Suppression of cancer cell migration and invasion by protein phosphatase 2A through dephosphorylation of mu- and m-calpains

The mu- and m-calpains are major members of the calpain family that play an essential role in regulating cell motility. We have recently discovered that nicotine-activated protein kinase C iota enhances calpain phosphorylation in association with enhanced calpain activity and accelerated migration and invasion of human lung cancer cells. Here we found that specific disruption of protein phosphatase 2A (PP2A) activity by expression of SV40 small tumor antigen up-regulates phosphorylation of mu- and m-calpains whereas C2-ceramide, a potent PP2A activator, reduces nicotine-induced calpain phosphorylation, suggesting that PP2A may function as a physiological calpain phosphatase. PP2A co-localizes and interacts with mu- and m-calpains. Purified, active PP2A directly dephosphorylates mu- and m-calpains in vitro. Overexpression of the PP2A catalytic subunit (PP2A/C) suppresses nicotine-stimulated phosphorylation of mu- and m-calpains, which is associated with inhibition of calpain activity, wound healing, cell migration, and invasion. By contrast, depletion of PP2A/C by RNA interference enhances calpain phosphorylation, calpain activity, cell migration, and invasion. Importantly, C2-ceramide-induced suppression of calpain phosphorylation results in decreased secretion of mu- and m-calpains from lung cancer cells into culture medium, which may have potential clinic relevance in controlling metastasis of lung cancer. These findings reveal a novel role for PP2A as a physiological calpain phosphatase that not only directly dephosphorylates but also inactivates mu- and m-calpains, leading to suppression of migration and invasion of human lung cancer cells.     

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 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.     

Protein kinase Ciota promotes nicotine-induced migration and invasion of cancer cells via phosphorylation of micro- and m-calpains

Nicotine is a major component in cigarette smoke that activates the growth-promoting pathways to facilitate the development of lung cancer. However, it is not clear whether nicotine affects cell motility to facilitate tumor metastasis. Here we discovered that nicotine potently induces phosphorylation of both mu- and m-calpains via activation of protein kinase Ciota (PKCiota), which is associated with accelerated migration and invasion of human lung cancer cells. Purified PKCiota directly phosphorylates mu- and m-calpains in vitro. Overexpression of PKCiota results in increased phosphorylation of both mu- and m-calpains in vivo. Nicotine also induces activation of c-Src, which is a known PKCiota upstream kinase. Treatment of cells with the alpha(7) nicotinic acetylcholine receptor inhibitor alpha-bungarotoxin can block nicotine-induced calpain phosphorylation with suppression of calpain activity, wound healing, cell migration, and invasion, indicating that nicotine-induced calpain phosphorylation occurs, at least in part, through a signaling pathway involving the upstream alpha(7) nicotinic acetylcholine receptor. Intriguingly, depletion of PKCiota by RNA interference suppresses nicotine-induced calpain phosphorylation, calpain activity, cell migration, and invasion, indicating that PKCiota is a necessary component in nicotine-mediated cell motility signaling. Importantly, nicotine potently induces secretion of mu- and m-calpains from lung cancer cells into culture medium, which may have potential to cleave substrates in the extracellular matrix. These findings reveal a novel role for PKCiota as a nicotine-activated, physiological calpain kinase that directly phosphorylates and activates calpains, leading to enhanced migration and invasion of human lung cancer cells.     

Involvement of mu- and m-calpains and protein kinase C isoforms in L8 myoblast differentiation

The objectives were to investigate the roles of different calpains and protein kinase C (PKC) isoforms in muscle differentiation. Concentrations of mu- and m-calpain increased significantly whereas PKCalpha and delta declined significantly during L8 myoblast differentiation. Both mu-calpain and m-calpain antisense oligonucleotides inhibited myotube formation and creatine kinase activity during L8 myoblast differentiation. These results implied that both mu- and m-calpain were involved in L8 myoblast differentiation. To investigate the involvement of calpain in regulation of PKC concentrations, mu-calpain antisense oligonucleotides were added to L8 myoblasts. PKCalpha remained unchanged and PKCdelta declined. By adding m-calpain antisense oligonucleotides instead, PKCalpha level remained unchanged and PKCdelta concentrations increased significantly during differentiation. These results suggest that PKCalpha, but not PKCdelta, is the substrate for mu-calpain and PKCalpha and delta are the substrates for the m-calpain. In addition, more phosphorylated myogenin was found in day 2 antisense oligonucleotides treated L8 cells. It is concluded that the decline of PKCalpha mediated by m- and mu-calpain is essential for L8 myoblast differentiation. The decline of PKC during myoblast differentiation may cause hypo-phosphorylation of myogenin, which in turn activates muscle-specific genes during myogenesis.     

