18 proteins.     

Autolysis of calpain large subunit inducing irreversible dissociation of stoichiometric heterodimer of calpain

Calpain, a calcium dependent cysteine protease, consists of a catalytic large subunit and a regulatory small subunit. Two models have been proposed to explain calpain activation: an autolysis model and a dissociation model. In the autolysis model, the autolyzed form is the active species, which is sensitized to Ca2+. In the dissociation model, dissociated large subunit is the active species. We have reported that the Ca2+ concentration regulates reversible dissociation of subunits. We found further that in chicken micro/m-calpain autolysis of the large subunit induces irreversible dissociation from the small subunit as well as activation. So we could propose a new mechanism for activation of the calpain by combining our findings. Our model insists that autolyzed large subunit remains dissociated from the small subunit even after the removal of Ca2+ to keep it sensitized to Ca2+. This model could be expanded to other calpains and give a new perspective on calpain activation.     

Milli-calpain from Sea Bass (Dicentrarchus labrax) White Muscle: Purification, Characterization of Its Activity and Activation In Vitro

A calcium-activated neutral cysteine protease was purified to homogeneity from Dicentrarchus labrax white muscle using three steps: hydrophobic interaction, anion exchange, and gel filtration chromatographies. The purified enzyme showed a native molecular weight of 124 kDa with an oligomeric structure (large subunit of 80 kDa and small subunit of 24 kDa). It has been classified as a milli-calpain from its calcium sensitivity. Activity was maximal at pH 7.0, 24°C in Tris buffer without NaCl as determined by means of a two-level experimental design and response surface methodology. Sea bass calpain is neither glycosylated nor phosphorylated and shared some common cleavage specificities and activation and autolysis mechanisms with other typical mammalian or invertebrates calpains. Calcium-induced activation and autolysis of calpain has been characterized together with the effect of the strontium cation acting as a calcium analog. On the basis of its in vitro properties, the contribution of the sea bass milli-calpain to the process of postmortem deterioration of fish muscle is discussed, even though further information such as in vivo regulation or in vitro effects on myofibrils is required. Received March 1, 2001; accepted July 9, 2001.     

The role of subunit autolysis in activation of smooth muscle Ca2+-dependent proteases

Ca2+-dependent proteases isolated from chicken gizzard and bovine aortic smooth muscle were compared with respect to subunit autolysis and the role of autolysis in modulating enzyme activity. The protease isolated from chicken gizzard was a heterodimer consisting of 80,000- and 30,000-dalton subunits. The protease isolated under identical conditions from bovine aorta consisted of 75,000- and 30,000-dalton subunits. In the presence of Ca2+, both enzymes underwent autolysis of their 30,000-dalton subunits with conversion to an 18,000-dalton species. In addition, the 80,000-dalton subunit of the gizzard protease was degraded to a 76,000-dalton form. The Ca2+ concentrations required for autolysis of the 30,000-dalton subunits were different for the two enzymes (i.e. gizzard: K0.5 Ca2+ = 335 microM; aortic: K0.5 Ca2+ = 1,250 microM) although in both cases, stimulation of autolysis by Ca2+ exhibited positive cooperativity. When compared with respect to kinetics of substrate degradation, the native forms of the smooth muscle Ca2+-dependent proteases (gizzard, GIIa = 80,000/30,000-dalton heterodimer; bovine aortic, IIa = 75,000/30,000-dalton heterodimer) exhibited a lag phase in product appearance. On the other hand, the autolyzed forms (gizzard, GIIb = 76,000/18,000-dalton heterodimer; bovine aortic, IIb = 75,000/18,000-dalton heterodimer) exhibited linear rates of substrate degradation. These results were analyzed in terms of autolysis of the 30,000-dalton subunits as determined by the conversion of this subunit to its 18,000 dalton form. For both enzymes, the time course for the autolytic transition, 30,000----18,000 daltons, and Ca2+-dependence of the apparent rate constants for this transition were found to correlate well with the lag phase in enzymatic activity. No such correlation could be established for the 80,000----76,000 dalton autolytic transition of the high molecular mass subunit of the gizzard protease. Our results suggest that catalytic activity of the Ca2+-dependent proteases isolated from gizzard and bovine aortic smooth muscle requires autolysis of the 30,000-dalton subunit. The native or unautolyzed forms of these enzymes appear to be proenzymes that can be activated by autolysis.     

The amino-terminal hydrophobic region of the small subunit of calcium-activated neutral protease (CANP) is essential for its activation by phosphatidylinositol

Ca2+-Activated neutral protease (CANP), that consists of 80K and 30K subunits, is converted to a low-Ca2+-requiring form by autolysis in the presence of Ca2+. Phosphatidylinositol greatly reduces the Ca2+-requirement for the autolysis of native CANP. However, this effect was not observed for CANP with a trimmed 30K subunit lacking the NH2-terminal hydrophobic and glycine-rich region. This suggests that the NH2-terminal hydrophobic region of the 30K subunit is important for the interaction of CANP with the cell membrane and that the calcium sensitivity of CANP is increased at the cell membrane through the effect of phosphatidylinositol.     

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.     