Conditional disruption of ubiquitous calpains in the mouse

Ubiquitous mu- and m-calpain proteases are implicated in development and apoptosis. They are heterodimers consisting of 80-kDa catalytic subunits encoded by capn1 and capn2, respectively, and a common 28-kDa regulatory subunit encoded by capn4. The regulatory subunit is required to maintain stability and activity of mu- and m-calpains; thus, genetic disruption of capn4 was predicted to eliminate both calpain activities. Germline disruption of capn4 caused embryonic lethality, hampering the use of those mouse models to explore physiological calpain functions. Here we describe a loxP/cre conditional capn4 targeted mouse model that enables tissue-specific and temporal deletion of calpain activity. Disruption of the floxed capn4 gene using a ubiquitous cytomegalovirus promoter driven Cre recombinase transgene led to midgestation embryonic lethality. Fibroblasts from these embryos lacked detectable regulatory subunit expression, had reduced levels of the mu- and m-calpain catalytic subunits, and had no detectable mu- and m-calpain activities. These defects were corrected with a capn4-encoding lentivirus.     

Characteristics of mitochondrial calpains

Calpains are considered to be cytoplasmic enzymes, although several studies have shown that calpain-like protease activities also exist in mitochondria. We partially purified mitochondrial calpain from swine liver mitochondria and characterized. Only one type of mitochondrial calpain was detected by the column chromatographies. The mitochondrial calpain was stained with anti-mu-calpain and calpain small subunit antibodies. The susceptibility of mitochondrial calpain to calpain inhibitors and the optimum pH differ from those of cytosolic mu- and m-calpains. The Ca(2+)-dependency of mitochondrial calpain was similar to that of cytosolic mu-calpain. Therefore, we named the protease mitochondrial mu-like calpain. In zymogram analysis, two types of caseinolytic enzymes existed in mitochondria and showed different mobilities from cytosolic mu- and m-calpains. The upper major band was stained with anti-mu-calpain and calpain small subunit antibodies (mitochondrial calpain I, mitochondrial mu-like calpain). The lower band was stained only with anti-calpain small subunit antibody (mitochondrial calpain II, unknown mitochondrial calpain). Calpastatin was not detected in mitochondrial compartments. The mitochondrial calpain processed apoptosis-inducing factor (AIF) to truncated AIF (tAIF), releasing tAIF into the intermembrane space. These results indicate that mitochondrial calpain, which differs from mu- and m-calpains, seems to be a ubiquitous calpain and may play a role in mitochondrial apoptotic signalling.     

Chicken skeletal muscle has three Ca2+-dependent proteinases

Chicken breast muscle has three Ca2+-dependent proteinases, two requiring millimolar Ca2+ (m-calpain and high m-calpain) and one requiring micromolar Ca2+ (mu-calpain). High m-calpain co-purifies with mu-calpain through successive DEAE-cellulose (steep gradient), phenyl-Sepharose, octylamine agarose, and Sephacryl S-300 columns, but elutes after mu-calpain when using a shallow KCl gradient to elute a DEAE-cellulose column. The mu- and m-calpains have 80 and 28 kDa polypeptides and are analogous to the mu- and m-calpains that have been purified from bovine, porcine and rabbit skeletal muscle. High m-calpain, which seems to be a new Ca2+-dependent proteinase, is still heterogeneous after the DEAE-cellulose column eluted with a shallow KCl gradient. Additional purification through two successive HPLC-DEAE columns and one HPLC-SW-4000 gel permeation column produces a fraction having six major polypeptides and 6-8 minor polypeptides on SDS-PAGE. A 74-76 kDa polypeptide in this fraction reacts in Western blots with monospecific, polyclonal anti-calpain antibodies that react with both the 80 kDa and the 28 kDa polypeptides of mu- or m-calpain. High m-calpain also is related to mu- and m-calpain in that it causes the same limited digestion of skeletal muscle myofibrils, has a similar pH optimum near pH 7.9-8.4, requires Ca2+ for activity, and reacts with the calpain inhibitor, calpastatin, and a variety of serine and cysteine proteinase inhibitors in a manner identical to mu- and m-calpain. High m-calpain differs from mu- and m-calpain in its elution off DEAE-cellulose columns and its requirement of 3800 microM Ca2+ for one-half maximal activity compared with 5.35 microM Ca2+ for mu-calpain and 420 microM Ca2+ for m-calpain. The physiological significance of high m-calpain in unclear. The presence of mu-calpain in chicken breast muscle suggests that all skeletal muscles contain both mu- and m-calpain, although the relative proportions of these two proteinases may vary in different species.     

Tobacco-specific nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone induces phosphorylation of mu- and m-calpain in association with increased secretion, cell migration, and invasion

Mounting evidence indicates that cigarette smoking not only promotes tumorigenesis but also may increase the spread of cancer cells in the body. However, the intracellular mechanism(s) by which cigarette smoking promotes metastasis of human lung cancer remains enigmatic. Nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) is an important component in cigarette smoke and is formed by nitrosation of nicotine. mu- and m-calpain (calpain I and calpain II) are major members of the calpain family, which are ubiquitously expressed in both small cell lung cancer and non-small cell lung cancer cells. Our findings indicated that NNK potently induces phosphorylation of both mu- and m-calpain in association with their activation and increased migration as well as invasion of lung cancer cells. Treatment of cells with PD98059 blocked phosphorylation of m- and mu-calpain and resulted in suppression of NNK-induced cell migration and invasion. p44 MAPK/extracellular signal-regulated kinase 1 (ERK1) and p42 MAPK/ERK2 were activated by NNK, co-localized with mu- and m-calpain in cytoplasm, and directly phosphorylated mu- and m-calpain in vitro. These findings suggest a role for the ERK1/2 kinases as NNK-activated physiological calpain kinases. Specific knock-down of mu- and/or m-calpain expression by RNA interference blocked NNK-stimulated migration and invasion, suggesting that mu- and m-calpain may act as required targets in a NNK-induced metastatic signaling pathway. Furthermore, NNK promotes secretion of active mu- and m-calpain from lung cancer cells through vesicles, which may have the potential to cleave substrates in the extracellular matrix. Thus, NNK-induced cell migration and invasion may occur, at least in part, through a novel mechanism involving phosphorylation of calpains that leads to their activation and secretion, which may contribute to metastasis and/or progression of lung cancer.     

Reduced cell migration and disruption of the actin cytoskeleton in calpain-deficient embryonic fibroblasts

The physiological functions and substrates of the calcium-dependent protease calpain remain only partly understood. The mu- and m-calpains consist of a mu- or m-80-kDa large subunit (genes Capn1 and Capn2), and a common 28-kDa small subunit (Capn4). To assess the role of calpain in migration, we used fibroblasts obtained from Capn4(-/-) mouse embryos. The cells lacked calpain activity on casein zymography and did not generate the characteristic calpain-generated spectrin breakdown product that is observed in wild-type cells. Capn4(-/-) cells had decreased migration rates and abnormal organization of the actin cytoskeleton with a loss of central stress fibers. Interestingly, these cells extended numerous thin projections and displayed delayed retraction of membrane protrusions and filopodia. The number of focal adhesions was decreased in Capn4(-/-) cells, but the cells had prominent vinculin-containing focal complexes at the cell periphery. The levels of the focal adhesion proteins, alpha-actinin, focal adhesion kinase (FAK), spectrin, talin, and vinculin, were the same in Capn4(+/+) and Capn4(-/-) cells. FAK, alpha-actinin, and vinculin were not cleaved in either cell type plated on fibronectin. However, proteolysis of the focal complex component, talin, was detected in the wild-type cells but not in the Capn4(-/-) cells, suggesting that calpain cleavage of talin is important during cell migration. Moreover, talin cleavage was again observed when calpain activity was partially restored in Capn4(-/-) embryonic fibroblasts by stable transfection with a vector expressing the rat 28-kDa calpain small subunit. The results demonstrate unequivocally that calpain is a critical regulator of cell migration and of the organization of the actin cytoskeleton and focal adhesions.     

Disruption of the murine calpain small subunit gene, Capn4: calpain is essential for embryonic development but not for cell growth and division

Calpains are a family of Ca(2+)-dependent intracellular cysteine proteases, including the ubiquitously expressed micro- and m-calpains. Both mu- and m-calpains are heterodimers, consisting of a distinct large 80-kDa catalytic subunit, encoded by the genes Capn1 and Capn2, and a common small 28-kDa regulatory subunit (Capn4). The physiological roles and possible functional distinctions of mu- and m-calpains remain unclear, but suggested functions include participation in cell division and migration, integrin-mediated signal transduction, apoptosis, and regulation of cellular control proteins such as cyclin D1 and p53. Homozygous disruption of murine Capn4 eliminated both mu- and m-calpain activities, but this did not affect survival and proliferation of cultured embryonic stem cells or embryonic fibroblasts, or the early stages of organogenesis. However, mutant embryos died at midgestation and displayed defects in the cardiovascular system, hemorrhaging, and accumulation of erythroid progenitors.     