The small subunits of human and mouse DNA polymerase epsilon are homologous to the second largest subunit of the yeast Saccharomyces cerevisiae DNA polymerase epsilon.

Human DNA polymerase epsilon is composed of a 261 kDa catalytic polypeptide and a 55 kDa small subunit of unknown function. cDNAs encoding the small subunit of human and mouse DNA polymerase epsilon were cloned. The predicted polypeptides have molecular masses of 59.469 and 59.319 kDa respectively and they are 90% identical. The human and mouse polypeptides show 22% identity with the 80 kDa subunit of the five subunit DNA polymerase epsilon from the yeast Saccharomyces cerevisiae. The high degree of conservation suggests that the 55 kDa subunit shares an essential function with the yeast 80 kDa subunit, which was earlier suggested to be involved in S phase cell cycle control in a pathway that is able to sense and signal incomplete replication. The small subunits of human and mouse DNA polymerase epsilon also show homology to the C-terminal domain of the second largest subunit of DNA polymerase alpha. The gene for the small subunit of human DNA polymerase epsilon (POLE2) was localized to chromosome 14q21-q22 by fluorescence in situ hybridization.     

Organization of genes expressing the blood-group-M-specific hemagglutinin of Escherichia coli: identification and nucleotide sequence of the M-agglutinin subunit gene

The organization of genes encoding the blood group M-specific hemagglutinin (M-agglutinin) of Escherichia coli strain IH11165 was studied with a cloned 6.5-kb DNA segment. This DNA segment contains at least five genes which code for the polypeptides of 12.5, 30, 80, 18.5 and 21 kDa. The 30-, 80- and 21-kDa polypeptides are synthesized as precursors that are approximately 2 kDa larger. The 21-kDa polypeptide was identified as the M-agglutinin subunit by its reactivity with anti-M-agglutinin serum. Nucleotide sequence analysis of the corresponding gene showed that the M-agglutinin precursor had a 24-amino acid (aa) signal sequence, while the mature protein is 146 aa residues long. Although the organization of the M-agglutinin gene cluster resembles those of other E. coli adhesins, there is no significant sequence homology between the M-agglutinin subunit and the subunits of the other potentially related proteins in E. coli.     

A second binding site revealed by C-terminal truncation of calpain small subunit, a penta-EF-hand protein

The subunits in calpain and in the related penta-EF-hand (PEF) proteins are bound through contacts between the unpaired EF-hand 5 from each subunit. To study subunit binding further, a tetra-EF-hand 18 kDa N- and C-terminally truncated form of the calpain small subunit was prepared (18k). This protein does not combine with the calpain large subunit to form active calpain, but forms homodimers in solution, as shown by ultracentrifugation. The X-ray structure of the 18k protein in the presence of cadmium was solved to a resolution of 2.0 A. The structure of the monomer is almost identical to the known structure of the calpain small subunit, but the 18k protein forms an oligomer in the crystal by the use of two binding sites. One of these sites is an artefact arising from the C-terminal truncation, but the other is a naturally occurring site that is fully exposed to water in intact purified calpain. The characteristics of this site suggest that it may be important in binding other protein modulators involved in the regulation of calpain and of PEF proteins.     

Alzheimer's disease amyloid precursor protein is present in senile plaques and cerebrospinal fluid: immunohistochemical and biochemical characterization

The amyloid fibrils deposited in cerebral vessel walls and senile plaques in Alzheimer's disease are polymeric forms of a 4 kDa fragment produced by proteolysis of a putative precursor protein (APP). Using antibodies to several fragments of the deduced precursor, we were able to demonstrate the presence of APP in senile plaques, brain extracts and cerebrospinal fluid. Membrane-associated APP is detected as a group of 105-135 kDa proteins while soluble APP is predominantly 105 kDa, does not react with an anti C-terminal antibody, and is 10 kDa shorter than the membrane-bound APP. Amino terminal sequence of the tissue 105 kDa protein indicates that APP begins at residue 18 of the cDNA sequence. These findings imply that i) two forms of APP are detected: membrane-bound and secreted, and ii) APP can be processed in situ.     

Identification of calpastatin and mu-calpain and studies of their association in pulmonary smooth muscle mitochondria

Using calpastatin antibody we have identified a 145 kDa major band along with two relatively minor bands at 120 kDa and 110 kDa calpastatin molecules in bovine pulmonary artery smooth muscle mitochondria. To the best of our knowledge this is first report regarding the identification of calpastatin in mitochondria. We also demonstrated the presence of micro-calpain in the mitochondria by immunoblot and casein zymogram studies. Immunoblot studies identified two major bands corresponding to the 80 kDa large and the 28 kDa small subunit of mu-calpain. Additionally 76 kDa, 40 kDa and 18 kDa immunoreactive bands have also been detected. Purification and N-terminal amino acid sequence analysis of the identified proteins confirmed their identity as mu-calpain and calpastatins. Immunoprecipitation study revealed molecular association between mu-calpain and calpastatin in the mitochondria indicating that calpastatin could play an important role in preventing uncontrolled activity of mu-calpain which otherwise may facilitate pulmonary hypertension, smooth muscle proliferation and apoptosis.