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.     

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.     

Mu-calpain is functionally required for alpha-processing of Alzheimer's beta-amyloid precursor protein

Alzheimer's beta-amyloid precursor protein (APP) is normally processed by an unidentified alpha-secretase. A unique feature of this protease is its high sensitivity to phorbol esters, yet the mechanism involved is unclear. We have previously reported that phorbol 12,13-dibutyrate (PDBu) activates calpain, a Ca2+-dependent protease, and PDBu-induced release of APPs (secreted APP) is sensitive to calpain inhibitors, suggesting that calpain is involved in APP alpha-processing. In the present study, we found that PDBu markedly promoted the expression of both mu- and m-calpains in cultured fibroblasts. Dose-response and time course studies revealed that mu-calpain was more sensitive to PDBu than m-calpain and the temporal course of the mu-calpain change coincides better with that of APPs release. Moreover, the stimulatory effect of PDBu on mu-calpain was selectively blocked by mu-calpain-specific siRNA (small interference RNA) and the blockage was accompanied by a concomitant decrease in APPs release. In contrast, m-calpain siRNA did not affect APPs release significantly. Measurement of amyloid beta protein (Abeta) release in the mu-calpain siRNA-treated cells indicated that Abeta40 and Abeta42 levels inversely changed in relation to APPs, and the changes in Abeta42 were more prominent than in Abeta40. Together, these data suggest that calpain, particularly mu-calpain, is a potential candidate for alpha-secretase in the regulated APP alpha-processing, and that changes in this protease can affect the outcome of the overall APP processing.     

Differentiation in cultures derived from embryonic chicken muscle: II. Phosphorylase histochemistry and fluorescent antibody staining for creatine kinase and aldolase

The presence or absence of five proteins (glycogen phosphorylase, aldolase A, aldolase C, creatine kinase M, creatine kinase B) in the various classes of cells found in primary cultures derived from embryonic chick breast muscle was investigated using cytological staining methods. Histochemical staining for phosphorylase and indirect fluorescent antibody staining for aldolase A and C as well as for creatine kinases M and B showed the following: All five proteins were found in the many myotubes present in standard medium cultures and in the very few myotubes found in cultures containing 5-bromodeoxyuridine (10^?5M). The elongated bipolar cells prevented from fusing in medium containing EGTA also contain all five proteins. The flattened myogenic cells that predominate in the 5-bromodeoxyuridine-treated cultures contain no phosphorylase or creatine kinase M, though many of them contain creatine kinase B and aldolases A and C. These results are interpreted as indicating that: (1) phosphorylase and creatine kinase M, but not aldolase A, are suitable all-or-none markers for terminal muscle differentiation; (2) the small amounts of creatine kinase M detected in electrophoreses of 5-bromodeoxyruridine-treated cultures can be accounted for by the few myotubes present and are not due to “protodifferentiation” of large numbers of cells; (3) proteins typical of differentiated muscle are produced only in cells that have passed through the last step in myogenesis that is susceptible to 5-bromodeoxyuridine inhibition, and (4) if fusion is blocked by reducing the concentration of calcium ions, accumulation of characteristic muscle proteins can continue in those cells that have initiated terminal differentiation.     

Association and dissociation of the calcium-binding domains of calpain by Ca2+

The calmodulin-like domain of calpain is important for the association of the calpain large and small subunits. We expressed the calmodulin-like domains of the large subunits of rabbit mu- and m-calpains and their small subunits in E. coli and purified them to homogeneity. Unlike the full-length subunits, the calmodulin-like domains are soluble in buffer containing Ca2+. We performed gel filtration chromatography of the purified proteins and found that all three calmodulin-like domains exist as homodimers in the absence of Ca2+ and dissociate into monomers upon the addition of Ca2+.     

Studies on Calpain Expression during Differentiation of Rat Satellite Cells in Primary Cultures in the Presence of Heparin or a Mimic Compound

We have synthesized dextran derivatives called RGTAs (for regenerating agents) that were designed to mimic some of the properties of heparin or heparan sulfate to interact with and protect heparin binding growth factors. Some of these growth factors have been described to be involved in myogenesis control. In previous studies, we have shown that muscle regeneration in adults could be greatly enhanced in vivo by treatment with RGTA. Since muscle regeneration occurs through the activation of satellite cells, in the present study we have used primary cultures of rat satellite cells and treated them with the heparan sulfate analogue RGTA or heparin in order to stimulate their growth and differentiation. We also studied the effect of these substances on calpain (calcium-activated neutral proteases) expression in these cultures. Indeed, several reports, principally based on fetal myoblast cultures or myogenic cell lines, have suggested that calpains might be involved in myoblast fusion during myogenic differentiation. We therefore studied the expression of microcalpain (mu-calpain), millicalpain (m-calpain), and calpain 3 in the course of differentiation of these satellite cell cultures in the absence or in the presence of heparin or of a mimic compound (the RGTA RG1282). RGTA and heparin were shown to have a dual effect on satellite cell proliferation and differentiation: RGTA stimulated proliferation with a maximum dose effect at 1 microgam/ml. Heparin used at concentrations similar to those of RGTA was less efficient at stimulating proliferation. Both substances were shown, however, to induce precocious and enhanced differentiation of satellite cells. We showed by quantitative RT-PCR analysis that mu-calpain, m-calpain, and calpain 3 mRNAs were expressed in satellite cell cultures in proliferating myoblasts (day 3) and differentiating cultures (days 7 and 12). The level of mu-calpain mRNA was increased by a factor of 3 during differentiation of satellite cells, whereas the level of m-calpain mRNAs was slightly increased at day 12 only, and calpain 3 mRNA was slightly reduced in these differentiating cultures. Interestingly enough, RGTA and heparin, which both strongly increased differentiation, reduced the expression of the mu- and m-calpains and slightly increased that of calpain 3 in differentiating cultures. These results showed that there was no correlation between the extent of myoblast differentiation and the level of calpain expression in satellite cells grown in primary cultures and underscored the differences between these adult cells and fetal myoblasts.     

Hydrostatic pressure and calcium-induced dissociation of calpains

The dissociation of mu- and m-calpains was studied by fluorescence spectroscopy under high hydrostatic pressure (up to 650 MPa). Increasing pressure induced a red shift of the tryptophan fluorescence of the calcium-free enzyme. The concentration dependence of the spectral transition was consistent with a pressure-induced dissociation of the subunits. Rising temperature increased the stability of calpain heterodimers and confirmed the predominance of hydrophobic interactions between monomers. At saturating calcium, the spectral transition was not observed for native or iodoacetamide-inactivated calpains, indicating that they were already dissociated by calcium. The reaction volume was about -150 ml mol-1 for both isoforms, and the dissociation constants at atmospheric pressure are approximately 10-12 M and 10-15 M for mu- and m-calpains, respectively. This result indicates a tighter interaction in the isoform that requires higher calcium concentration for activity.     

The mechanisms of reduction of protein mixed disulfides (dethiolation) in cardiac tissue

Dethiolation of proteins (reduction of protein mixed disulfides) by NADPH-dependent and glutathione (GSH)-dependent enzymes, and by nonenzymatic reaction with GSH, was studied by electrofocusing methodology with glycogen phosphorylase b and creatine kinase as substrates. Phosphorylase b was not rapidly dethiolated by reduced glutathione alone, but a cardiac extract catalyzed rapid dethiolation by both an NADPH-dependent and a GSH-dependent process. In contrast, creatine kinase was actively dethiolated by GSH. This GSH-dependent dethiolation was not enhanced by a soluble extract of bovine heart. Creatine kinase was also not dethiolated by an NADPH-dependent process. Partial purification of the phosphorylase dethiolases showed that the NADPH-dependent dethiolase had both a high-molecular-weight and a low-molecular-weight component The properties of these components were similar to those of thioredoxin and thioredoxin reductase. These two components were sensitive to inhibition by phenylarsine oxide and inhibition was reversed by addition of a dithiol. In contrast, GSH-dependent dethiolation required a single component of low molecular weight. This process was less sensitive to phenylarsine oxide inhibition. These studies show that two cytosolic proteins, phosphorylase b and creatine kinase, were dethiolated by different mechanisms. Phosphorylase b was dethiolated by both NADPH-dependent and GSH-dependent enzymes found in a soluble extract of bovine heart. In contrast, creatine kinase was rapidly dethiolated nonenzymatically by GSH alone.